Capillary lumens are highlighted in green, the GBM in yellow. In B and C cell nuclei of podocytes are shown in violet, the PBM in light brown. **(A)** A group of 6 podocytes in the process of detachment. Only podocytes 1 to 4 still have contact with the GBM, whereas 5 and 6 are attached to the podocytes beneath by junctions (red circles). Within this cluster of podocytes, a communicating system of pseudocysts is seen that starts at bare areas of GBM (red dots). It may readily be suggested that the filtrate entering through bare GBM has to pass these spaces to reach Bowman's space; openings into Bowman's space are not hit in this section. Note that additional detaching podocytes (colored in various shades of violet) are gathered in another cluster. **(B)** Cluster of some 10 detaching or detached podocytes forming a cell bridge between the tuft and Bowman's capsule. The unlabeled cell within the parietal epithelium cannot be assigned by any plausible criterion to either parietal cells or to podocytes. The innermost cells are still partially attached to the GBM (arrows). The cells are interconnected to each other and still contain abundant pseudocysts that form a continuous chamber system starting from bare GBM (red dots) extending to the outermost layer. Note that the walls of many pseudocysts are undergoing shedding (asterisks). **(C)** Podocytes after almost complete detachment from the GBM are assembled as a cluster in Bowman's space interconnected to each other by cell contacts (red circles). The inner row of podocytes shows assemblies of small pseudocysts, the outer row appears as rounded cells. The outermost cells have contacts to Bowman's capsule (arrows). Male rats, growth stimulation with FGF-2 for 13 weeks. TEMs. Bars: **(A)** 5µm, **(B)** 4µm, **(C)** 2µm.

Initially, those clusters of podocytes are connected to the GBM by the innermost cells (**Figure 4A**). In later stages, they made contact via the outermost cells with the parietal epithelium (**Figures 4B,C**and **5**),thereby forming multicellular"bridges" between the GBM and the parietal epithelium. During this process, podocytes change in shape. Apparently by shedding of the pseudocysts, i.e., of their thin walls, the detached podocytes turned into groups of rounded cells without any matrix in between (**Figures 4** and **5**). Mitotic figures were never encountered in these cells.

In even later stages, these bridging clusters of detached podocytes initiate the formation of tuft adhesions to Bowman's capsule, i.e., firm cell and matrix connections between the GBM and the parietal basement membrane, as described previously (10). Concomitantly,the tuft itself underwent considerable shrinkage, including wrinkling of the GBM, loss of capillaries, and mesangial cells (**Figure 5**; to be described in detail in another context).

The detachment of podocytes that have come to protrude into the urinary orifice has already been described in various models and interpreted as the consequence of high shear forces within the urinary orifice acting on the cell bodies (5, 14). Here, we found that the final changes accounting for detachment are likely also rooted in the destabilization of FP attachment to the GBM, as described above, and that these podocytes frequently detach in groups and travel in groups along the tubule (not shown).

#### **QUANTIFICATION OF GLOMERULAR INJURIES**

Crucial steps of the above-described sequence in damage progression were quantified in 1µm sections. **Figure 1A** shows an entire glomerular profile indicating the injuries that were counted and compared. As seen in **Table 1** and in agreement with the clinical data of the former study (10), the damage was significantly more severe in males than in females.

**FIGURE 5 | Clustering of podocytes in Bowman's space is associated with shrinkage of the mesangio-capillary area**. The GBM is highlighted in yellow, capillary lumens in green, the PBM and cell nuclei of parietal cells in brown, podocyte cell nuclei in violet. Glomerular profile with a tuft that is broadly connected to Bowman's capsule by a large cellular crescent that surrounds the tuft from three sides. The mesangio-capillary area (delimited by the GBM) is small compared to the area occupied by podocytes. Note that podocyte cell bodies (visualized by their cell nuclei) have almost completely disappeared from central tuft areas being all contained within the crescent. Most of the podocytes have retained pseudocysts. The cluster of podocytes on top of the tuft does not show any contacts to the tuft nor to the PBM, whereas the major part of the crescent displays extensive contacts to the GBM as well as to Bowman's capsule. Nuclei of cells, which by no plausible criterion can be assigned as podocytes or parietal cells are uncolored. Note that the GBM is heavily wrinkled at several sites and the mesangial area appears to be collapsed with disappearance of capillaries at several loci (asterisks). Male rat, growth stimulation with FGF-2 for 13 weeks. TEM. Bar: 10µm.

# **DISCUSSION**

#### **GENERAL**

It is commonly agreed that the loss of podocytes underlies the development of progressive renal failure. Research of the past 20 years has shown that podocytes are lost by a unique mechanism, i.e., by detachment from the GBM as viable cells and excretion in the urine. This fact, in conjunction with their inability to replicate, puts podocytes in a precarious situation, unlike any other cell (5). Thus, the precise knowledge of why and how podocytes undergo detachment from the GBM is of great importance when searching for strategies to delay the progression to end-stage renal failure.

The present study has addressed this question in a model of excessive glomerular growth initiated by long-term stimulation with FGF-2 (10). Endothelial and mesangial cells proliferated

A glomerular profile was considered positive for the respective injury if a minimum of (i) three large pseudocysts, (ii) two areas of small pseudocysts (indicative of local detachments), (iii) four podocytes forming a cluster of connected cells in Bowman's space, and (iv) one adhesion was encountered.The weighted damage score includes all changes in a graded way (see Materials and Methods). \*p < 0.05, \*\*p < 0.001.

in this condition; podocytes participating solely by hypertrophy could not cope with the overall glomerular growth, underwent structural derangements and,finally,detached from the GBM. This growth-stimulating rat model is similar to a recently published transgenic rat model (15) in which the ability of podocytes for hypertrophy was restricted resulting, after growth stimulation by uninephrectomy, in a mismatch of endothelial and mesangial cell proliferation on the one hand and podocyte hypertrophy on the other, followed by podocyte loss through detachment. Both models mirror what happens in degenerating progressive glomerular diseases including all forms of secondary FSGS.

#### **THE DETACHMENT OF PODOCYTES FROM THE GBM**

The difficulty in the interpretation of the changes encountered in conjunction with podocyte detachment consists of separating the sequence of changes progressing to detachment from the sequence terminating in complete FPE, thus reinforcing attachment to the GBM. Changes belonging to either of these developments may be found at different sites of the same podocyte. This emphasizes the most important insight of this study: podocyte damage is local, even in the sense that the same podocyte undergoes detachment in one part of the cell, whereas at another part, it successfully reinforced its attachment to the GBM. This fact supports the view that, at least in this model, overall derangements of podocyte cell function are not the crucial causes underlying detachment but local imbalances between the forces accounting for attachment and those for detachment appear to play the major role.

This triad of hypertrophy, hypertension, and hyperfiltration has been convincingly shown to account for the progression of glomerular diseases under a great variety of circumstances (8, 9). These three factors are causally interconnected with each other but it remains an open question whether one of them plays a dominant role. Likely all three are involved in a mutually reinforcing process (16, 17). An elegant study showing in two mouse models of glomerulosclerosis that the development of glomerular injuries was significantly curtailed after unilateral ligation of the ureter emphasizes the effects of changes in filtration dynamics (18), whereas prevention of glomerular growth in a model with established hyperfiltration and glomerular hypertension also resulted in a marked decrease in sclerosis development (19), showing a major impact of growth. Also, in human beings, glomerular hypertrophy was found to be the decisive cofactor in minimal change disease accounting for progression to FSGS (20). The present study suggests that excessive hypertrophy of podocytes may be a major factor in progression to what extent hyperfiltration and glomerular hypertension develop in this model is unknown.

Therefore, damage development in the FGF-2 model may be taken to reflect the combined effects of all three factors.

Let us look in detail to a hypothetical local scenario that may start the process of detachment. A certain podocyte has reached its ultimate limit of hypertrophy that allowed it to cover a correspondingly increased area of GBM with normal FPs. In case the rheological situation with high filtrate flows overtaxes the available slit area, the only protective option remaining consists of sealing the slits by occluding junctions. This represents a most crucial event. The formation of cell–cell junctions between FPs in glomerular disease models has been described previously by several groups, characterizing them as an "occluding" type (21–27). More recent work has shown that they are a certain type of tight junctions (28, 29). Moreover, it was shown that tight junctional proteins increase several-fold in puromycin-induced nephrosis (29).

If sealing the slits is successful, compensation by adjacent podocytes is required, with these being exposed to higher local filtrate flows. If these podocytes have some reserve capacity to hypertrophy, they may adequately manage the situation.

In the case that the sealing strategy fails, a fatal situation will develop. Damage to the slit membrane will increase the shear stress on the involved FPs and may initiate its local detachment, thereby increasing the bare area of GBM. This will lead to local increases in flow (local hyperfiltration) and worsen the unstable situation. Thus, as soon as any detachment has started locally, the final struggle against the detachment of the entire cell has begun. If there is any chance to reverse this situation, it probably consists of spreading of neighboring, uninvolved podocytes on the GBM covering the denuded area. If this maneuver also fails, detachment of the first podocyte will proceed and may include adjacent podocytes.

The process of detachment seems to follow a rather stereotyped structural sequence. Circumscribed bare areas of GBM in between two adherent FPs of the adjacent podocyte (as the result of initial local detachments) frequently form the floors of domelike cavities overarched by attenuated cytoplasm of broadened primary processes. These spaces may be considered as a second type of pseudocyst associated, in contrast to the large pseudocysts, with bare GBM. They merge with each other, later with the large pseudocysts, finally forming a communicating system of extracellular spaces that starts above scattered areas of bare GBM and opens into Bowman's space through the narrow openings of the subpodocyte spaces. In many previous studies, this system of empty spaces has been termed "podocyte vacuolation," considered to consist of intracellular vacuoles ("blebs") (23,30–34),indicating a prenecrotic stage. Here, we show that for the most part, these are extracellular spaces bordered by intact cell membranes of viable podocytes.

It may readily be suggested that sites of bare GBM allow increased filtrate flows and are the sites of protein leakage, as frequently suggested and shown previously (22, 35–37). The filtrate has to find a way out of these spaces along routes offering some amount of resistance to the outflow. The resulting expansile forces on the walls of these spaces induce progressive bulging and detachment, leaving behind increasing areas of bare GBM. Consequently, the flow of the filtrate through the system of connected pseudocysts likely exerts expansile forces on the concerned podocyte, tending to lift it off the GBM (**Figures 4** and **5**).

Even if started locally at a single podocyte,the process of detachment generally comprises a group of podocytes. This may be another explanation for the observation by Ichikawa et al. (38) that damage to a podocyte always damages neighboring podocytes. In our view, the detachment of a podocyte, once started, will often take others along and may be difficult to arrest.

The interpretation of the sealing of filtration slits by "FPE" as a protective measure of podocytes against detachment conflicts with the widely held view that FPE represents the common initial derangement of podocytes leading to proteinuria potentially progressing to more severe glomerular damage (39). As discussed previously (5), FPE is necessarily accompanied by transient protein leakage as a consequence of the motility associated with the extensive local rearrangements. The transient character of this kind of proteinuria has recently been found in studies after Rac-1 stimulation (40–42) that is known to elicit FPE (39, 43). Progression of the damage to FSGS has not been found in these studies supporting our view of a protective impact.

#### **THE FATE OF DETACHED PODOCYTES**

In static structural studies, it is impossible to follow individual podocytes as they are translocated from one site to another. However, we can observe that along the course of the disease, podocytes are regularly encountered at other sites than where they are found under normal conditions. From such comparisons, we may deduce the routes how they came from site A to site B. We do not think that such movements of podocytes are effected by any active motility of the podocytes themselves, We think that detachment from the GBM exposes them to the rheological forces of filtrate flow pushing them toward Bowman's capsule and the urinary orifice.

The fraction of podocytes that detach directly in front of the urinary orifice generally will be swept into the tubular lumen (5, 10, 14). Surprisingly, during their passage along the tubule, they did not undergo anoikosis but, as known from several studies (e.g., 3 and 4), may have reached the renal pelvis as living cells.

Podocytes detaching from most other sites of the tuft may form massive cell accumulations within Bowman's space interconnected by occluding junctions with the innermost cells keeping contact to the GBM. During this gradual process of detachment, podocytes change in shape; they shed all the peripheral material forming the thin walls of pseudocysts, finally appearing as rounded cells. Frequently, such cell clusters establish, with their outermost cells, contacts to parietal epithelial cells, thus forming purely cellular crescents connecting the tuft and Bowman's capsule.

These observations are in full agreement with recent *in vivo* studies by multiphoton microscopy in two other models with podocyte detachment, i.e., unilateral ureter ligation (UUO) and Adriamycin nephropathy (44). In these studies, the formation of multicellular clusters of podocytes in Bowman's space was observed that made contact to Bowman's capsule; also, podocytes in single file moving down the tubule were seen. The authors also observed that the podocyte clusters made contacts to parietal cells by cell projections and nano-tubes. We found by TEM abundant occluding junctions between podocytes as well as between podocytes and parietal epithelial cells suggesting that they are responsible for the mechanical cohesion. As also verified in serial sections, the outermost cells of the podocyte clusters are fully separated from the GBM but fixed to inner cells of the cluster by occluding junctions. It appears that as soon as they come into contact with parietal cells, they establish junctional contacts to parietal cells, as well.

The further evolution of such clusters of detached podocytes attaching to Bowman's capsule, finally turning into tuft adhesions and crescents has been shown and discussed previously (10, 45, 46).

#### **Relationships to pathologies seen in human renal diseases**

It is quite obvious that the clustering of podocytes within Bowman's space and the shrinkage and collapse of the mesangiocapillary area have equivalents in human renal diseases, known as "cellular crescent," "cellular lesion," and "collapsing glomerulopathy"(CGP). The origin of the abnormal cells in these pathologies is under debate (47–50). In the present degenerative animal model, the cells clustering on the outer aspect of the tuft within Bowman's space are clearly derived from podocytes and do not proliferate. CGP combines the collapse of the mesangio-capillary compartment with hypertrophy and seemingly hyperplasia of the cells that take the place of podocytes on the outside of the GBM, assembled in clusters looking like crescents (51–54). The shrinkage of the mesangio-capillary area with wrinkling of the GBM and collapse of capillaries in CGP look identical to what is seen in the present animal model. Also, the clustering of cells in Bowman's space in CGP may look very similar to the clustering of detached podocytes in our animal model.

The great number of such cells seen in a cross-sectional profile of CGP seems incompatible with detachment as the only mechanism. However, as far as we know, the total number of podocytes on the surface of a collapsed tuft in CGP has never been estimated by stereological methods. Though a fully reliable calculation is presently not possible, we tried the following approach applying the recently published method by Venkatareddy et al. (55) to estimate podocyte number from a single glomerular profile in a single section. Since we do not have own biopsy material from a case of CGP, we took the glomerulus shown in **Figure 3** from the paper by Jhaveri et al. (56) and made the following calculation. In this glomerular profile, we counted 90 podocyte cell nuclei. From the listed magnification, the tuft diameter is about 100µm (healthy tufts are usually in the range 180–220µm, glomerular tufts are shrunken in CGP). The crosssectional area is, therefore, 7854µm2 and the area density of nuclei is 90/7854 = 0.01146. Using the estimated caliper diameter for human podocyte nuclei of 8.2µm (given in 55), we obtain the volumetric podocyte density of 0.01146/8.2 = 0.001397 podocytes/µm3 . The 100µm tuft diameter yields an estimated tuft volume of 523,585µm3 (much smaller than a typical tuft volume of 3,000,000µm3 ). Multiplying the density by the tuft volume gives an estimate of 731 podocytes in the tuft. To the degree that any podocytes are binucleate, this will be an overestimate of cell number.

Thus, it seems possible that the seemingly great number of podocytes on a shrunken tuft in CGP is all derived from detachment, gathering on the shrunken surface without any cell proliferation. This is supported by the fact that mitotic figures are generally not found in these cell assemblies in CGP. Major support in favor of detachment comes from the work of Bariety et al. (57) and also from findings in a transgenic mouse model of HIV-associated nephropathy (58).

#### **CONCLUSION**

Loss of podocytes represents the central event underlying the glomerular disease progression (4, 46). Here, in a model of continuous growth stimulation by FGF-2, we show that loss of podocytes occurs by detachment from the GBM as viable cells. This is a gradual process that starts focally at randomly distributed sites. The local origin strongly supports the view that the forces starting detachment consists of local rheological disturbances. The detachment of a podocyte, once started, may be difficult to arrest. However, podocytes have developed protective mechanisms against detachment, most important the sealing of the filtration slits followed by FPE.

#### **ACKNOWLEDGMENTS**

We thank Dr. KevinV. Lemley for extremely useful help, discussion, and insights. We gratefully acknowledge the continuous support of our work by the "Gotthard Schettler Gesellschaft fuer Herz-und Kreislaufforschung."

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 September 2014; paper pending published: 06 October 2014; accepted: 17 November 2014; published online: 12 December 2014.*

*Citation: Kriz W, Hähnel B, Hosser H, Rösener S and Waldherr R (2014) Structural analysis of how podocytes detach from the glomerular basement membrane under hypertrophic stress. Front. Endocrinol. 5:207. doi: 10.3389/fendo.2014.00207*

*This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Kriz, Hähnel, Hosser, Rösener and Waldherr. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Podocyte dedifferentiation: a specialized process for a specialized cell

# **Carl James May \*, Moin Saleem and Gavin IainWelsh**

Academic Renal Unit, University of Bristol, Bristol, UK

#### **Edited by:**

Maciej Wojciech Jankowski, Medical University of Gdansk, Poland

#### **Reviewed by:**

Elena Torban, McGill University, Canada David Andrew Long, University College London, UK

#### **\*Correspondence:**

Carl James May, Academic Renal Unit, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK e-mail<EMAIL_ADDRESS>
The podocyte is one of the two cell types that contribute to the formation of the glomerular filtration barrier (GFB). It is a highly specialized cell with a unique structure. The key feature of the podocyte is its foot processes that regularly interdigitate. A structure known as the slit diaphragm can be found bridging the interdigitations. This molecular sieve comprises the final layer of the GFB. It is well accepted that the podocyte is the target cell in the pathogenesis of nephrotic syndrome. In nephrotic syndrome, the GFB no longer restricts the passage of macromolecules and protein is lost into the urine. A number of phenotypic and morphological changes are seen in the diseased podocyte and in the literature these have been described as an epithelial–mesenchymal transition (EMT). However, there is a growing appreciation that this term does not accurately describe the changes that are seen. Definitions of type-2 EMT are based on typical epithelial cells. While the podocyte is known as a visceral epithelial cell, it is not a typical epithelial cell. Moreover, podocytes have several features that are more consistent with mesenchymal cells. Therefore, we suggest that the term podocyte disease transformation is more appropriate.

**Keywords: nephrotic syndrome, podocytes, proteinuria, epithelial–mesenchymal transition, dedifferentiation**

# **INTRODUCTION**

The podocyte is thought to be the target cell in the pathogenesis of nephrotic syndrome. The term "podocytopathy" is being increasingly used to describe disease that has arisen due to insult or injury to the podocyte. Minimal change nephropathy (MCN), focal segmental glomerulosclerosis (FSGS), diffuse mesangial sclerosis (DMS), and collapsing glomerulopathy (CG) are all thought to be podocytopathic nephrotic diseases (1). Irrespective of the cause, podocytes demonstrate dramatic morphological differences when there is nephrotic range proteinuria (2). Actin cytoskeleton rearrangement, slit diaphragm loss, and more cuboidal morphology are all hallmark features of the diseased podocyte (2). The field has tended to refer to this loss of typical phenotype as an epithelial–mesenchymal transition (EMT) event (3–5).

However, EMT is a highly plastic and reversible process: indeed, the word "transition" was chosen deliberately to reflect this transient nature. The phenotypic and morphological changes seen in diseased podocytes are only reversible if the insult is not very severe. In minimal change disease, the disease phenotype is reversible following glucocorticoid therapy. However, in FSGS, the changes in morphology and phenotype are not only irreversible but progressive (6). Therefore, using the term EMT implies an innate reversibility, which is not strictly accurate in the case of the podocyte. Therefore, it is important to reassess the extent to which the term EMT accurately describes the transposition from the healthy to the diseased podocyte morphology. Additionally, the podocyte is not a typical epithelial cell. Despite podocytes also being known as visceral epithelial cells, they retain several mesenchymal features (spindle shaped morphology and high levels of matrix interaction) and lack archetypal epithelial markers (cell–cell contacts that are based predominantly on P rather than

E-cadherin). Therefore, the podocyte fails to demonstrate the emblematic features of EMT. If the change in podocyte morphology and phenotype seen in disease does not fulfill the accepted criteria for an EMT event and is not necessarily transient in nature, then the usefulness of this term in this context must be questioned.

Immortalized podocytes *in vitro* respond to the classic inducer of EMT, TGF-B1. Following TGF-B1 treatment, human podocytes *in vitro* demonstrate increased levels of α-SMA, cadherin switch from P-cadherin to N-cadherin, and expression of the main effector transcription factors of EMT: SNAIL and SLUG (7). Similar phenotypic changes are seen in mouse podocytes *in vitro* when exposed to TGF-B1. Again suppression of P-cadherin along with suppression of ZO-1 and nephrin with concomitant upregulation of desmin, fibronectin, and collagen I is observed (8). *In vitro*, following 24 h exposure to TGF-B1, human podocytes lose their highly arborized morphology and adopt a more cobblestone-like morphology. The existence of similar phenotypic changes in both mouse and human podocytes in response to TGF-B1 is indicative of an evolutionarily conserved disease mechanism.

The pathological effects of such phenotypic changes within a cell-type, such as the podocyte, are severe. Foot process effacement is linked to a diminished ability to restrict urinary protein loss. This leads to runaway proteinuria and nephrotic syndrome. Effaced podocytes have less contact with the glomerular basement membrane, making podocyte loss much more likely. Additionally, the increased synthesis of extracellular matrix (ECM) components such as fibronectin and collagen I may contribute to GBM thickening. These are all hallmark features of diabetic nephropathy (9).

FSP1 is a fibroblast (mesenchymal) marker, which has been found in podocytes from patients with diabetic nephropathy. Healthy podocytes do not express FSP1; therefore, *de novo* expression of a mesenchymal marker is a pathological change in phenotype. Moreover, the frequency of FSP1+ podocytes in the urine has been linked to disease severity (8). FSP1+ podocytes have also been found in the glomeruli of FSGS patients (10).

The term EMT is over simplistic in this context, and does not encapsulate the process that is seen in the disease state *in vivo* and disease models *in vitro*. Broadly speaking, the morphological and phenotypic changes seen in diseased podocytes appear to be an EMT type event. However, in order for the assertion that this is an EMT event to be accurate, one must first consider to what extent the changes seen correlate with the definition of EMT.

#### **THE PODOCYTE: A SPECIALIZED CELL**

The podocyte is a highly specialized cell that is situated on the outer surface of the GBM. It comprises three structurally and functionally unique segments. These are known as the cell body, the major process, and the foot process (11). The cell body, the major processes, and the foot processes share a common actin cytoskeleton contractile apparatus similar to that found in smooth muscle cells or pericytes (11). The foot processes extend from the major processes and cover the GBM. Neighboring foot processes interdigitate, and where this occurs a modified cellto-cell junction known as the slit diaphragm is formed (12). The slit diaphragm forms the final layer of the glomerular filtration barrier (GFB) and has both charge and size selective properties (13).

The foot processes are the main functional unit of the podocyte. These contain loops of filamentous actin (F-actin) that can be assembled, disassembled, and bundled together in response to the changing requirements of the foot process. The tensile strength of F-actin and its concentration in the foot processes enable the podocyte to withstand the pressure of glomerular flow (14). It is this ability of actin, to be soluble as a monomer and then rapidly polymerize to provide structure and support that allows the redistribution of the podocytes foot processes. Bivalent molecules, such as α-actinin-4 and dystrophin can link bundled actin fibers for added strength (15). There are more than 100 proteins that are involved in the regulation of actin filament formation and breakdown indicating that these are essential procedures (16). The importance of the actin cytoskeletal regulation in the proper functioning of the podocyte is indicated by the number of socalled "nephrotic" genes whose protein products act on the actin cytoskeleton (17) (Table S1 in the Supplementary Material).

Such is the importance of the podocyte and the slit diaphragm in particular that there are multiple monogenic mutations that cause nephrotic syndrome as shown in Table S1 of Supplementary Material.

Structural and functional defects in the GFB result in an inability to restrict urinary protein loss. Nephrotic syndrome is defined by the triad of proteinuria, hypoalbuminemia, and edema. Proteinuria is defined by presence of non-physiological levels of a mixture of proteins in the urine (>200 mg/l) (20). However, in clinical practice, the albumin–creatinine ratio is more likely to be employed as it accounts for differences in urine dilution, i.e., a level of >30µg/mg demonstrates proteinuria (21). Structural and functional defects in the GFB result in an inability to restrict urinary protein loss.

Podocytes are terminally differentiated cells meaning that they are unable to proliferate. This lack of podocyte proliferation limits their capacity to recover from any damage. Therefore, podocyte injury is thought to be central to nephrotic syndrome pathogenesis. The range of podocyte injuries that can play a role in nephrotic syndrome pathogenesis is collectively referred to as podocytopathies. Many of these podocytopathies are caused by mutations in key genes. These genes are central to podocyte function; encoding either slit diaphragm proteins, transcription factors, or signaling mediators. The genetic causes of nephrotic syndrome are discussed in detail in an excellent review by Hildebrandt (17). In addition to genetic podocytopathies, there are also reactive podocytopathies. In these reactive podocytopathies, the podocyte is damaged by mediators in the microenvironment (1). These extrinsic podocyte stressors can be from several sources. They can be viral, toxic, immune-mediated, mechanical, or metabolic disorder derived.

#### **EPITHELIAL–MESENCHYMAL TRANSITION**

It is well accepted that a loss of the highly specialized podocyte structure limits the capacity of the final layer of the GFB to restrict urinary protein loss. The changes seen in diseased podocytes could be due to an EMT event.

It has been posited in the literature that since podocytes develop from mesenchymal cells via a mesenchymal–epithelialtransition (MET) event, it is reasonable to assume that the loss of mature podocyte characteristics is the reverse of this process (22). Glomerular development consists of four stages as follows: the vesicle stage, the *S*-shaped body stage, the capillary loop stage, and the maturation stage (23). The early podocytes can first be distinguished as a layer of cells at the proximal end of the *S*-shaped body. During this stage, the podocytes develop from the columnar epithelial cells (24). In turn, the columnar epithelial cells arise from the metanephric mesenchyme via a MET event (25). At this point, the podocytes express specific podocyte markers such as Wilms' tumor suppressor (WT1) and nephrin (26, 27). As the capillary loops form, the podocyte progenitors lose their lateral cell–cell contacts and begin to migrate. As the capillary loop stage progresses, the podocyte foot processes form. At this point, expression of the slit diaphragm proteins, nephrin, podocin, and CD2AP can be seen (28–30).

Epithelial-to-mesenchymal transition is a tightly regulated process by which epithelial cells lose their hallmark epithelial characteristics and gain the features of mesenchymal cells. EMT can be initiated in response to circulating mediators such as TGF-B1 (31, 32). During the process of EMT, the podocytes should lose their epithelial polarity, the cell-to-cell junctions (the slit diaphragm) will be altered, and the actin cytoskeleton will be rearranged (33). Following stimulation with TGF-B1, podocytes lose expression of nephrin and ZO-1 (34). As important slit diaphragm proteins, the loss of nephrin and ZO-1 expression is detrimental to the function of the podocyte. Not only does TGF-B1 lead to loss of epithelial characteristics in podocytes (as evidenced by the loss of nephrin expression) but also an increase in mesenchymal characteristics. Desmin is one such mesenchymal marker that is upregulated by TGF-B1, moreover, desmin upregulation by podocytes is seen in glomerular diseases where podocyte damage is a key feature (35).

These changes have severe consequences for the structure and function of the podocyte cell. The loss of epithelial cell–cell junctions during EMT is best represented by a loss of the slit diaphragm in the diseased podocyte. As previously described, podocytes demonstrate a clear change in their morphology and phenotype in the diseased state. Expression of the slit diaphragm is lost and the foot processes are effaced. However, does the loss of essential podocyte features seen in disease and disease models, both *in vivo* and *in vitro*, respectively, represent a typical EMT event?

Epithelial–mesenchymal transition has been categorized into three types: type 1, associated with implantation, embryo formation, and organ development, type 2, associated with wound healing tissue regeneration and organ fibrosis, and type 3, which is associated with cancer metastasis and progression (36). The distinctions between these subtypes of EMT are outside the scope of this review but are extensively covered in a review by Kalluri and Weinberg (36). For the purposes of this review, EMT will now be a reference to type-2 EMT. The criteria for type-2 EMT are listed in **Table 1**.

The changes in morphology and phenotype between healthy and diseased podocytes only partially satisfies most of the criteria for type-2 EMT and does not fit at all with the rest. The table demonstrates quite clearly the inaccuracies of describing this process as EMT. For instance, one of the criteria for type-2 EMT is a cadherin switch from E-cadherin to N-cadherin. While the podocytes do undergo a cadherin switch in the diseased state, this is from P-cadherin to N-cadherin. No evidence could be found in the literature for podocytes expressing DDR2 or HSP47. Neither there is clear evidencefor the nuclear translocation of TWIST. Otherwise, it is the lack of typical epithelial phenotype in podocytes that precludes them from matching this definition of EMT.

First of all, podocytes are atypical epithelial cells. While it is the case that podocytes demonstrate epithelial features, such as clear apical–basal cell polarity, they also exhibit mesenchymal features such as vimentin and intermediate filament expression (16). They also express features of differentiated mesenchymal cells, in particular smooth muscle actin, akin to a pericyte phenotype (42).

High migration capacity is a mesenchymal feature, while low invasive capacity is an epithelial feature, again demonstrating the dichotomous nature of the podocyte. The healthy podocyte maintains a dynamic range within which motility is regulated (43). It has been shown that plasma from patients with active FSGS significantly increases podocyte motility *in vitro* (44). The insult caused by the disease is increasing the mesenchymal characteristic of the podocyte. These hypermotile podocytes are not invasive due to the location of the podocytes on the outside of the GBM. The flux of hyperfiltrate through the GFB leads to a loss of podocytes in the urine. Indeed, as mentioned previously, viable urinary podocytes have been found with mesenchymal fibroblastic markers such as FSP1 (25).

**Table 1 | Hallmark characteristics of EMT**.

The dedifferentiation of podocytes seen in podocytopathic nephrotic syndromes only partially satisfies some of the criteria for a type-2 EMT transition. Clearly, the morphological and phenotypic changes seen in diseased podocytes are not obtained via a type-2 EMT event.

Podocytes express P-cadherin instead of E-cadherin; again this is atypical for an epithelial cell (7, 45). The generation of a spindle-like morphology is indicative of a mesenchymal phenotype. However, normal podocytes both *in vitro* and *in vivo* have a highly arborized structure that is consistent with the aforementioned spindle-like morphology. Following dedifferentiation, the podocyte actin cytoskeleton rearrangement causes foot process effacement leading to the morphology seen in **Figure 1**. This effaced morphology could be described as analogous to the typical epithelial "cobblestone" morphology. In this way, following dedifferentiation, podocyte morphology goes from the mesenchymal-like highly arborized morphology to the epithelial-like "cobblestone" morphology. The epithelial and mesenchymal characteristics of the podocyte are summarized in **Figure 1**.

Therefore, when describing podocyte dedifferentiation, one must be careful to consider to what differential state the podocyte is reverting.A partial reversal of the mature, partially mesenchymal state to a more immature epithelial state seen during the *S*-shaped body, and capillary loop stages of development could be termed as MET. However, complete dedifferentiation to the "mesenchymal rest" state of development could also conceivably occur, and be more accurately described as EMT, though in neither case is the terminology adequately nuanced.

The terminology "EMT" was adapted from the original term "epithelial–mesenchymal transformation" in order to emphasize the transient nature of this mechanism and the potential for its reversal (46). Clearly, the podocyte is not the average epithelial cell and its dedifferentiation does not entirely fit with the definition of EMT outlined in this article. This is more than simply a semantic argument. Evidently, mediators such as TGF-B1 can instigate EMT and induce podocyte dedifferentiation that is at least reminiscent of EMT. However, by constraining the conceptualization of this process by only thinking in terms of EMT, novel mediators and disease processes causing podocyte dedifferentiation may be missed. In light of the criticisms of this term to describe podocyte dedifferentiation in disease as EMT, perhaps the term podocyte disease transformation (PDT) is more appropriate. With transformation reflecting that PDT is less transient than EMT.

#### **GENETIC FORMS OF NEPHROTIC SYNDROME AND EMT**

Single gene defects causing NS tend to cause irreversible podocyte damage. This is to be expected since many of the genes that are known to cause nephrotic syndrome express proteins that are key components of the slit diaphragm (nephrin and podocin) or are essential to the specialized architecture of the podocyte (such as alpha actinin IV). However, the incorporation of transition in this term is deliberate. The diseased podocyte phenotype can be transient. Nephrotic syndrome is often treated with glucocorticoids and/or calcineurin inhibitors both of which have been shown to have direct effects on the actin cytoskeleton of the podocyte (47–49). One key renal developmental gene, known to cause NS when mutated, has been shown to result in features of podocyte dedifferentiation. This is WT1, a transcription factor both highly expressed and necessary for renal development (50), yet only expressed in the podocyte in the mature kidney. Mouse models of the human disease resulting from WT1 mutations, Denys–Drash Syndrome (DDS), reveal loss of podocyte ZO-1 expression and upregulation of podocyte TGF-B (51). Furthermore, loss of WT1 and re-expression of PAX2 and cytokeratin in podocytes have been described in cellular lesions in FSGS biopsies, implying an epithelial switch during acquired disease (52). Our own data examining the phenotype of conditionally immortalized podocytes from children with DDS confirms re-expression of PAX2, but also clear mesenchymal features suggestive of complete dedifferentiation (unpublished results). Thus, WT1 may be a key regulator of podocyte EMT both developmentally and as a target in disease.

The extracellular domain of nephrin forms the protein scaffold of the slit diaphragm (53). There are eight IgG-like motifs in the extracellular domain each of which contains two cysteine residues that are bound to each other via disulfide bridges. In addition, there are three "free" cysteine residues per nephrin molecule that are available to form disulfide bridges with bordering nephrin molecules (54). In this way, nephrin forms the scaffold for the slit diaphragm. The extracellular domain of nephrin interacts with other proteins in order to maintain the integrity of the barrier. Two such proteins are Neph 1 and Neph 2 (55). Clearly, the loss of most of the extracellular domain, as in patients with the Finmajor mutation, has massive implications for the formation of the slit diaphragm (56). The cytoplasmic domain of nephrin, of which part is missing in patients with the Fin-minor mutation, plays a role in the maintenance of the structural and functional capabilities of nephrin (56). The cytoplasmic domain also connects nephrin, and hence the slit diaphragm to the actin cytoskeleton of the foot process (57). It has been suggested that nephrin can bind directly to the actin cytoskeleton of the foot process via its cytoplasmic domain, while Yuan and colleagues have shown that nephrin is at least capable of binding to actin (58).

Podocin comprises 383 amino acids and has a hairpin structure such that both the C and N terminus are cytoplasmic (59). It is a raft-associated constituent of the foot process membrane that is localized at the insertion of the slit diaphragm itself (29). Within the raft, podocin can form oligomers, which lead to invagination of the foot process membrane, to which CD2AP and nephrin are recruited (60). In fact, a fully functioning podocin is required for nephrin transport to the membrane (61). This is the foundation of the slit diaphragm assembly (62). Between 10 and 28% of all nonfamilial childhood, SRNS cases are caused by recessive podocin mutations, such is the importance of this protein (62).

CD2AP is an adaptor molecule that possesses a coiled coil domain and 3 Src homology 3 (SH3) domains (59, 63). It also has an actin binding site at its NH2 terminus and is believed to contribute to dynamic actin assembly (64, 65). CD2AP is capable of interacting with nephrin and in complex with nephrin and podocin is able to recruit PI3K to the plasma membrane (63).

#### **CONCLUSION**

In reactive podocytopathies, the podocyte is injured by a circulating factor (66). It is clear, however, that the podocytes undergo a set of phenotypic and morphological changes during nephrotic syndrome. This process has been likened to EMT. TGF-B1 is a potent inducer of EMT. The research described in this review has relied heavily on *in vitro* work centered on the response of

common actin cytoskeleton. Neighboring foot processes regularly interdigitate. The gap between these interdigitations is bridged by a specialized cell–cell junction known as the slit diaphragm **(C)**. Following insult, the podocyte foot processes are effaced **(B)**. This concomitantly causes a loss of the slit diaphragm. The actin cytoskeleton is rearranged and the podocyte is no longer able to restrict urinary protein loss. A number of insults can cause the podocyte to lose its essential morphology by Patrakka et al. (68). **(D)** Epithelial and mesenchymal features of the podocyte are shown. Epithelial and mesenchymal characteristics as defined by Voulgari and Pintzas (40). The podocyte clearly possesses features that are both epithelial and mesenchymal. Based on this observation, it is not accurate to describe the podocyte as an epithelial cell. Hence, describing podocyte dedifferentiation as EMT is also an oversimplification.

podocytes to TGF-B1 treatment. As demonstrated, the podocytes do not undergo a typical type-2 EMT in response to TGF-B1. The new term "podocyte disease transformation" has been coined in order to distinguish this process from EMT.

A new technique known as transcriptome *in vivo* analysis (TIVA), has been developed to analyze the transcriptome of single cell populations *in vivo* without destroying the tissue (67). This technique could be employed to further study the changes in podocytes in disease models.

The traditional definition of a podocyte as an epithelial cell is clearly simplistic, and in fact, this is a uniquely differentiated cell, fit for a specific functional purpose that fulfills features of a partial mesenchymal and partial epithelial cell. It reaches this mature phenotype via immature mesenchymal and then epithelial stages, and therefore, dedifferentiation in disease could result in regression to either of these states. It will be important in understanding podocyte disease to understand the drivers of these changes, and recognizing the developmental features that are correlated with specific clinical conditions.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fendo.2014.00148/ abstract

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 22 July 2014; paper pending published: 28 July 2014; accepted: 01 September 2014; published online: 01 October 2014.*

*Citation: May CJ, Saleem M and Welsh GI (2014) Podocyte dedifferentiation: a specialized process for a specialized cell. Front. Endocrinol. 5:148. doi: 10.3389/fendo.2014.00148*

*This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 May, Saleem and Welsh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### **Agnieszka Bierzynska1 , Katrina Soderquest 2 and Ania Koziell 2\***

1 Academic Renal Unit, School of Clinical Sciences, Bristol University, Bristol, UK

2 Division of Transplantation Immunology and Mucosal Biology, Department of Experimental Immunobiology, Faculty of Life Sciences and Medicine, King's College London, London, UK

#### **Edited by:**

Barbara Lewko, Medical University of Gdansk, Poland

#### **Reviewed by:**

Kazunori Sango, Tokyo Metropolitan Institute of Medical Science, Japan Keiko Naruse, Aichi-Gakuin University, Japan

#### **\*Correspondence:**

Ania Koziell, Division of Transplantation Immunology and Mucosal Biology, Department of Experimental Immunobiology, Faculty of Life Sciences and Medicine, 5th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK

e-mail<EMAIL_ADDRESS>
After decades of primarily morphological study, positional cloning of the NPHS1 gene was the landmark event that established aberrant podocyte genetics as a pivotal cause of malfunction of the glomerular filter.This ended any uncertainty whether genetic mutation plays a significant role in hereditary nephrotic syndromes (NS) and confirmed podocytes as critical players in regulating glomerular protein filtration. Although subsequent sequencing of candidate genes chosen on the basis of podocyte biology had less success, unbiased analysis of genetically informative kindreds and syndromic disease has led to further gene discovery. However, the 45 genes currently associated with human NS explain not more than 20–30% of hereditary and only 10–20% of sporadic cases. It is becoming increasingly clear both from genetic analysis and phenotypic data – including occasional response to immunosuppressive agents and post-transplant disease recurrence in Mendelian disease – that monogenic inheritance of abnormalities in podocyte-specific genes disrupting filter function is only part of the story. Recent advances in genetic screening technology combined with increasingly robust bioinformatics are set to allow identification and characterization of novel disease causing variants and more importantly, disease modifying genes. Emerging data also support a significant but incompletely characterized immunoregulatory component.

**Keywords: podocyte nephropathy, podocytes, genetic predisposition to disease, gene mutation, nephrotic syndrome, nephrotic genes**

# **NEPHROTIC SYNDROME AND THE GLOMERULAR FILTRATION BARRIER**

Nephrotic syndrome (NS) is one of the commonest kidney conditions to affect children and adults. It manifests as excessive leak of protein into the urine, with interstitial edema occurring through albumin loss, aggravated by salt and water retention. Moreover, secondary knock-on effects on lipid metabolism, hemostasis, and the endocrine system through loss of key binding proteins serve to augment morbidity and mortality. NS represents a heterogeneous group of conditions, some idiopathic, others with a clear genetic basis. It may be highly kidney specific, associated with extra-renal developmental malformations or occur as a complication of systemic disease. The mainstay of treatment is immunosuppression, also effective in about 8–10% of genetic cases. Non-responders generally progress to renal failure with kidney transplantation, the only life-saving treatment.

# **THE ROLE OF PODOCYTES**

Despite the apparent heterogeneity, the unifying feature in NS is malfunction of the glomerular filtration barrier (GFB). This highly sophisticated macromolecular sieve with size and charge restricting characteristics is the primary ultra-filter of plasma by the kidney. It allows free flow of water and small solutes but restricts the passage of molecules >15 kDa; proteinuria occurs through loss of these normal permselective properties. The GFB comprises three layers: fenestrated endothelium, glomerular basement membrane (GBM) and podocytes, specialized terminally differentiated epithelial cells connected by slit diaphragms (SD), unique intercellular junctions interposed between interdigitating foot processes (1). Endothelial cells and podocytes have a negatively charged surface glycocalyx, which together with GBM sialoproteins and heparan sulfate gives the GFB an overall negative charge at physiological pH. Although size is the primary determinant of molecular filterability, recent detection of mutations in the main component of the podocyte glycocalyx, podocalyxin, in familial NS (2) supports additional charge selection through electrostatic repulsion.

Although damage to any of the three layers can result in significant proteinuria and kidney disease (3), podocytes are considered pivotal for maintaining barrier integrity. They encircle the glomerular capillaries creating a compact interdigitating network on the urinary side of the GFB. The connecting SDs integrate structural components of tight, adhesion, gap, and neuronal junctions to meet the diverse functional requirements of macromolecular filtering under high pressure while subjected to rapid changes in mechanical shear stress (4). SDs connect with the actin cytoskeleton to initiate signaling pathways that regulate podocyte function, namely plasticity of foot processes, mechanosensation, calcium flux, endocytosis, cell polarity, and cell survival. It is perhaps not surprising that podocyte gene mutations link to these key cellular functions. Podocytes also react very stereotypically to injury irrespective of whether this is acquired or resulting from an intrinsic

developmental defect with reorganization of the actin cytoskeleton,foot process effacement,molecular re-characterization of SDs, apoptosis, and detachment from the GBM (5). These dramatic morphological changes correlate with dysregulation of specific markers of podocyte differentiation including WT1, PAX2, and nephrin again signifying an underlying molecular basis. Additionally, de-differentiated podocytes can attempt healing sometimes by excessive proliferation, with the eventual outcome of repair or cell death attributable to complex interplay of poorly defined genetic and epigenetic mechanisms (6–8).

Clinical data support the hypothesis that the predominant cellular lesion targets podocytes; GBM gene defects usually result in insidious plasma protein leak whereas defects in podocyte/slitdiaphragm genes cause precipitous leak and NS. Moreover, there is evidence for a potential role for immune-regulation, particularly in childhood. Podocytes express cytokine and chemokine receptors as well as Toll Like receptors (TLRs) (9) and can respond to immune stimuli both *in vivo* and *in vitro*. Moreover, emerging evidence indicates that podocyte injury in NS may sometimes result from an unknown circulating factor, cytokine imbalance, or immune complex injury, with rare genomic variants potentially dictating susceptibility or resistance to immune triggers and the degree of subsequent response.

#### **ADVANCES IN MOLECULAR CHARACTERIZATION OF PODOCYTES**

Initial evidence supporting a molecular basis for NS came from positional cloning in familial cases and experimental animal models. Inherited diseases are frequently caused by mutations in genes with restricted expression patterns that generally do not cause early embryonic lethality but instead manifest at the time when gene function becomes critical for a specific tissue and subsequent survival (10). This is certainly the case for *NPHS1* and *NPHS2* (11, 12), the first podocyte genes cloned in NS encoding nephrin, an immunoglobulin superfamily member, and podocin an integral membrane protein both exclusively expressed at the podocyte SD supporting a key role in protein filtration. Mutations are associated with autosomal recessive NS manifesting at birth or early life during post-natal glomerular maturation and resultant increase in glomerular filtration.

Detection of *WT1* mutations in human syndromic NS (13), together with murine transgenic approaches using *WT1, CD2AP* (14), and *NEPH1* (15) underscored the contribution of developmentally regulated podocyte genes. Subsequent identification of mutations in *ACTN4* (16) and *INF2* (17) emphasized the central role of the actin cytoskeleton whereas gain of function mutations in *TRCP6* linked podocyte disease to abnormalities of cellular calcium flux and associated signaling pathways (18). Inherited defects of mitochondrial (19) and lysosomal components (20) have also been shown to lead to profound podocyte dysfunction, suggesting that they have high energy requirements and turn over. This also lead to the discovery that lysosomes participate in autophagy,increasingly recognized as protecting podocytes against injury (21, 22) Other genes such as *CD2AP* that participate in endocytosis and/or actin assembly have also been detected in glomerular disease providing further important clues about podocyte biology.

Familial and candidate gene studies have provided important information about the molecular basis of fundamental podocyte functions such as slit-diaphragm signaling, regulation of actin cytoskeleton dynamics, and cell–matrix interactions. However, until recently, only limited information about the overall genetic landscape was available, which posed a challenge to correct interpretation of genetic findings. Previously, podocytes could be extracted with high purity, but low cell yields during isolation hampered complete characterization of the transcriptome and proteome. Primary cells are difficult to grow in culture, so immortalized podocytes provided a useful tool to study gene function but incompletely mirrored their *in vivo* counterparts at molecular level. However, recent development of a double fluorescent reporter mouse model and optimized bead perfusion protocol, combined with FACS sorting, microarray, proteomics, and unbiased mathematical biology has yielded >5223 differentially regulated genes within the podocyte transcriptome and >1280 translated proteins (23). These studies have revealed that the podocyte proteome is enriched with plasma membrane, cytoskeleton, and neuronal-type proteins. Subsequent functional analysis of transcriptome and proteome data resulted in seven gene clusters: endoplasmatic reticulum, ubiquitination, cytoskeleton, nuclear elements, mitochondria, peroxisome and protein transport, and cell junction. Interestingly, nuclear and translational proteins were depleted, reflecting the post-mitotic nature of podocytes and alternative splicing was reduced, perhaps to ensure discreet cellular regulation. This has also enabled more meaningful RNA sequencing, which has identified eight miRNA's preferentially expressed in podocytes with significantly reduction fold change of miRNA target genes supporting miRNA-mediated mRNA degradation.

Improved description of the podocyte genome has also resulted in more effective interpretation of data from next generation sequencing (NGS) and to detect new variants or genes that cause or contribute to NS. NGS allows rapid sequencing of the whole exome or genome, and is becoming an increasingly powerful and cost effective method of analyzing the genetic contribution to any disorder. An unbiased approach can be taken to highlight new genes, new pathways, and new disease mechanisms through rigorous statistical genetics followed by functional experiments. This is especially relevant to NS where there is already known association with complex genetic determinants and this is likely to prove important mechanistically. However, as with any rare diseases, finding causal variants in NS suffers from twin problems of moderately low sample size and disease heterogeneity. In presumed Mendelian NS, the approach is still to search for genetic variants that segregate with disease within a family. In sporadic NS, we hypothesize that the condition will be caused by a rare variant in a small number of cases (limiting sample size and statistical power) and that the variant and, indeed the gene, may vary between cases. Added to this, although it is often assumed that there will be a heterozygous, homozygous, or compound heterozygous change that is causal, in common with related glomerular disorders, there is evidence for modifier effects in NS, at the genetic (24) and environmental level (25). This introduces the concept that NS is more complex than the Mendelian one podocyte gene, one disease situation as previously thought as variants are not always completely penetrant (26), synergistic activation between genes may

occur and in non-heritable disease, an environmental trigger is needed to cause disease. There is now clear evidence that in at least one podocyte gene, the pathogenicity of a particular allele depends on the trans-associated mutation (27). These concepts are supported by increasing evidence that expected phenotype– genotype correlations are not always adhered to and pathological changes may develop focally (not in all glomeruli) and segmentally (only in parts of a glomerulus), a pattern suggestive of an initial insult precipitated by an environmental stressor, perhaps a viral infection, that leads to localized cell injury (28).

Attempts to analyze NGS data in rare yet moderately complex diseases such as NS initially tend to focus on three main issues, namely how to best divide the genome for maximum signal, which statistical test to use and how to computationally assess the function of any variants found. Unlike common complex conditions, variants in conditions such as NS are too rare to provide meaningful sample sizes for statistical testing on their own and must be collapsed or aggregated to the level of gene, network, or other biologically meaningful unit. That is, testing for enrichment of variants in NS versus a control population will likely involve counting the number of rare protein altering variants in each gene or other biological unit in cases and controls and finding genes with an excess of these rare variants, rather than searching for an enrichment of any one variant as would be the case in a common complex Genome Wide Association Study (GWAS) approach. In addition to deciding on the biological unit by which to combine variants, decisions also need to be made about the statistical techniques used to combine and then analyze variants (29, 30). There are then several databases and algorithms by which the functional deleteriousness of potential variants can be assessed [(31): CADD; (32): ClinVar; (33): HGMD; (34): SIFT; (35): PolyPhen]. These might be used throughout the analysis to remove samples with known causal variants before statistical testing, or choose likely deleterious variants during testing. They can also be used to examine the likelihood that new variants might be causal prior to investing resources in replication studies or functional testing *in vitro* or *in vivo.* Nonetheless, understanding genotype/phenotype relationships without powerful systems biology tools in place has become increasingly complicated as emerging NGS data reveal not only considerable genetic heterogeneity but also evidence showing that factors such as epigenetic modifications, imprinting, non-coding RNAs, and RNA editing may play an important role in determining phenotype (36).

#### **PODOCYTE-SPECIFIC GENES: THE OLD AND THE NEW**

Mutations in 45 genes have been associated with familial and sporadic NS to date (**Figure 1**). These disrupt function either through SD disassembly, damaging cell architecture or metabolism, disturbing cell–matrix interactions, and/or impeding signaling pathways. All are expressed in podocytes, but there is increasing evidence to suggest that other systems, including immunoregulatory, play a role. Moreover, mutations in podocyte genes currently only explain 20–30% of familial and 10–20% of sporadic NS. From a clinical perspective, the renal phenotype rarely correlates absolutely with genotype as histology overlaps, suggesting a final common pathway to glomerular damage. Nonetheless, mode of inheritance and age of onset can give important clues. Gene mutations detected in early life are biased toward developmental genes, podocyte/SD/GBM malformation, and autosomal recessive inheritance whereas in later life, mutations are more frequently autosomal dominant (AD), preferentially affecting genes that participate directly or indirectly in regulation of the actin cytoskeleton. Moreover, combined gene defects in more than one podocyte gene may play a role in the development of NS, for example, mutations in both *NPHS1* and *NPHS2* can cause a tri-allelic hit modifying phenotype (37), and bi-allelic trans-heterozygosity has been described for *CD2AP* and *NPHS2* in sporadic NS (38). Additionally,*ADCK4* appears to modify CoQ10 (39) Furthermore, R229Q, a nonneutral *NPHS2* polymorphism that may predispose to NS in adults appears pathogenic only when associated with 30 *NPHS2* mutations in trans- (27), or deleterious mutation in another podocyte gene (37). Another emerging layer of complexity is miRNAs, for example,miR-193a downregulates the expression of WT1 in transgenic mice resulting in rapid and progressive NS (40). Other genes regulate podocyte differentiation and SD function by interacting synergistically on common enhancers or repressors, e.g., LMX1B, which combinatorially regulates *NPHS2* with FoxC (41). This underlines the likely diversity of genetic interaction within podocytes, supporting a requirement for additional environmental factors and/or modifier genes for full phenotypic expression.

**Table 1** summarizes all genes currently associated with human NS. Broadly speaking, these can be categorized on the basis of function substantiated by the predicted effect of mutations. SD associated and adaptor proteins normally communicate between the SD and the podocyte cytoskeleton, forming multimeric signaling complexes associated with lipid raft micro-domains coordinating actin remodeling, cell survival, and endocytosis. The prototypes are nephrin (*NPHS1*), and podocin (*NPHS2)*, which interact with proteins such as CD2AP (42) and TRPC6 to adapt between SD and cytoskeleton through a number of cytoskeletal linkers including F-actin and synaptopodin (43). More than 236 mutations in *NPHS1* have been described (HGMD® Professional 2014.2), and 171 mutations in *NPHS2* (www.lovd.nl/NPHS2, HGMD® Professional 2014.2). *CD2AP* haploinsufficiency causes NS, and is associated with trans-heterozygosity (38)*.* Gain of function TRPC6 mutations cause AD NS mainly but not exclusively in adult life, by increasing calcium influx and dysregulating the actin cytoskeleton via aberrant signaling (18). Nuclear proteins regulate transcription, podocyte differentiation and homeostasis; mutations result in dysregulation of downstream targets often initiating catastrophic collapse of the entire podocyte-stabilizing system. *WT1* mutations cause Denys–Drash (intersex, Wilm's tumor, NS) and Frasier syndromes (intersex, NS, gonadoblastoma) and AD adult onset NS (44). *LMX1B*, a homeobox transcription factor, regulates SDs and is essential for the maintenance of the actin cytoskeleton. Haploinsufficiency causes Nail–Patella syndrome, a rare AD NS that can occur as just kidney specific disease (45). The most recent gene to be cloned is *WDR73,* its exact function in the podocyte is as yet unknown but mutations result in Galloway–Mowat syndrome, a rare association between microcephaly, diaphragmatic hernia, and SRNS (46). Proteins regulating the actin cytoskeleton are also targeted. *ACTN4* (α-actinin-4) causes AD adult onset NS with incomplete penetrance. Here, mutant proteins show stronger affinity for F-actin and impair cytoskeletal function. Another actin regulating gene, *INF2* belongs to the formin family and normally homodimerizes to inhibit actin depolymerization.

Mutations are detected in a significant proportion of familial but not sporadic NS. Mutations in *PLCe1* (phospholipase C epsilon) mostly cause early-onset NS by dysregulating the cytoskeleton and signaling, and may be variably penetrant with some missense mutations resulting in later onset disease responding to immunosuppression (47). *PLCe1* mutations may also co-exist with trans-heterozygote mutations in other podocyte genes. *PTPRO* encodes a protein tyrosine phosphatase receptor located on the apical side of podocyte foot processes that controls the glomerular pressure/filtration rate (48). Only two splice-site mutations have been detected in *PTPRO* and cases partially responded to immunosuppression (49). Recently, mutations in *ANLN* (anillin), another F-Actin binding protein,were detected in familial NS (50), and other mutation screens of inherited disease have identified *ARHGDIA* (51) and *ARHGAP24* (52), underpinning a key role for RHO GTPases and Rho–Rac signaling in regulating the podocyte

cytoskeleton. *MYO1E* (53) and *MYH9* (24), mutated in autosomal recessive NS regulate actin cytoskeletalfunction and cell shape. *APOL1* mutations also result in increased NS in African Americans but the mechanism remains unclear (54). Mutations affecting protein expressed in the GBM may also disrupt GFB functions sufficiently to cause NS. *LAMB2* mutations, encoding laminin β2, cause Pierson's syndrome characterized by early-onset NS with or without microcoria, depending on the severity of the mutation (55). *ITGA3* and *ITGB4* encode integrins thought to be mainly passive GBM stabilizers, with mutations causing hyperglycosylation that prevents formation of functional integrins manifesting as NS and skin defects (56). COL4A3, A4, and A5 are key GBM proteins that cause Alport's syndrome; recent data suggest caution in interpreting COL4A variants detected in apparentfamilial NS (57). Mitochondrial and other rare metabolic syndromes also present with NS, by disrupting podocyte metabolism.

**Table 1 | Genes directly associated nephrotic syndrome**.

(Continued)

#### **Table 1 | Continued**

AR, autosomal recessive; AD, autosomal dominant; CNS, congenital nephrotic syndrome; NS, nephrotic syndrome; ?, inheritance neither clearly autosomal dominant or recessive, possibly complex.

Initial identification of monogenic defects correlating with morphological findings in podocytes in health and disease provided important mechanistic insights. However, this is now too simplistic, a model; podocyte genetic mechanisms are becoming increasingly complex, perhaps not surprising considering their super-specialist role within the GFB. Multiple levels of control involving modifier genes and di-genic and multi-genic inheritance are undoubtedly necessary to deal with epigenetic events and environmental effects (58). It is also likely that some gene variants are not directly causative, but rather modifiers of phenotype that may even arise from primary immune injury or presence of a circulating permeability factor. The advent of NGS and increasingly sophisticated bioinformatics approaches pave the way for exciting exploration of these alternative mechanisms, which undoubtedly play a key role in podocyte pathobiology.

# **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 September 2014; paper pending published: 06 November 2014; accepted: 09 December 2014; published online: 23 January 2015.*

*Citation: Bierzynska A, Soderquest K and Koziell A (2015) Genes and podocytes – new insights into mechanisms of podocytopathy. Front. Endocrinol. 5:226. doi: 10.3389/fendo.2014.00226*

*This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2015 Bierzynska, Soderquest and Koziell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Podocyte pathology and nephropathy – sphingolipids in glomerular diseases

# **Sandra Merscher \* and Alessia Fornoni \***

Peggy and Harold Katz Family Drug Discovery Center and Division of Nephrology, Department of Medicine, University of Miami, Miami, FL, USA

#### **Edited by:**

Gavin Iain Welsh, University of Bristol, UK

#### **Reviewed by:**

Tadeusz Pawelczyk, Medical University of Gdansk, Poland Karlhans Endlich, Universitätsmedizin Greifswald, Germany

#### **\*Correspondence:**

Sandra Merscher, Peggy and Harold Katz Family Drug Discovery Center and Division of Nephrology, Department of Medicine, University of Miami, 1580 NW 10th Avenue, Batchelor Building, Room 628, Miami, FL 33136, USA e-mail<EMAIL_ADDRESS>Alessia Fornoni, Peggy and Harold Katz Family Drug Discovery Center and Division of Nephrology, Department of Medicine, University of Miami, 1580 NW 10th Avenue, Batchelor Building, Room 633, Miami, FL 33136, USA

e-mail<EMAIL_ADDRESS>
Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay–Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann–Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in postreperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed.

#### **Keywords: sphingolipid, podocyte, kidney disease, glomerular disease, S1P, ASMase, SMPDL3b, ceramide**

Sphingolipids, more precisely sphingomyelin, cerebroside, and cerebrosulfatide were first described in 1884 by Johann L. W. Thudichum who named them for their enigmatic ("Sphinx-like") properties (1). They are important components of the lipid rafts in plasma membranes of mammalian cells and thus contribute to the proper function of cells. Within the kidney, the function and survival of major cell constituents of the glomerular filtration barrier, i.e., podocytes, heavily depends on the integrity of lipid rafts. Podocytes are differentiated cells of the kidney glomerulus consisting of a cell body, major processes, and foot processes (FP). The FP of podocytes are linked to the glomerular basement membrane with their actin cytoskeleton. Processes from neighboring podocytes form a characteristic interdigitating pattern that leaves filtration slits in between them. The latter are bridged by the slit diaphragm (SD) that together with the glomerular basement membrane and the fenestrated endothelium plays an important role in the selective permeability of the filtration barrier of the glomerulus (2–4). Integrity of this filtration barrier is important in order to prevent the loss of protein into the urine (proteinuria) and mutations in genes coding for SD proteins cause proteinuria-associated nephropathies (5–9). Research of the past two decades has revealed an essential role of sphingolipids in glomerular disorders with podocyte involvement.

This review will focus on different types of sphingolipids and sphingolipid metabolites that have been implicated in the pathogenesis of sphingolipidoses of genetic and non-genetic origin with podocyte involvement. We will also discuss sphingolipid signaling in podocytes and its influence on the actin cytoskeleton.

# **BIOLOGY OF SPHINGOLIPIDS**

Sphingolipids are a diverse class of lipids with a varying degree of hydrophobic and hydrophilic properties. The hydrophobic region of sphingolipids consists of a longchain sphingoid base with generally 18 carbons, such as sphingosine, which is linked to a fatty acid via an amide bond. The hydrophilic region consists in the simplest case of a hydroxyl group in the case of ceramide. Fatty acids may vary in their composition but palmitic (C16:0) and stearic (C18:0) are most commonly present. More complex sphingolipids have sugar residues (glycosphingolipids) and phosphates as side chains (phosphosphingolipids) (**Figure 1**).

Glycosphingolipids such as GM1 and phosphosphingolipids such as sphingomyelins are commonly found in eukaryotes,

in some prokaryotes, and in viruses as components of plasma membranes and membranes of organelles such as lysosomes, endosomes, endoplasmatic reticulum (ER), and others. The fluidity of the plasma membrane is tightly regulated by ordered packing of cholesterol between the phospholipid molecules, mainly sphingomyelin (SM) and thus sphingolipids have an important structural function. Sphingolipids are localized in the outer leaflet of the plasma membrane where they are asymmetrically distributed. Lipid rafts or raft-related caveolae are sphingomyelin-rich microdomains of the membrane, which are also enriched with cholesterol and membrane-associated proteins. The formation of lipid rafts is critical for proper cell function, protein–protein interactions, and signal transduction. For example, conversion of SM to ceramide, locally at the plasma membrane, by sphingomyelinases (SMases) will have a direct effect on the biophysical properties of the membrane and cell function as ceramide accumulation will lead to the displacement of cholesterol from the plasma membrane thus altering lipid rafts and signaling properties (10–12). Likewise, an interruption of raft-dependent cell signaling or even cell death can occur as a consequence of a cellular depletion of cholesterol that can be achieved by the use of cholesterol-depleting agents such as beta-cyclodextrin, methyl-beta-cyclodextrin, or 2-hydroxy-propyl-beta-cyclodextrin.

In recent years, it has become clear that, besides being integral part of membranes and having a structural function, sphingolipid metabolites such as ceramides, sphingosine, sphingosine-1-phosphate (S1P), and others play also important roles as second messengers in many biological processes including cell growth (13), differentiation, migration, and apoptosis (14). Complex sphingolipids were shown to interact with growth factor receptors, extracellular matrix, and neighboring cells (15). In addition, studies in yeast mutants revealed that sphingolipids have an important role in cellular stress responses as sphingolipid mutants yeast grew normally under usual culture conditions but were unable to survive if challenged or stressed (16).

Ceramide represents the centerpiece of the sphingolipid metabolic pathway reviewed in (17). Ceramide can be synthesized *de novo* starting with the condensation of l-serine and palmitoyl-CoA by serine palmitoyl transferase (SPT) to generate 3-ketodihydrosphinganine. The latter is then reduced by 3-ketosphinganine reductase to sphinganine, which in turn is N-acylated by ceramide synthetase (CS) to produce dihydroceramide. Finally, dihydroceramide is converted to ceramide by the enzyme dihydroceramide desaturase. Ceramide can also be generated by hydrolysis from sphingomyelin (SM) by SMases producing ceramide and phosphocholine. For sphingolipid biosynthesis, ceramide can be converted to sphingomyelin. This reaction is catalyzed by sphingomyelin synthetase (SMS), an enzyme that transfers the phosphocholine head group from phosphatidylcholine (PC) onto ceramide simultaneously generating diacylglycerol (DAG). Finally, ceramide can be generated by breakdown of glycosphingolipids and galactosylceramide to dihydroceramide and subsequent hydrolyzation (**Figure 2**).

Once ceramide is generated, it can accumulate in the cell or it may be further metabolized. Phosphorylation by ceramide kinase will generate ceramide-1-phosphate whereas deacylation by neutral or acid ceramidases will generate sphingosine, which can be phosphorylated by sphingosine kinase to yield S1P. Endproducts of the ceramide catabolic pathway are ethanolamine-1-phosphate and C16 fatty aldehydes, which are generated from S1P lyase from S1P.

# **SPHINGOLIPIDS IN GLOMERULAR DISEASES**

In the past decade, it has become clear that there is an association between the accumulation of sphingolipids in the kidney and glomerular disease. The accumulation of sphingolipids in form of glycosphingolipids, ceramide, and ceramide metabolites has been described in several models of experimental and clinical nephropathy and is characteristic of some rare genetic glycosphingolipid (GSL) storage disorders. The observation that intracellular accumulation of sphingolipids in glomerular cells such as podocytes is also observed in the absence of genetic mutations and is associated with the development and progression of kidney disease suggests the existence of "acquired" sphingolipid storage disorders.

Most mammalian GSLs are synthesized from glucosylceramide (**Figure 2**) and are primarily present in the outer leaflet of the plasma membrane where they have important functions in mediating cell–cell interactions and modulating activities of proteins in their proximity. They are usually not uniformly distributed within the plasma membrane but cluster in lipid rafts (18, 19). Gangliosides are sialic acid-containing glycosphingolipids in which one or more *N*-acetlyneuraminic acids (NANA) is linked to the sugar chain and are essential components of plasma membranes (20). Gangliosides with one NANA include GM1, GM2, GM3, gangliosides with two NANAs are GD1a, GD1b, GD2, GD3, and the gangliosides GT1b and GQ1 are characterized by three and four NANAs, respectively. Gangliosides were first identified in nervous tissue but are also abundantly present in the kidney (21). GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, and GT1b are gangliosides present in normal rat glomeruli (22–24). GM3, GD3, and disialosyllactosylceramide (*O*-acetyl GD3) are the most abundant gangliosides present in kidney and 9-*O*-acetyl GD3 is a podocyte specific ganglioside (25, 26).

**FIGURE 2 | Sphingolipid metabolism**. Ceramide is the centerpiece of the sphingolipid metabolic pathway and can be synthesized de novo from L-serine and palmitoyl-CoA (green), through hydrolysis of sphingomyelin (yellow), or through hydrolysis of glycosphingolipids and sulfatites (purple). Ceramide can also be synthesized from sphingomyelin through the action of sphingomyelinases, or from ceramide-1-phosphate through the action of ceramide-1-phosphate phosphatase. Finally, ceramide can be further catabolized (blue) to sphingosine and sphingosine-1-phosphate, which are biologically active metabolites and finally to ethanolamine-1-phosphate and

**SPHINGOLIPID ACCUMULATION AND GLOMERULAR DISEASE OF GENETIC ORIGIN**

Sphingolipidoses are inherited disorders leading to defects in the sphingolipid metabolism resulting in the accumulation of excess glycosphingolipids and phosphosphingolipids. It is interesting to note that different metabolites will tend to accumulate in different cell types, therefore leading to highly variable clinical–pathological findings.

Gaucher disease type 1 (OMIM #230800) is the most prevalent GSL storage disease and is characterized by an accumulation of glucocerebroside (GlcCer) in the affected tissues and cells, mainly in red blood cells, liver, and spleen. Gaucher disease is of autosomal recessive inheritance and it is caused by mutations in the acid beta-glucosidase 1 (*GBA1*) gene on chromosome 1q22 in the vast majority of patients (**Table 1**). This gene encodes for the enzyme that cleaves the beta-glucosidic linkage of glycosylceramide and mutations in this gene lead to accumulation of GlcCer. However, in some patients GlcCer accumulation is due to a lack of saposin C. Enzyme replacement therapy with macrophage-targeted recombinant human glucocerebrosidase is successfully used to treat patients with Gaucher disease (27–29) and drugs that block GlcCer

C16 fatty aldehydes. SPT, serine palmitoyl transferase; 3-KSR, 3-ketosphinganine reductase; CS, ceramide synthetase; DES, dihydroceramide desaturase; SMase, sphingomyelinase; SMS, sphingomyelin synthetase; PC, phosphatidylcholine; DAG, diacylglycerol; C1PPase, ceramide-1-phosphate phosphatase; CK, ceramide kinase; CDase, ceramidase; CS, ceramide synthase; SK, spingosine kinase; S1PP, spingosine-1-phosphate phosphatase; GCS, glycosylceramide synthase; GCase, glycosylceramidase; GalCS, galactosylceramide synthase; Gal-CDase, galactosylceramidase.

synthesis are currently being tested in clinical trials (30). Although the presence of renal pathology in Gaucher disease is rather rare, it has been described in some patients (31) and it is associated with the accumulation of GlcCer in form of Gaucher bodies in glomerular mesangial and endothelial cells and in interstitial cells of the kidney (31). Sphingolipid activator proteins (saposins A, B, C, and D) are glycoproteins that are encoded in tandem and are derived from a common precursor protein (prosaposin, PSAP). Saposins stimulate the degradation of GSLs by lysosomal enzymes. Defects in saposins are associated with the accumulation of lipids in affected tissues in lysosomal storage disorders. Humans with saposin C deficiency exhibit the clinical presentation of Gaucherlike disease (32). Combined deficiency of Saposin C and D in mice led to accumulation of GSLs and ceramide in brain and kidney due to decreased β-glucosidase activity (33).

Tay–Sachs disease (OMIM #272800) is a genetic disorder with autosomal recessive inheritance that is caused by mutations in the alpha subunit of the hexoseaminidase A (*HEXA*) gene on chromosome 15q23. Sandhoff disease (OMIM #268800) is caused by mutations in the beta subunit of the hexosaminidase gene B (HEXB) on chromosome 5q13 (**Table 1**). Both disorders are

**Table 1 | Sphingolipid accumulation in glomerular diseases of genetic and non-genetic origin**.

characterized by an accumulation of the ganglioside GM2 in the affected tissues and are clinically indistinguishable from each other. GM2 accumulation occurs mainly in the brain and liver but has also been found in the kidney (34, 35). Unlike in humans, targeted inactivation of the *Hexa* and *Hexb* gene in mice revealed phenotypical differences between the two models. Whereas *Hexa* knockout mice showed GM2 accumulation in the brain and membranous cytoplasmatic bodies in neurons in the absence of neurological manifestations, *Hexb* knockout mice showed profound neurological disturbances. These differences, which are not found in patients with the two diseases, are due to differences in the ganglioside degradation pathway between humans and mice (36).

Fabry disease (OMIM #301500) is caused by mutations in the gene encoding alpha-galactosidase A (*GLA*) on chromosome Xq22, which leads to the systemic accumulation of globotriaoslyceramide (Gb3) (**Table 1**) and related glycosphingolipids in body fluids and affected tissues (37), mainly brain, heart, and kidney. Elevated levels of Gb3 are detected in plasma or urine of patients with Fabry disease (38, 39) and more recently, highly increased levels of globotriaosylsphingosine (lyso-Gb3) were also described (40, 41). In the kidney, Gb3 accumulation occurred mainly within lysosomal, ER, and nuclear markers of renal cells (42). The renal pathology observed in Fabry disease includes hypertrophic podocytes with foamy appearing vacuoles, characteristic inclusion bodies of glycolipids in podocytes (zebrabodies), and mesangial widening (43). Progressive podocyte injury due to accumulation of Gb3 and related glycosphingolipids was shown to be associated with albuminuria and foot process effacement. Thereby, podocyte Gb3 inclusion volume density and foot process effacement increased with age when compared with controls and correlated directly with proteinuria (44, 45). Enzyme replacement

therapy using recombinant human α-GalA is the primary treatment for patients with Fabry disease and was shown to attenuate renal complications, halt the progression of renal pathology, and prevent renal failure in patients with Fabry disease (44, 46). Studies in the alpha *GalA* knockout mouse, a mouse model of Fabry disease, revealed reduced levels of glycosylceramide and ceramide in plasma, liver, spleen, kidney, and heart possibly a consequence of Gb3 accumulation. The observation that enzyme replacement therapy in this model normalized glycosylceramide levels possible via increased Gb3 degradation further supported the hypothesis that Gb3 accumulation contributes to the phenotype observed in these mice (46). Thereby, targeting of recombinant α-GalA requires the expression of endocytic receptors, megalin, sortilin, and mannose-6-phosphate receptor (M6PR), which are expressed in human glomerular podocytes (47). Interestingly, lentiviral knockdown of α-GalA in human podocytes led to intracellular Gb3 accumulation, which was associated with a loss of mTOR kinase activity and dysregulated autophagy suggesting a link between autophagy and glomerular injury in Fabry disease (48).

Hereditary inclusion body myopathy 2 (HIBM2) (OMIM #600737) is a genetic disorder with autosomal recessive inheritance that is caused by mutations in the gene encoding UDP-acetylglucosamine 2-epimerase/*N*-acetylmannosamine kinase (GNE) on chromosome 9p13 (**Table 1**). GNE is a key enzyme in the sialic acid biosynthetic pathway, which catalyzes the first two steps in NANA biosynthesis, which are main components of gangliosides (49). The disease is a progressive neuromuscular disorder but no renal disorders have been reported in patients with HIBM2. Interestingly, knockin mice carrying a homozygous M712T Gne/Mnk mutation died perinatally in the absence of myopathic features but were characterized by a renal phenotype. The renal pathology observed included glomerular hematuria,segmental splitting of the glomerular basement membrane, proteinuria, and podocytopathy, including effacement of podocyte FP, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration to homozygous knockin mice was associated with improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities (50). Likewise, mice with a V572L point mutation in the GNE kinase domain show no apparent myopathies or motor dysfunctions but exhibited renal impairment accompanied by massive albuminuria. Histologically, kidneys of the mutant mice showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis, and abnormal podocyte foot process morphologies. This phenotype was partially prevented by the administration of *N*-acetylneuraminic acid (Neu5Ac) to the mutant mice (51). These studies indicate that hyposylation of podocyte glycoproteins including podocalyxin may contribute to albuminuria, foot process effacement, and splitting of the glomerular basement membrane and that treatment with sialic acid may represent a new strategy to prevent or treat glomerular phenotypes associated with HIBM2.

Farber disease or Farber lipogranulomatosis (OMIM #228000) is a genetic disorder with autosomal recessive inheritance that is caused by mutations in the gene encoding acid ceramidase (*ASAH1*) on chromosome 8p22, the enzyme, which is responsible for the degradation of ceramide into sphingosine and free fatty acids (**Table 1**). Lipid accumulation is mainly seen in the joints, tissues, and the central nervous system, but also in liver, heart, and kidneys. Ceramide accumulation in the kidney leading to a particular phenotype of lipogranlulomatosis was described (52).

Niemann–Pick is a genetic disorder with autosomal recessive inheritance that is caused by mutations in the *NPC1* gene on chromosome 18q11 (OMIM #257220), by mutations in the *NPC2* gene on chromosome 14q24 (OMIM #607616), and by mutations in the sphingomyelin phosphodiesterase-1 (*SMPD1*) gene on chromosome 11p15 (OMIM #257200). Mutations in these genes lead to the accumulation of lipids in form of cholesterol (NPC1 and NPC2 mutations) and sphingomyelin (SMPD1 mutation) (**Table 1**). Acid sphingomyelinase (ASMase) deficiency in Niemann–Pick disease due to mutations in the *SMPD1* gene leads accumulation of sphingomyelin in the affected tissues including the kidney. The occurrence of lipid-laden macrophages resembling foam cells was described in the bone marrow, liver, and kidney in patients with Niemann–Pick disease and in SMPD1 knockout mice (53, 54). Enzyme replacement therapy using recombinant human ASM in *SMPD1* knockout mice led to significant improvements in the organs of the reticuloendothelial system but neurological deficits remained (55).

Nephrotic syndrome of the Finnish type (OMIM #256300) is a genetic disorder caused by homozygous or compound heterozygous mutations in the gene *NPHS1* encoding for the SD protein nephrin (**Table 1**). Nephrotic syndrome of the Finnish type occurs in association with deposits of disialoganglioside *O*-acetyl GD3 (56). Accumulation of galactosylceramides, mainly sulfatides, was also described in nephrotic syndrome of non-genetic, idiopathic orgin (57). However, what causes the accumulation of *O*-acetyl GD3 in nephrotic syndrome remains unclear. Saposins do not seem to play an important role as mRNA expression in diseased kidneys was found normal and no mutations in the PSAP gene were found in cDNA clones (56). It has been suggested that tumor necrosis factor alpha (TNFα) and CD95 may play an important role in the pathogenesis of nephrotic syndrome. TNFα and CD95 were found significantly increased in patients with nephrotic syndrome (58). It was shown in lymphoid and myeloid tumor cells that accumulation of GD3 induced Fas (APO-1/CD95)-mediated apoptosis in a caspase-independent manner that was the consequence of the disruption of the mitochondrial transmembrane potential. This phenotype was prevented by pharmacological inhibition of GD3 synthesis (59). In other studies, an important role of membrane-associated ASMase was suggested in Fas-mediated apoptosis as activation of ASMase leads to the generation of free ceramide, which then can be converted to GD3 (60, 61). A similar pathway has been recently described in TNFα-mediated apoptosis (62, 63). Significant 9-*O*-acetyl GD3 accumulation, together with increases in GM2 and GM4 gangliosides in glomerular cells was also observed after low level and long term lead exposure and was associated with decreased apoptosis in glomerular cells suggesting that GD3-O acetylation could represent a new strategy to attenuate apoptosis in renal glomerular cells and contribute to cell survival as observed during lead exposure (64).

# **SPHINGOLIPID ACCUMULATION IN GLOMERULAR DISEASE OF NON-GENETIC ORIGIN**

Diabetic kidney disease (DKD) is the most common cause of end-stage renal disease and renal failure in the US and podocyte injury and the consequent loss of podocytes (podocytopenia) is an important feature of DKD in patients with type 1 and type 2 diabetes (65–69). Increased levels of sphingolipids such as glycosphingolipids (70), ceramide (71, 72), sphingosine (73), and sphinganine (72, 73) have been described in the plasma of patients with diabetes. More recently, it has become clear that the intracellular sphingolipid composition in podocytes and other cells of the kidney glomerulus may contribute to the pathogenesis and progression of the disease (**Table 1**).

Several studies investigated the effects of streptozotocin (STZ) induced diabetes in rats on intracellular sphingolipid accumulation and its association with glomerular cell proliferation and glomerular hypertrophy. Accumulation of S1P was observed in rat glomeruli after 4 days of diabetes induction and was associated with an increase in neutral ceramidase and sphingosine kinase activity, the two enzymes involved in the conversion of ceramide to S1P (74). In another study, accumulation of GlcCer and GM3 occurred in the kidneys of rats 16 days after STZ-induced diabetes (75) whereas reduced GM3 and sialic acid content was detected in the glomeruli of rats 15 days after STZ-induced diabetes (76). Increased ceramide production due to increased expression of SPT, a key enzyme in the ceramide *de novo* synthesis pathway (**Figure 2**), was described in tubular epithelial cells and microvascular endothelial cells and was associated with increased apoptosis. Rapamycin treatment significantly reduced apoptosis and proteinuria indicating an important function of the Akt/mTOR pathway in STZ-induced DKD (77). We recently showed that the expression of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is increased in glomeruli from patients with DKD, in DKD sera treated human podocytes and in glomeruli of diabetic mice (db/db). Because SMPDL3b is a protein with homology to ASMase, we hypothesized that SMPDL3b may activate SM metabolic pathways leading to the accumulation of sphingolipids other than sphingomyelin. Increased SMPDL3b expression was associated with increased RhoA activity and apoptosis but prevented αVβ3 integrin activation via its interaction with soluble urokinase plasminogen activating receptor (suPAR) in human podocytes cultured in the presence of sera from patients with DKD and in *db/db* mice (78). Because ceramide, sphingosine, and S1P are known sphingolipid metabolites to accumulate in apoptotic cells, we determined the ceramide content in kidney cortexes of db/db mice and found ceramide levels to be decreased in kidney cortexes of these mice. We therefore concluded that increased SMPDL3b levels may lead to increased cellular sphingosine or S1P content in the kidneys of db/db mice as it is the case in glomerular mesangial and tubular cells in *db/db* mice (79,80), and in adipocytes of *ob/ob* mice (81). Taken together, these studies indicate a possible link between sphingolipid accumulation in form of S1P, GlcCer, and GM3 and glomerular proliferation and hypertrophy in DKD whereas the accumulation of ceramide and other ceramide metabolites such as sphingosine may contribute to podocytopenia observed in DKD. Thus, targeting sphingolipids and sphingolipid metabolites may represent a new strategy to treat patients with DKD.

Puromycin aminonucleoside (PAN) induced nephropathy is a model for human minimal change disease. Following PAN injection in rats, significant decreases in kidney GD3 and *O*-acetyl GD3 occurred in a dose- and time-dependent manner and preceded the development of proteinuria indicating a possible causative effect (82). Because sialoglycoproteins contribute significantly to the negative charge of the glomerular filtration barrier, it seems possible that decreases of GD3 and *O*-acetyl GD3 contribute to decreases in the negative charge of the filtration barrier and to the changes in glomerular permeability observed in PAN-induced nephropathy (83). Likewise, it was shown that PAN treatment of human podocytes led to a loss of sialic acid which was accompanied by increased generation of superoxide anions, a phenotype that was prevented by sialic acid supplementation (84).

HIV-associated nephropathy (HIVAN) is the classic renal disease associated with HIV infection. HIV-1 infection of renal tubular and glomerular podocytes leads to dedifferentiation and increased proliferation of podocytes (85, 86). Because podoyctes do not express HIV-1 receptors, it has been suggested lipid raft mediated endocytosis may facilitate the viral entry (87) underlining an important role for sphingolipids in mediating viral entry into the host cell. Most of our understanding of the pathogenesis of HIVAN has come from the Tg26 transgenic mouse model in which the gag/pol-deleted HIV-1 provirus is expressed. Transgenic mice show glomerular epithelial cell dedifferentiation and proliferation that is associated with proteinuria and renal failure. Renal histology revealed focal segmental glomerulosclerosis (FSGS) and microcystic tubular dilatation, resembling human HIVAN (85, 88). Studies in human podocytes in culture and transgenic mice showed that stable expression of Nef was sufficient to induce increased proliferation and loss of contact inhibition (89–92). In addition, recent studies have shown a strong association between

HIVAN and the *APOL1* gene on human chromosome 22 (93) and, although not found in glomeruli, significant accumulation of Gb3 was found in renal tubular epithelial cells of HIV transgenic mice (94) (**Table 1**) indicating a possible role for (sphingo-) lipid metabolism in HIVAN.

Focal segmental glomerulosclerosis is a glomerular disease that is characterized by proteinuria and progression to end-stage renal disease. FSGS is the leading cause of nephrotic syndrome and the most common cause of primary glomerular disease in adults (95). Several mutations in genes coding for proteins that are expressed in podocytes have been shown to cause FSGS. We will focus in this paragraph on non-genetic forms of FSGS, mainly FSGS recurrence after transplantation, which occurs in about one third of patients (96–98) and on primary (idiopathic) forms of FSGS. We recently reported an important role of sphingomyelin-like phosphodiesterase 3b (SMPDL3b) gene in FSGS. Studying 41 patients at high risk for recurrent FSGS, we showed that the number of SMPDL3bpositive podocytes in post-reperfusion biopsies was decreased in patients who developed recurrent FSGS. As mentioned above, SMPDL3b is a protein with homology to ASMase and we hypothesized that decreased expression of SMPDL3b may lead to decreased ASMase activity and accumulation of sphingomyelin contributing to the pathogenesis of FSGS (**Table 1**). Indeed,we were able to show that human podocytes treated with the sera from patients with FSGS had decreased SMPDL3b expression and decreased ASMase activity. In addition, this was associated with actin cytoskeleton remodeling and apoptosis, a phenotype that was prevented by overexpression of SMPDL3b in podocytes or by treatment with rituximab, a monoclonal antibody directed against CD20 that we have found to also bind SMPDL3b in podocytes. The percentage of cells characterized by actin cytoskeleton remodeling in form of a loss of stress fibers correlated with proteinuria suggesting an important role of sphingomyelin in the pathogenesis of FSGS (99). Because decreased SMPDL3b expression in podocytes *per se* does not cause actin cytoskeleton remodeling and apoptosis, it seems possible that accumulation of sphingomyelin renders podocytes more susceptible to apoptosis and may act as a master modulator of danger signaling in podocytes. Supporting an important role of SMPDL3b in actin cytoskeleton remodeling and apoptosis, it was recently demonstrated that administration of rituximab to baboons after xeno-kidney transplantation from pigs delayed, but did not prevent, the post-transplant occurrence of proteinuria. As in the case of our study investigating the role of SMPDL3b in FSGS, this study demonstrated that rituximab was also able to prevent pig podocyte injury and prevented decreases in SMPDL3b expression in podoytes after exposure to naive baboon sera in association with preservation of cell viability (100). Finally, sequestration of plasma membrane lipids by cyclodextrin was shown to prevent suPAR-mediated αVβ3 integrin activation in podocytes (101), a pathway that may be causative of proteinuria in FSGS. Because circulating suPAR levels are elevated in FSGS patients, associate with decreased SMPDL3b expression, and suPAR-dependent αVβ3 integrin activation in podocytes (78, 99, 101, 102), whereas cyclodextrin protects podoctyes from injury in DKD where SMPDL3b expression is increased in podocytes (78, 103), we investigated if SMPDL3b expression modulates the podocyte injury phenotype in these two kidney diseases. We

demonstrated that contrary to what is observed in FSGS, increased SMPDL3b expression in DKD prevented αVβ3 integrin activation via its interaction with suPAR and led to increased RhoA activity rendering podocytes more susceptible to apoptosis (78). These observations suggest that SMPDL3b and thus sphingomyelin or sphingomyelin catabolites are important modulators of podocyte function shifting suPAR-mediated podocyte injury from a migratory to an apoptotic phenotype. Therefore, modulating sphingolipids in podocytes may represent a new strategy to prevent or treat podocyte injury in FSGS and DKD.

### **SPECIAL CONSIDERATIONS FOCUSING ON S1P AND S1P RECEPTORS IN RENAL DISEASE**

Sphingosine-1-phosphate is generated by phosphorylation of sphingosine by sphingosine kinases (SPHK1, SPHK2) in response to various stimuli including growth factors, cytokines, G-proteincoupled receptor agonists, antigens, and others (**Figure 2**). Examples of factors that can transiently increase levels of S1P are TNFα and factors such as angiogenic growth factor, platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) all of which have been implicated in the pathogenesis of glomerular diseases. S1P signaling governs important cellular processes that determine cell fate. Thereby, extracellular S1P signaling is mediated via binding of S1P to G-protein-coupledreceptors (GPCRs). A family of five GPCRs termed S1P1–S1P5 [reviewed in Ref. (104, 105)] has been identified to date. Depending on the receptor subtype being expressed in the target cell, exogenous S1P can bind, and regulate a variety of important cellular functions including cell survival, cytoskeletal rearrangment, mitogenesis, cell differentiation, migration, and apoptosis. In the kidney, the receptors S1P1 (EDG1), S1P2 (EDG5), S1P3 (EDG3), and S1P5 (EDG8) are expressed in glomerular mesangial cells (106, 107) and whereas expression of S1P1, S1P2, S1P3, and S1P4, but not S1P5, was shown to be expressed in an immortalized mouse podocyte cell line (108). Increases in S1P synthesis mediated by sphingosine kinase, the use of S1P1 receptor agonists, such as FTY720 (an unselective S1P receptor agonist) and SEW2871 (a selective S1P1 receptor agonists) or by SPHK1 gene delivery were shown to protect from renal ischemia reperfusion injury (109–112), which is associated with increased ceramide expression (113–115), from DKD (108), and from various forms of glomerulonephritis (116–118) (**Table 1**). In addition, FTY720 and KRP-203, another S1P1 receptor agonist, have proven highly effective in preventing graft rejection in preclinical models of renal transplantation (119, 120). Whereas the activation of the S1P/S1P1 receptor pathway seems to be beneficial in the context of kidney disease, it was suggested that excessive S1P/S1P2 receptor pathway in renal tubular cells in DKD may play an important role in Rho kinase activation and renal fibrosis (80). Such mechanism could also explain activation of RhoA and increased apoptosis in podocytes in DKD as we previously described (78). In patients with lupus nephritis (LN), an inflammation of the kidney caused by systemic lupus erythematosus (SLE), a disease of the immune system, circulating S1P levels are increased (121). Likewise, S1P and dihydro-S1P levels in serum and kidney tissues from a mouse model of LN were elevated and treatment of these mice with a specific SPHK2 inhibitor, ABC294640 improved renal injury (122).

It was suggested that in the case of renal inflammatory disease, extracellular S1P induces COX-2 expression via activation of S1P2, subsequently leading to Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells. Although research of the past two decades has greatly advanced our understanding of the role of S1P and S1P/S1P receptor signaling in the pathogenesis and/or treatment of kidney diseases, more studies are needed to obtain a better and more detailed understanding of their physiological and pathophysiological significance *in vivo*. Certainly, targeting S1P/S1P receptor signaling pathways may represent a novel strategy to treat renal diseases.

# **SPECIAL CONSIDERATIONS FOCUSING ON THE ACTIN CYTOSKELETON IN RENAL DISEASE**

The kidney glomerulus is a highly specialized structure ensuring the selective ultrafiltration of plasma so that essential proteins are retained in the blood (3). Podocytes are glomerular epithelial cells consisting of a cell body, major processes, and FP. FP from neighboring cells are bridged by a 40-µm wide extracellular structure known as the SD (123, 124). Podocyte injury is an important feature of several renal diseases, including FSGS and DKD, in which independent of the underlying disease, a reorganization of the FP structure with fusion of filtration slits and apical displacement of the SD occurs (3, 125, 126). The SD is also required to control actin dynamics, response to injury, endocytosis, and cell viability. These observations make actin the common denominator in podocyte function and dysfunction (127, 128). Regulation of the podocyte actin cytoskeleton is therefore of critical importance for sustained function of the glomerular filter (129, 130). The connection of the actin cytoskeleton to the SD is mediated by several podocyte proteins such as CD2AP, Nephrin, ZO-1, and Podocin (131–134). Lipid rafts in podocytes are critical for the dynamic functional organization of the SD. Nephrin is partially associated with podocyte lipid rafts and co-immunoprecipitates with a podocyte specific 9-O-acetylated ganglioside. Injection of an antibody against the 9-O-acetylated ganglioside causes morphological changes of the filtration slits, resembling FP effacement (135) further underlining the important of intact lipid rafts and sphingolipids in the organization of the SD. Other sphingolipids such as S1P have also been implicated in cytoskeletal remodeling. S1P was shown to induce rapid reorganization of the actin cytoskeleton resulting in stress-fiber formation in 3T3 fibroblast, which was accompanied by transient tyrosine phosphorylation of focal adhesion kinase (FAK) and of the cytoskeleton-associated protein paxillin in association with RhoA activation in 3T3 fibroblasts (136). In renal mesangial cells, the serine/threonine protein kinase LIM kinase-1 (LIMK-1) was identified, which is involved in the regulation of cytoskeletal organization, as a ceramide-induced protein (137). Shiga toxin is a bacterial toxin that induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Shiga-toxin-mediated intracellular signals were shown to induce cytoskeleton remodeling in renal tubular epithelial carcinoma cells (138). VEGF and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. However, recently a new role for FLT1, i.e., the soluble form of FLT, sFLT has been described in podocytes where it binds to the glycosphingolipid GM3 in lipid rafts, promoting adhesion, and rapid

actin reorganization (139). Taken together, these studies underline the important function of sphingolipids in the formation of lipid rafts in podocytes thus contributing to the maintenance of a functional SD under physiological conditions.

#### **CONCLUDING REMARKS**

Sphingolipids play an important role in modulating podocyte function in glomerular disorders of genetic and non-genetic origin. Several genetic diseases are characterized by genetic mutations in genes that code for enzymes involved in the sphingolipid metabolism and are characterized by the accumulation of sphingolipids and sphingolipid metabolites in glomerular cells resulting in glomerular pathology. Thus, targeting sphingolipid metabolism in glomerular disease may prove beneficial in the treatment of proteinuric kidney diseases with glomerular involvement. Enzyme replacement therapy has proven to ameliorate disease progression in sphingolipid associated disorders of genetic origin such as Gaucher and Fabry disease. However, less is known about sphingolipid associated disorders of non-genetic origin. While ManNAc and rituximab are promising available therapeutic strategies for sphingolipid associated disorders of non-genetic origin, additional therapeutic strategies specifically targeting proteins such as SMPDL3b remain to be developed. Because sphingolipidoses of non-genetic origin seem to be more complex, additional research needs to be completed in order to elucidate the exact mechanisms by which sphingolipids cause injury to renal cells and thus contribute to the pathology of glomerular diseases.

#### **ACKNOWLEDGMENTS**

Sandra Merscher and Alessia Fornoni are supported by US National Institutes of Health (NIH, http://www.nih.gov/) (DK090316), by the Diabetes Research Institute Foundation, by the Nephcure Foundation, by the Peggy and Harold Katz Family Foundation, and by the *Diabetic Complications Consortium* (DiaComp). Sandra Merscher is supported by Stanley J. Glaser Foundation Research Award. Alessia Fornoni is also supported by the NIH/NIDDK (5U24DX076169) and by the National Center for Advancing Translational Sciences (1UL1TR000460).

#### **REFERENCES**

reduction therapy for Gaucher disease type 1. *Blood* (2010) **116**:893–9. doi:10.1182/blood-2010-03-273151

**Conflict of Interest Statement:** Sandra Merscher and Alessia Fornoni are inventors on pending or issued patents aimed to diagnose or treat proteinuric renal diseases. They stand to gain royalties from their future commercialization. Alessia Fornoni is consultant for Hoffman-La Roche, Alexion, and Mesoblast on subject matters that are unrelated to this publication.

*Received: 08 May 2014; paper pending published: 09 June 2014; accepted: 14 July 2014; published online: 30 July 2014.*

*Citation: Merscher S and Fornoni A (2014) Podocyte pathology and nephropathy – sphingolipids in glomerular diseases. Front. Endocrinol. 5:127. doi: 10.3389/fendo.2014.00127*

*This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Merscher and Fornoni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Role of protein kinase C in podocytes and development of glomerular damage in diabetic nephropathy

# **Beina Teng, Michelle Duong, Irini Tossidou, XuejiaoYu and Mario Schiffer \***

Department of Medicine/Nephrology, Hannover Medical School, Hannover, Germany

#### **Edited by:**

Barbara Lewko, Medical University of Gdansk, Poland

#### **Reviewed by:**

Undurti Narasimha Das, UND Life Sciences, USA Chris R. J. Kennedy, Ottawa Hospital Research Institute, Canada

#### **\*Correspondence:**

Mario Schiffer, Department of Medicine/Nephrology, Hannover Medical School, Carl Neuberg Street 1-OE6840, Hannover 30625, Germany e-mail: schiffer.mario@ mh-hannover.de

# **INTRODUCTION**

Diabetic nephropathy (DN) is the most common cause of the endstage renal disease (ESRD) (1). The glomerular changes in DN are characterized by excessive accumulation of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and mesangial expansion, which ultimately progress primarily to glomerulosclerosis and secondarily to tubulointerstitial fibrosis. The various cell types involved include glomerular endothelial cells, mesangial cells, podocytes, and tubular epithelia, which are all targets of hyperglycemic injury. However, accumulating evidence suggests that the extent of injury and loss of podocytes is a major prognostic determinant in both, type I and type II DN (2–6). As the terminally differentiated podocytes are believed to play a critical role in maintaining the integrity of the glomerular filtration barrier, effaced podocytes may contribute to the development of albuminuria, a hallmark of DN. Although primarily structure proteins were thought to be the key elements that compose the slit diaphragm initially, it has become clear that the slit diaphragm protein complex is a highly dynamic functional protein complex and is able to initiate cascades of signaling pathways that affect podocyte function (7). More recent data indicate that podocytes express receptors for many circulating hormones and growth factors, which also suggest that a more complex cross-talk between the kidney and other organs affected by diabetes may occur in health and disease (8).

Among various signaling kinases, the protein kinase C (PKC) family seems to play a critical role in the pathogenesis of DN (9). The activation of PKCs in the kidney is a well-known pathway of the diabetic milieu. The PKC family is involved in a variety of signal transduction pathways, cell proliferation, differentiation, cell cycle, and apoptosis. However, the role of PKCs on podocytes in DN has not yet been fully defined. This present review will give an overview of the general role of PKCs and summarize the

The early glomerular changes in diabetes include a podocyte phenotype with loss of slit diaphragm proteins, changes in the actin cytoskeleton and foot process architecture. This review focuses on the role of the protein kinase C (PKC) family in podocytes and points out the differential roles of classical, novel, and atypical PKCs in podocytes. Some PKC isoforms are indispensable for proper glomerular development and slit diaphragm maintenance, whereas others might be harmful when activated in the diabetic milieu. Therefore, some might be interesting treatment targets in the early phase of diabetes.

**Keywords: proteinuria, diabetic podocytopathy, glomerulosclerosis, nephrin endocytosis, effacement**

recent research into the regulatory role of PKCs in podocytes under diabetic conditions.

# **PKC SUBFAMILIES AND ISOFORMS**

All PKC isoforms contain a highly conserved catalytic domain and a regulatory domain. The catalytic domain consists of several motifs and is essential for the ATP/substrate-binding and catalysis, whereas the N-terminal regulatory domain contains an auto inhibitory pseudo-substrate domain and two discrete membrane targeting modules, C1 and C2. The sequence of pseudo-substrate contains an alanine in place of the serine/threonine phospho-acceptor site, but otherwise resembles a PKC substrate (10).

Protein kinase C isoforms are subdivided into three subfamilies based on differences of structure in their N-terminal regulatory domain. The isoforms α, βI, βII, and γ belong to conventional PKCs (cPKCs). The regulatory domains of cPKCs contain a C1 domain that functions as diacylglycerol (DAG)/phorbol 12-myristat 13-acetat (PMA) binding motif and a C2 domain that binds anionic phospholipids in a calcium-dependent manner (10). The novel PKCs (nPKCsδ, ε, η, and θ) also have two C1 domains and a C2 domain. The nPKC C2 domains lack the critical calcium-coordinating acidic residues, which is the distinct difference between cPKC and nPKC. The nPKCs can be maximally activated by agonists that promote DAG accumulation or by PMA, but they are insensitive to calcium. Atypical PKCs (aPKCs, ζ, and λ/ι) lack a calcium-sensitive C2 domain; however, they contain an atypical C1 domain, which binds PIP3 or ceramide, but not DAG or PMA. The activity of aPKCs is primarily regulated by protein–protein interaction and phosphorylation catalyzed by phosphoinositide-dependent kinase-1 (PDK-1). Although a few PKC isoforms are expressed in a tissue-specific manner, most are ubiquitously expressed.

# **REGULATION OF PKCs**

Unless it is post-translationally or co-translationally phosphorylated, PKCs are incapable of being activated by DAG or other cofactors (11, 12). PKC is translated as a catalytically inactive protein, is converted into an active enzyme by an initial phosphate addition and then into a mature form by further phosphorylation (11, 13, 14). In addition to phosphorylation on serine and threonine residues, PKCs also undergo phosphorylation by tyrosine kinases. PKCs are regulated by two sequential and equally critical mechanisms: phosphorylation triggered by PDK-1 and binding to DAG and/or other cofactors such as phosphatidylserine (PS) or phorbol ester (PE). Each mechanism regulates the structure, subcellular localization, and function of PKCs (15).

As a consequence of increased glycolytic flux, chronic hyperglycemia elevates the *de novo* synthesis of DAG and thus leads to an increased activation of DAG dependent classic and novel PKC isoforms in cultured bovine aortic endothelial cells and smooth muscle cells (16). Furthermore, high-glucose induced cellular levels lead to an increased generation of advanced glycation end products (AGEs), which initiate several signaling events by activating PKC, MAP kinase, and transcription factors such as nuclear factor-κB (NF-κB). This would increase the activity of various growth factors, such as TGF-β, and thereby alter expression of ECM proteins (17). In addition, under high-glucose conditions PKC is activated by higher concentrations of reactive oxygen species (ROS) generated following AGE:RAGE (AGE receptor) interactions (15, 18). In turn, the ROS are generated via NADPH-oxidase activated by PKC, ROS/PKC therefore can act in a cyclical manner to activate one another (19).

# **CONVENTIONAL PKCs IN DIABETIC NEPHROPATHY**

Among all the PKC isoforms, the role of PKCα in the pathogenesis of DN has been investigated intensively, and several studies have demonstrated that PKCα-deficient mice show a better outcome after streptozotocin (STZ) induced diabetes with less proteinuria and preserved nephrin expression (20, 21). Studies from our group underline the involvement of PKCα in proteinuria development in DN. The expression of PKCα in podocytes of patients with DN was increased. Mice were treated after STZ-induced diabetes with the synthetic PKCα inhibitor (GÖ6976), which prevented proteinuria development and led to preserved nephrin expression. Furthermore, we could show a central role for PKCα in endocytosis of the slit diaphragm component nephrin (22, 23). Quack et al. further concluded that proteinuria of diabetic mice is as a result of increased endocytosis of nephrin, which is mediated by a complex consisting of PKCα, protein interacting with c kinase-1 (PICK1), and beta-arrestin2. They found rising glucose levels go along with increased binding of beta-arrestin to nephrin *in vitro* as well as in *in vivo*, but only with preceding PKCα phosphorylating activity on nephrin. This fact and the indispensability of PICK1 suggest PKCα and PICK1 as possible drug targets in early stages of DN (23). These studies show that expression of PKCα is regulated by glucose concentration in the external milieu of the podocyte and that PKCα is directly involved in the maintenance of the slit diaphragm. High levels of PKCα in podocytes led to enhanced endocytosis of nephrin and instability of the slit diaphragm.

In addition, Menne and colleagues demonstrated that the highglucose induced downregulation of nephrin is probably caused by the PKCα mediated reduction of the transcriptional factor Wilms tumor 1 (WT-1), which has previously been described as a directly interacting binding partner of the nephrin promoter (24, 25). These findings are consistent with earlier studies, suggesting one of the PKC isoforms might be pivotal for the regulation of nephrin transcription and expression in podocytes (26), but not for CD2AP and Podocin as they remain at levels similar to those in non-diabetic kidneys (27).

Langham et al. showed that in diabetic patients treated with perindopril, an ACE inhibitor, nephrin levels preserved resembling those of non-diabetic subjects (28) and subsequently could be one factor contributing to the anti-proteinuric effects of ACE inhibitors. Another study suggests that the AGEs, which can be inhibited by aminoguanidine, are also implicated in the downregulation of nephrin in diabetes (29). Interestingly, several groups could demonstrate the reduction of PKC activity under the treatment with ACE inhibitors and aminoguanidine in diabetic subjects that could explain the nephrin protective effects (30, 31).

Most previous studies with specific PKCβ inhibitor ruboxistaurin (LX333531) *in vivo* and *in vitro* indicated that PKCβ isoform is primarily responsible for the high-glucose-induced renal effects in diabetes (32–36). Meier et al. tested this hypothesis by inducing DN in PKCβ deficient mice and did not find a significant preventive effect of PKCβ deficiency on albuminuria. In contrast to non-albuminuric diabetic PKCα −/− mice, the loss of the basement membrane proteoglycan perlecan and the podocyte slit diaphragm protein nephrin were not prevented in the PKCβ −/− mice under diabetic conditions (20, 37). However, the hyperglycemia-induced renal and glomerular hypertrophy as well as increased expression of ECM proteins was reduced in PKCβ deficiency diabetic mice.

In summary, the two important physiological features of DN, renal hypertrophy and albuminuria, are regulated through different PKC isoforms; PKCα is involved in the development of albuminuria and maintenance the glomerular filtration barrier structure, whereas the PKCβ-isoform contributes to hyperglycemiainduced renal fibrosis.