The detection rate of 2.5% is identical to a recent pediatric study from the UK, where disease-causing mutations in COL4A3- 5 genes were identified in six out of 255 individuals with an SRNS phenotype (19). Other studies found even higher rates of COL4 mutations particularly in adult SRNS cohorts, but these included heterozygous variants in COL4A3/4 which are unlikely to cause severe and progressive proteinuric kidney disease (17, 18). In line with other recent studies, all patients with COL4 gene mutations in our cohort showed FSGS on biopsy and no extrarenal disease manifestations were reported (17–19). These findings support the notion that genetic abnormalities collagen type IV formation may result in a phenotype of isolated nephrotic range proteinuria.

### **CLCN5-associated nephropathy**

A novel mutation in the CLCN5 gene (c.2000delC) was detected by exome sequencing in a family from the PodoNet cohort with apparently isolated nephrotic range proteinuria that had been classified as familial SRNS on clinical grounds. Both affected children were screened negative in 31 glomerulopathy-related genes. Mutations in CLCN5, which encodes a voltage-gated chloride ion channel expressed selectively in the renal tubule, are the major cause of Dent disease. Dent disease is an X-linked proximal renal tubular disorder; the key features include lowmolecular weight proteinuria, hypercalciuria, nephrocalcinosis, and/or nephrolithiasis.

The genetic finding triggered clinical investigation for a putative tubular defect, which revealed asymptomatic hypercalciuria in only one of two children studied. In the absence of nephrocalcinosis and nephrolithiasis, Dent disease was not considered as a potential differential diagnosis of nephrotic-range proteinuria before. The genetic classification of the disease has important therapeutic and prognostic implications for the affected children. The children had been (mis)-classified as familial SRNS based on the observed nephrotic range proteinuria (>1 g/m2 per day). Our case illustrates the genetic and phenotypic heterogeneity of proteinuric kidney diseases. The remarkable phenotypic variability of mutations in CLCN5 suggests that this gene should be included in NGS panel screening for hereditary proteinuric disorders.

### TREATMENT AND LONG-TERM OUTCOME OF SRNS

### Responsiveness of SRNS Patients to Intensified Immunosuppression

Whereas, the initial treatment of idiopathic nephrotic syndrome with oral glucocorticoids is well-established, we observed a large variety of the intensified immunosuppressive protocols and algorithms applied following the diagnosis of primary steroid resistance (**Figure 5**). In an analysis of immunosuppressive therapies applied in 612 SRNS patients in the first year after diagnosis of steroid resistance (2), 62% of children were treated with a single treatment protocol, 28% with two, and 10% with

three or more different immunosuppressive drug combinations as a polypragmatic therapeutic approach. While approximately three quarters of patients received Calcineurin inhibitors as first line therapy, others were administered intravenous steroid pulses, mycophenolate mofetil, oral or intravenous cyclophoshamide, or Rituximab (**Figure 5**). More than 80% of patients were cotreated with oral steroids initially, and more than 70% with renin angiotensin aldosterone system (RAS) antagonists.

Applying a standardized set of criteria to define treatment response and remission, we observed only 41% of included 612 SRNS patients to respond to any intensified immunosuppression with a relevant proteinuria reduction to IIS (24% with complete, 17% with partial remission), whereas 59% of patients remained unresponsive—often to several therapeutic interventions (**Figure 6**). The highest rates of complete (30%) or partial (19%) remission were achieved with CNI based protocols. These response rates are in the lower range of previously reported treatment experience, where complete remission was observed in 31% to 89% and partial remission in 19 to 38% of patients (9, 21– 25). The variation of the response rates may be related to the selection and composition of the individual study cohorts and the chosen set of response criteria.

The efficacy of CNI-based therapies in the PodoNet cohort was by far superior to steroid pulses, cyclophosphamide, and MMF monotherapy, all of which did not show any therapeutic effect in around 85% of patients as first line therapy (**Figure 6**) and were completely non-efficacious as second- or third-line therapies in CNI resistant patients. These findings confirm previous studies (26–31) and provide strong evidence against the use of these therapeutics in SRNS. Notably, B-cell depleting therapy with Rituximab induced complete remission in 44%, and partial remission in 15% of patients (1).

RAS inhibition has been demonstrated to lower proteinuria by 40–50% in patients with SRNS (32, 33). In the PodoNet cohort, RAS inhibition alone was associated with partial proteinuria remission in 21% and even maintained complete remission in 27% of patients (1). Hence, the frequent co-administration of RAS antagonists with immunosuppressive agents is a major confounder to the efficacy analysis of the latter.

Among the 602 cohort patients available for pharmacotherapy analysis 82% had sporadic disease, 74 (12%) confirmed genetic disease and 36 (6%) familial disease with negative genetic screening in all known podocytopathy genes.

### Do Patients With Genetic SRNS Respond to Immunosuppression?

Anecdotal clinical observations in previous case series and small retrospective studies suggested that individual patients with pathogenic mutations in podocytopathy genes might show some responsiveness to intensified immunosuppression. In total, 11 hereditary SRNS patients reported in literature developed complete remission and 17 partial remission of proteinuria while on calcineurin inhibitor and usually concomitant RAS antagonist therapy (4, 34–38). A non-immunological antiproteinuric action

(green), others as multi-drug resistant (red).

(2)].

of Ciclosporin A (CsA) mediated by stabilization of the actin cytoskeleton has been suggested based on experimental findings (39).

The PodoNet registry allowed to evaluate the largest cohort of children with hereditary podocytopathies to date exposed to calcineurin inhibitor and other immunosuppressive therapy. Complete or partial proteinuria responsiveness to pharmacotherapy was observed in 45% of children with sporadic disease and in 47% of those with familial, gene screening negative disease, whereas only 13% of patients with hereditary podocytopathies displayed any, mostly transient responsiveness to pharmacotherapy (2).

Transient complete remission was documented in only two out of 74 patients (2.7%) diagnosed with genetic disease in whom extended treatment and response data were available: one child with a WT1 mutation reportedly achieved complete remission for 2 weeks after start of CsA, followed by subnephrotic range proteinuria for > 11 years. Another child with NPHS2 associated disease achieved complete remission for 4–6 weeks, followed by a relapse and subsequently persistent proteinuria with progression to ESKD within 4 years. Transient partial remission with reduction of proteinuria to the non-nephrotic range was observed in response to CsA in another 8 patients (10.8%) with genetic disease (4 NPHS2, 3 WT1, 1 COQ6). Five of these returned to nephrotic-range proteinuria within <2.5 years, and four progressed to CKD stage 3–5 within <5 years. Importantly, 4 of 8 patients were co-treated with RAS antagonists. It is impossible to differentiate whether the transient responsiveness was related to calcineurin inhibition, supportive antiproteinuric RAS antagonist therapy or the natural course of disease with diminishing proteinuria due to worsening renal function. Notably, almost all patients with genetic disease and apparent CsA responsiveness progressed to ESKD soon despite ongoing therapy.

Hence, the findings in the PodoNet cohort argue against a relevant nephroprotective effect of calcineurin inhibition—or other immunosuppressive therapies—in children with genetic forms of SRNS and support the notion that such patients should be spared immunosuppressant side effects. On the other hand, the knowledge about the heterogeneity of underlying genetic disease mechanisms might help to develop specific podocyteprotecting treatment strategies in order to minimize proteinuria. A promising example of an innovative gene specific treatment

option is successful use of CoQ10 in children with SRNS due to genetic defects leading to CoQ10 deficiency (15, 16, 40). In the light of these findings and given the progress in the speed, comprehensiveness and cost efficacy brought about by nextgeneration sequencing, genetic screening should be considered in all SRNS patients at the time of diagnosis of steroid resistance prior to starting intensified immunosuppressive treatment.

Finally, the efficacy of other supportive and/or preventive treatment strategies including antiproteinuric therapies and treatment of hyperlipidemia have not yet been studied in detail in the PodoNet or other cohorts and await exploration in future studies.

### Long-Term Outcome and Risk Factors for ESKD

Historically, the prognosis of SRNS was mainly staged according to histopathologic findings, with limited predictability of medium- and long-term disease outcomes (41–46). Providing comprehensive clinical and biochemical information with up to 15 years of follow-up, the PodoNet database allows to assess the prognostic effect of early treatment responsiveness in the context of genetic and histopathologic findings, which provide a rationale for a evidence-based reclassification of SRNS into sporadic immunosuppression-responsive (32%), sporadic multidrug-resistant (24%), genetic, and familial SRNS (30%)(**Figure 7**).

The average overall ESKD-free survival of patients with primary SRNS in the PodoNet cohort was 74% at 5 years, 58% at 10 years and 48% at 15 years, in keeping with previous cohort studies in which 5-year renal survival ranged from 65 to 92% and 15-year survival from 34 to 72% (43, 45, 47–49).

Responsiveness to intensified immunosuppression was highly predictive of long-term outcomes: Whereas complete remission during the first treatment year was associated with 94% 15 year renal survival, only 37% of the multidrug resistant patients were not in end-stage disease 15 years after disease onset (2). A fraction of patients achieved partial remission; it is controversial whether such intermediate response patterns reflect pharmacological effects on the immune system or non-specific proteinuria-lowering effects of RAS antagonists and possibly calcineurin inhibitors. Notably, partial proteinuria reduction was associated with superior long-term renal survival compared to patients with multidrug resistant proteinuria. The other key determinant of long-term renal outcome was the identification a genetic disease cause (2): Three quarters of patients diagnosed with a hereditary podocytopathy progressed to end-stage kidney disease within 15 years, as compared to only 4% of SRNS patients with sporadic disease and responsiveness to immunosuppressive

analysis [with copyright permission (2)].

therapy (**Figure 8**). Long-term renal outcomes were remarkably similar in children with different genetic disease entities. The 10-year renal survival rate was 72% for NPHS2-associated nephropathy, 77% for WT1-associated disease and 71% for the less common podocytopathies (2).

Remarkably, the outcome of children with proven genetic disease was also inferior compared to patients with multidrugresistant disease in whom no genetic diagnosis could be established. The prognostic values of genetic diagnosis and immunosuppression responsiveness were found to be independent by multivariate analysis: ESKD risk was increased by 150% in patients in whom a genetic diagnosis was established whereas complete remission reduced renal risk by 87% in patients who achieved complete remission and by 50% in those who responded partially to immunosuppressive therapy (2).

Traditionally, the diagnostic categorization and prognostic evaluation in SRNS was based on histopathologic diagnosis. As expected, we found strong associations between histopathologic findings at time of diagnosis and long-term renal survival: ESKDfree survival was significantly higher for MCN than FSGS (79% v. 37% at 15 years) (**Figure 9**), and the overall ESKD risk of children diagnosed with FSGS was 4-fold higher compared to MCN (2). Children diagnosed with DMS showed the poorest outcomes, with a 20-fold increase of ESKD risk relative to MCN with progression to ESKD in 80% within 5 years after initial SRNS manifestation. Importantly, the prognostic value of FSGS and DMS prevailed even when adjusting for CKD stage at diagnosis, responsiveness to intensified immunosuppression, and the presence or absence of a genetic diagnosis (2). For instance, a patient with a genetic podocyte disorder, multidrug resistance, and a given eGFR will still have a nearly threefold higher ESKD risk with the diagnosis of FSGS compared with MCN. Hence, the independence of genetic and histopathological findings in the multivariate risk analysis suggests that histopathological assessment is still relevant in the genetic era.

Additional independent risk factors of progression to ESKD were identified as age <1 year and older than 5 years at disease onset and advanced CKD at initial presentation (2).

### Which Lessons Have We Learned From the Podonet Registry So Far?

The understanding of the underlying molecular, genetic and pathophysiological mechanisms of SRNS has profoundly advanced during the past decade. Established in 2009, the PodoNet registry has grown to one of the largest pediatric SRNS cohorts with more than 2000 children feeding clinical and genetic research. While the large size of the cohort is a major asset, the incompleteness of data reporting and biosample availability has been a limitation to data analysis and interpretation. Also, in the early years of the Network technological limitations

and restricted resources hampered genetic screening. These limitations were partially overcome in later years thanks to improved funding and the central implementation of podocyte gene panel sequencing.

The discovery of numerous novel genetic podocytopathies and the progress in diagnostic screening technology have already changed clinical practice during the time span of the PodoNet project. Initially, children underwent a diagnostic algorithm and were selectively screened for individual SRNS genes by means of Sanger sequencing based on clinical criteria and depending on age at disease onset. With the advent of NGS technologies genetic screening has become much more rapid, comprehensive and cost-effective and consequently rapidly transgressed from an optional adjunct to a key component of the diagnostic workup in SRNS. This transformation is well-reflected in the output of PodoNet as well as other study consortia (10).

The PodoNet network has been an active player in this field by contributing to gene discoveries, developing and systematically applying podocytopathy gene panels, and analyzing phenotypes and outcomes by comprehensive assessment of clinical, genetic, and histopathological findings as well as responsiveness to pharmacotherapies.

Our studies allow concluding that genetic screening should be initiated as soon as the diagnosis of steroid resistance has been made, in parallel to renal biopsy (**Figure 10**).

We obtained evidence that genetic forms of SRNS are largely non-responsive to intensified immunosuppressive therapy, which, in consequence, should be avoided in patients with genetic disease. Future research will address in detail the short- and longterm usefulness of RAS inhibition and other supportive treatment strategies in genetic podocytopathies.

Our studies also have highlighted the lacking specificity of the most common histopathological diagnoses with respect to identifying underlying etiologies. On the other hand, the histopathological diagnosis was found to retain some prognostic value even when the genetic status of a patient is known.

In immune-mediated forms of SRNS, calcineurin inhibition was demonstrated to be the most efficacious second-line therapy following the diagnosis of steroid resistance. Children resistant to calcineurin inhibitors are usually also resistant to other immunosuppressive agents. Moreover, initial responsiveness to calcineurin inhibitors is uniquely predictive of long-term preservation of kidney function.

The insights obtained from the PodoNet cohort will facilitate the development of rational evidence based clinical practice recommendations for SRNS. In view of the major prognostic differences associated with genetic findings and pharmacotherapeutic responsiveness, we propose to sub-classify SRNS patients as sporadic immunosuppression-responsive, sporadic multidrug-resistant, genetic, and familial SRNS.

### AUTHOR CONTRIBUTIONS

AT, BL-Z, FS contributed substantially to the acquisition, analysis, and/or interpretation of the data, participated actively in preparing the manuscript and approved the submitted final version.

### ACKNOWLEDGMENTS

The PodoNet project has been made possible by support received from E-Rare (German Ministry of Education and Research), the EU 7th Framework Programme (EURenOmics, grant 2012-305608), the Polish Ministry of Science and Education (grant N402631840), the German Research Foundation (Scha 477/11-1), and the Scientific and Technological Research Council of Turkey (TUBITAK) (grant 108S417).

multicentre trial by the Arbeitsgemeinschaft fur Padiatrische Nephrologie. Pediatr Nephrol. (2008) 23:1483–93. doi: 10.1007/s00467-008- 0794-1

**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.

Copyright © 2018 Trautmann, Lipska-Zi˛etkiewicz and Schaefer. 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) and the copyright owner(s) 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.

## Treatment of Genetic Forms of Nephrotic Syndrome

#### *Markus J. Kemper1 and Anja Lemke2 \**

*1AK Nord Heidberg, Asklepios Medical School GmbH, Hamburg, Germany, 2Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany*

Idiopathic steroid-resistant nephrotic syndrome (SRNS) is most frequently characterized by focal segmental glomerulosclerosis (FSGS) but also other histological lesions, such as diffuse mesangial sclerosis. In the past two decades, a multitude of genetic causes of SRNS have been discovered raising the question of effective treatment in this cohort. Although no controlled studies are available, this review will discuss treatment options including pharmacologic interventions aiming at the attenuation of proteinuria in genetic causes of SRNS, such as inhibitors of the renin–angiotensin–aldosterone system and indomethacin. Also, the potential impact of other interventions to improve podocyte stability will be addressed. In this respect, the treatment with cyclosporine A (CsA) is of interest, since a podocyte stabilizing effect has been demonstrated in various experimental models. Although clinical response to CsA in children with genetic forms of SRNS is inferior to sporadic SRNS, some recent studies show that partial and even complete response can be achieved even in individual patients inherited forms of nephrotic syndrome. Ideally, improved pharmacologic and molecular approaches to induce partial or even complete remission will be available in the future, thus slowing or even preventing the progression toward end-stage renal disease.

 This concept was supported by the response to immunosuppression in many patients and by the fact that recurrence after renal transplantation occurred, possibly due to the presence of a humoral factor, e.g., produced by the immune system (1) However, it is now known that a significant proportion of patients with SRNS [FSGS, but also diffuse mesangial sclerosis (DMS)] have an inherited cause of the nephrotic syndrome (NS), e.g., affecting structural proteins such as nephrin or podocin (2). At first sight, immunosuppression in such patients with inherited structural defects of the podocytes makes no sense. Yet, clinical observations, typically made in patients who were treated with immunosuppression as the genetic result was not yet available, show that a subset of patients does achieve partial or complete remission associated with such treatment. This raises the issue of optimal treatment in this cohort. Should all patients receive immunosuppressive treatment and if so, for how long and in what form? Are their clinical characteristics that can guide treatment? In general, prospective studies addressing this issue have never been performed and evidence pro -and contra- have been generated by retrospective series. Also, treatment options beyond immunosuppression (supportive and pharmacologic) have never been addressed prospectively, which is not surprising due to the rarity, severity, and heterogeneity of the diseases involved.

*Rachel Lennon, University of Manchester, United Kingdom Frederick Jeffrey Kaskel, Children's Hospital at Montefiore, United States*

*Universitätsklinikum Köln, Germany*

#### *\*Correspondence:*

*Edited by: Max Christoph Liebau,*

*Reviewed by:*

*Anja Lemke<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 01 November 2017 Accepted: 12 March 2018 Published: 26 March 2018*

#### *Citation:*

*Kemper MJ and Lemke A (2018) Treatment of Genetic Forms of Nephrotic Syndrome. Front. Pediatr. 6:72. doi: 10.3389/fped.2018.00072*

### General Considerations

Several factors have to be considered before choosing treatment for genetic causes of SRNS. First, the age of presentation and severity of initial symptoms is of utmost importance. In this respect, patients with congenital and infantile NS are probably the most problematic group, because children often present with severe symptoms, sometimes antenatally (3). Historically, the so called "Finnish" type of NS is a good example and aggressive treatment led to a dramatic improvement of survival and longterm-outcome (4). Although patients presenting with infantile NS (onset in the first year of life) have a high risk of carrying a monogenic mutation (5), it should be noted that some patients can have a good prognosis reaching remission with supportive treatment alone (6); individual patients may in fact have minimal change disease responding to steroids. Thus, not only genetic testing but also renal biopsy should be considered in this group.

Second, genetic testing results need to be considered for treatment. Although only mutations in few genes are frequent, there are now panels available testing for 30 or more genes and it can be assumed that there will be new genetic causes in the future. This implies a clinical heterogeneity, not only among identical but also between different genotypes. In this respect, it needs to be at least mentioned that there is often a delay in getting results of genetic testing, which often takes weeks, sometimes months (and sometimes years because new variants are to be detected). Some patients with a negative initial test result may have a yet undiscovered monogenetic cause, and this has implications in choosing a therapeutic approach.

The last problem arises from the fact that there is no universal consensus regarding the definition of treatment response. A (rapid) complete remission of proteinuria is the ideal situation and will generally be accepted without any discussion. However, the definition of partial remission is much more problematic. Fluctuations of proteinuria are difficult to evaluate as they can occur with and without treatment. Most authors would agree that a reduction of proteinuria, for instance, by 50% can be regarded as partial remission, others would demand a concomitant increase of serum-albumin with cessation of edema, which obviously is of greater clinical relevance. There is also no consensus on how to exactly assess the impact of treatment on glomerular filtration rate (GFR) and renal survival.

### Supportive and Non-Immunologic Treatment for Genetic Causes of NS Congenital and Infantile NS

The prognosis of congenital NS has improved substantially in recent years. No differences in mortality and transplant outcome between Finnish and non-Finnish patients with *NPHS1* mutations was noted in a recent registry report on 170 patients (4). Finnish patients started dialysis much earlier because of early bilateral nephrectomy, while in non-Finnish, many other interventions were performed (but not reported on in detail). Despite this, outcome of *NPHS1* patients on renal replacement therapy in fact compared to patients with congenital anomalies of the kidney and urinary tract (CAKUT). No details of treatment approaches and mortality prior end-stage renal disease are presented however. These may influence morbidity and mortality in patients with *NPHS1* and other genetic causes, however and include the following options.

#### *Albumin Infusions*

In severe forms of congenital but also infantile NS regular (mostly daily) albumin infusions have been recommended to decrease edema, increase urine output, and enhance nutrition (7). This strategy requires sufficient renal function; otherwise, fluid overload may occur with potentially severe consequences, such as cardiac failure or pulmonary edema. Since infusions have to be performed regularly, often daily, a central venous access is usually necessary with the associated risks of infection, thrombosis, and hospitalization. Of interest, a recent report by Reynolds et al. (7) showed that after adequate training, administration of albumin can be performed at home, which has an important impact on quality of life. Unfortunately, in reports on regular albumin infusions, other treatments have been used as well (see below). In a yet unpublished French study, 96% of patients received albumin infusions initially daily, with a subsequent reduction in frequency in many patients. It was even discontinued in 10 patients. However, in this report, data about concomitant drug treatment are not available (Berody et al. accepted by NDT, complete citation expected to be available in February).

#### *Nephrectomy*

Unilateral or even bilateral nephrectomy has been used as therapeutic option to decrease or stop proteinuria. Bilateral nephrectomy is probably the most aggressive approach, which will on the one hand completely stop proteinuria, normalize protein and lipid status, and improve nutritional state, but on the other, make (peritoneal) dialysis treatment inevitable (3). Unilateral nephrectomy has been advocated by some authors to reduce proteinuria in children with congenital NS, again often in addition with medical treatment (indomethacin and captopril). In one study (8), serum albumin (sAlb) increased from 11 to 18 g/l after 6–12 months and the number of albumin infusions could be reduced later. This series of five patients also documented an increase in height standard deviation score.

#### *Renin–Angiotensin–Aldosterone System (RAAS) Inhibitors (With/Without Indomethacin)*

A further more conservative approach is drug treatment in order to reduce GFR and thus decrease proteinuria. The value of inhibiting the RAAS by ACE inhibitors (ACEI) and angiotensin receptor blockers (ARBS) in proteinuric renal diseases has been established for many years (9), starting from studies of IgA nephritis. Therefore, in clinical practice, RAAS inhibitors are widely used frequently even in the absence of hypertension. The mechanisms of action relate to decreasing intraglomerular pressure as well as anti-TGFβ properties leading to deceleration of the progression of renal insufficiency. The Cochrane group (9) included RAAS inhibitors in their recommendations for treatment of SRNS but no large studies concerning their use in congenital or infantile NS are available. In one (8), RAAS inhibitors were combined with indomethacin and unilateral nephrectomy. Licht et al. (10) used a stepwise approach: five patients with different causes of congenital NS were treated with captopril and indomethacin serum protein and growth improved in four children. Unilateral nephrectomy was only deemed necessary in two patients during the subsequent course.

Although published evidence is limited, these studies support a stepwise approach in a clinically stable patient with congenital/ infantile NS starting with the use of RAAS inhibitors. In neonates and infants, captopril has been most frequently used and can be titrated best. In severe cases, a combination with indomethacin seems justified; if edema are controlled by this approach (unilateral), nephrectomy can be avoided.

#### Non-Immunologic Treatment in Pediatric and Adolescent Genetic SRNS

Presentation of SRNS after the neonatal period usually leads to a different approach, because the underlying genotype and histological lesions are typically different; *NPHS2* mutations are probably the most frequent single genetic cause (2). Bilateral or unilateral nephrectomy is only practiced in individual severe cases. Treatment with steroids usually has been initiated prior diagnosis and—per definition—failed. Since results of genetic testing are not available immediately in many patients, further treatment, e.g., with calcineurin-inhibitors will be considered and initiated, except maybe in syndromic SRNS. In most published registries (11–13), patients with genetic nephrotic syndrome had received immunosuppressive treatment.

As in congenital and infantile NS, treatment with RAAS inhibitors are a possibility to decrease proteinuria, slow progression of chronic kidney disease, and treat hypertension. RAAS inhibitors are often used in combination with immunosuppressants in glomerulonephrits and NS, although few systematic studies are available evaluating the isolated or combined treatment. As mentioned before, RAAS inhibitors are recommended by the Cochrane group (9) because of two studies. Yi et al. (14) treated SRNS patients with fosinopril and prednisone and compared to a group receiving prednisone alone. Proteinuria decreased significantly in both groups but more in the fosinopril-treated patients. Bagga et al. (15) studied the effect of enalapril at high and low doses showing that there is a dose-related reduction in proteinuria. The benefits of a combined treatment of ACEI and ARBS was suggested by small prospective study in eight patients with SRNS (16). One case report documented complete remission with captopril; regular albumin infusions could be stopped at the age of 15 months (17). Combined antiproteinuric therapy with RAAS inhibitors was also able to induce complete remission in a patient with Nail–Patella syndrome and NS (18). Although all these studies are small and data on genetic causes are not always provided, the long experience with RAAS inhibitors in children would probably be in favor for early use of these drugs in pediatric patients, especially since they are tolerated well and can also be used for associated hypertension. Nevertheless, more controlled data are desirable. Of interest, a current controlled study comparing sparsetan (a dual acting angiotensin receptor blocker and highly selective endotheline Type A receptor antagonist) with irbesartan has been initiated in FSGS to assess the impact also in genetic forms of NS (19).

### OTHER OPTIONS

In individual patients with FSGS (mainly with permeability factor associated NS), treatment with galactose had been reported to be beneficial. Trachtman et al. (20) evaluated treatment of FSGS with a TNF- α inhibiting antibody adalimumab and galactose, the latter being an intersting non-immunologic treatment option also for genetic forms of FSGS. In the cited study, 2 out of 7 patients with FSGS had a 50% reduction in proteinuria after galactose, confirming previous case reports. Data on genetic testing in patients are not available, so no definite conclusion about the utility of galactose in genetic SRNS can be drawn. Unfortunately, a controlled trial with a monoclonal anti-TGF-β antibody (fresolimumab) in SRNS did not lead to a significant reduction in proteinuria (21).

Similarly, the use of vitamin D analogs and stimulation of the calcium-sensing receptor has been assessed. Experimental studies have shown that stimulation of the calcium-sensing-receptor enhances podocyte stability and thus cinacalcet (or vitamin D) may be an option to improve proteinuria in NS (22). So far, data on cinacalcet are not available, but a recent meta-analysis in IgA nephropathy suggested an effect of vitamin D supplementation (23). However, a recent randomized controlled trial of vitamin D supplementation in steroid-sensitive NS did not reduce relapse rate, arguing against a direct podocyte stabilizing effect of vitamin D (24). Although definitive conclusions of these alternative approaches cannot be drawn, future studies into the field of podocyte stabilization by non-immunosuppressive drugs are warranted and could have an important impact on treatment in genetic SRNS.

### Immunosuppression in Hereditary SRNS Evidence from Experimental Studies

Several experimental studies have suggested that immunosuppressants have a direct glomerular effect leading to podocyte stabilization and thus have efficacy beyond their immunological actions. Some agents, such as cyclosporine also have a hemodynamic (nephrotoxic) effect leading, e.g., to reduction of GFR, thereby reducing proteinuria (25).

The initial studies confirming a stabilization of the glomerular cytoskeleton by cyclosporine independent of the immunosuppressive action was provided by Faul et al. (26) and confirmed by various other studies that will not be described in detail (27). Also, steroids, levamisole, mechanistic target of Rapamycin (mTOR) inhibitors, and even Rituximab have received attention in experimental studies although clinical data are virtually nonexistent. For instance, glucocorticoids have been shown to protect and enhance recovery of cultured murine podocytes *via* actin filament stabilization (28, 29). Levamisole, a drug that has never been used in SRNS, was able to induce expression of glucocorticoid receptor (GR) and to activate GR signaling and also protected against podocyte injury in a cell model (30). Also, low-dose rapamycin, an inhibitor of the mTOR, diminished disease progression in an experimental model of FSGS (31). Finally, rituximab, a B-cell-depleting antibody, may bind directly on sphingomyelin phosphodiesterase acid-like 3b protein (SMPDL3b), and thus could have an effect at the cellular level (32).

In summary, there is now emerging evidence that immunosuppressants, especially cyclosporine A (CsA), have a stabilizing effect at the podocyte levels aside from their immunological actions in experimental models and thus may be valuable therapeutic options in the treatment also of genetic forms of SRNS.

### CLINICAL DATA ON IMMUNOSUPPRESSANTS IN GENETIC NS

Most authors would agree that children with *NPHS2* mutations do not respond to treatment with steroids (33). However, in their first report on mutations in the *Plectin1* (*PLEC1) gene*, the authors mention two patients who responded to prednisolone treatment (34). This could not be confirmed from a French series (35); interestingly, the authors report on three unaffected and unrelated patients with homozygous PLCE1 mutations but without clinical disease. Thus, the natural history of SRNS with PLEC1 mutations may be different and modifier genes and environmental factors may play a role. There are no data, whether patients with SRNS and other mutations have a documented partial or complete remission after initial steroid treatment.

To our knowledge, no data on treatment with mycophenolate mofetil (MMF) are available in genetic NS. Unfortunately, in a randomized US study comparing MMF with cyclosporine, genetic testing was not performed (36). In contrast, data on treatment with calcineurin inhibitors (mainly CsA) in cohorts with genetic SRNS have been published; in most of these studies, genetic diagnosis was confirmed *post hoc*, sometimes after years. Unfortunately, response to treatment interventions is often not detailed and sometimes interpretations are superficial.

In two studies from Buscher et al. (12, 13), the vast majority of patients with genetic FSGS did not achieve remission with cyclosporine. In the first study on 91 patients, mutation in 1 of 6 genes studied were detected in 52% of patients. None of the patients with mutations showed a complete response to CsA, but two patients with Wilms tumor suppressor gene 1 (*WT1*) mutation showed partial remission. The response rate of CSA was significantly better without mutations in podocyte genes (68 vs. 17%, *p* < 0.005). In the second, larger retrospective multicenter study by Buscher et al., 131/231 patients with SRNS had an identified genetic diagnosis, including 60 patients with congenital NS; of these, 63 (48%) patients had received CSA. 2 patients (1 with congenital NS, CNS) entered complete and 5 partial remission (none with CNS). In summary, this series documents a partial or complete response to CSA in 19% of hereditary SRNS (with no details on genotype stated) and 1.7% of congenital NS.

In a recent report from the Podonet consortium, 906/1,354 patients with SRNS were treated with immunosuppressants (380 with one, 173 with two, and 59 with three or more different, respectively) (11). Among 74 patients with documented genetic diagnosis, two patients had complete and 8 partial remission, respectively. Another four patients entered partial remission with a combination of CSA and RAAS inhibitor. Thus, in this series, a total of 14/74 (19%) patients with genetic SRNS seemed to have a response to immunosuppression.

In our own retrospective single center study (37) of nine SRNS patients with an identified genetic diagnosis (excluding patients with CNS, *WT1* mutations, and syndromic NS), we observed a partial (2 patients with *NPHS2* mutations) or complete (one patient with compound heterozygous *NPHS1* and one with a dominant *ACTN4* mutation, respectively) remission. Thus 4/9 (44%) showed some response to CSA; **Figure 1** demonstrates evolution of serum-albumin levels in two patients with a complete and partial response, respectively.

Currently, no controlled data are available as to how long CSA treatment in genetic forms of SRNS should be continued, if no response is documented. The series by Klaassen et al. (12, 13, 37), however, show that most patients with complete remission responded after a median of 2 months, so that this treatment period seems to be a minimum. It is currently unknown, whether patients with distinct mutation, e.g., in *WT1* or *NPHS2* show a differential response.

### EXPERIENCES IN SPECIFIC GENETIC DISORDERS

### *WT1*-Mutations

The Wilms Tumor Suppressor Gene 1 (WT1) is a transcription factor with many functions, among them transcriptional as well as tumor-suppressor activities. WT1 plays a pivotal role in early urogenital and kidney development (38), in adults, it continues to be a key regulator of podocyte function (39). Mutations in *WT1* mostly occur as spontaneous heterozygous germline mutations, but familiar cases are also described. Type and location of specific mutations allow for limited prediction of clinical course, histology, and comorbidities (40). By far, not all patients show all symptoms of the classical syndromal descriptions associated with *WT1* mutation: Denys–Drash syndrome described in patients with missense mutations, it includes DMS, SRNS rapidly progressing to end-stage renal disease, XY disorder in sex development with complete gonadal dysgenesis, and a high risk of developing Wilms' tumor. Frazier Syndrome is typically caused by mutations affecting the canonic donor splice site of intron 9, patients present with streak gonads, and are at high risk of developing gonadoblastoma.

There is evidence from studies with small patient numbers that early initiation of treatment with CsA in combination with RAAS inhibitors can lead to favorable response of NS in patients with *WT1* mutations (41). Gellermann et al. report on three children with WT1 mutations and FSGS in whom long-term reduction of proteinuria could be achieved through treatment with CSA in combination with steroids and RAAS inhibitors while maintaining normal renal function (42). Wasilewska et al. describe a patient with *WT1* mutation and DMS on histology in whom nephrotic range proteinuria resolved after initiation of treatment with CSA and enalapril (43). Buscher et al. report two patients affected by NS due to mutations in *WT1* showing a partial response to CsA with a reduction of proteinuria and normalization of sAlb (13). Unfortunately, in their 2016 study, Buscher et al. do not give details on their patients with *WT1* mutations treated with CSA (12). Further studies are needed to define in more detail which patients with *WT1* mutations (type of mutation and histological changes) can benefit from immunosuppressive treatment with

CSA. Mechanisms mediating the benefits of CSA, other than stabilizing podocyte cytoskeleton, in patients with disturbed WT1 expression also need further elucidation (41). Collection of patients with *WT1*-mutations in a prospective registry has been initiated by the German Society of Pediatric Nephrology (GPN).

### Coenzyme Q10 (CoQ10)-Deficiency

CoQ10, also known as ubiquinone, is involved in many essential cellular processes, especially in the mitochondria. Its biosynthesis requires at least 15 genes. So far, mutations in eight of these genes have been found to cause primary CoQ10 deficiency, which results in diseases with variable age of onset. Associated clinical phenotypes are ranging from a multisystem disease to nephropathy or isolated central nervous system disease (myopathy or cerebellar ataxia) (44). Nephropathy can result in steroid resistant NS and loss of renal function in an isolated form (45) or in combination with sensorineural deafness (46) or other neurological symptoms (47).

In contrast to most mitochondrial respiratory chain disorders, for which there is no effective treatment, patients with primary CoQ10 deficiency partially respond to oral CoQ10 supplementation (48). *In vitro* data and first clinical experiences suggest that high-dose oral treatment with CoQ10 has the potential to stop the progression of encephalopathy, muscular symptoms, and NS and even induce remission, if initiated early enough. Heeringa et al. (46) describe proteinuria and hearing loss in patients with coenzyme Q10 biosynthesis monooxygenase (*COQ6)*-mutations. *In vitro*, apoptosis caused by *COQ6*-knockdown was partially reversed by CoQ10 treatment. The authors report a positive response to treatment with CoQ10 and RAAS inhibitors in two children with mild disease. Ashraf et al. (49) describe that mutations in the aarF domain containing kinase 4 gene (ADCK4) leads to CoQ10 deficiency causing SRNS. Knockdown of *ADCK4* in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Indeed, one individual with *ADCK4*-mutation was successfully treated with CoQ10 supplementation. Once severe kidney or neurological damage is established, this cannot be reversed (47, 48).

### Gene Therapy in Genetic Forms of SRNS

So far, no successful gene therapeutic approaches have been reported for hereditary forms of SRNS. However, a mouse model for NPHS2 has recently been reported (50) and may have important consequences also for the development of specific molecular treatment approaches. Other investigations have indicated a role of specific miRNAs in the etiology of FSGS, which may be amenable to specific treatment (51). Taken together, studies into the molecular biology of SRNS have a yet undiscovered potential to develop new treatment modalities and potentially a cure for certain genetic forms of SRNS.

### SUMMARY

Although no adequate systematic treatment studies have been performed in patients with genetic forms of NS, choice of treatment needs to consider clinical factors (e.g., severity of clinical presentation and age of presentation). In a clinically stable patient with congenital/infantile NS, use of an RAAS inhibitor could be

first choice. In neonates and infants, captopril has been most frequently used and can be titrated best. In severe cases, a combination of RAAS inhibitor with indomethacin seems appropriate in order to avoid (unilateral or even bilateral) nephrectomy, which is an effective option in severely affected individuals. In steroidresistant NS patients, most patients with Mendelian forms seem resistant to immunosuppressive treatment. Yet, several studies have shown that in some patients, a complete or partial remission with CSA can be achieved also in hereditary SRNS, including patients with infantile nephrotic syndrome (**Figure 2**). Therefore, in our opinion, a therapeutic trial with CSA in genetic SRNS is justified, since especially CSA has been shown to exert a relevant podocyte stabilizing effect, which may reduce proteinuria in some

### REFERENCES

patients. In the future, more clinical studies of optimal treatment of genetic SRNS are necessary, also evaluating the impact of confounding factors, such as genotype/phenotype correlations, impact of attenuation of proteinuria on rate of progression into ESRD, and others. Ultimately, gene therapy will hopefully be available in the future offering a specific cure of (some) genetic causes of steroid resistant NS.

### AUTHOR CONTRIBUTIONS

MK wrote the first draft of the manuscript; AL wrote sections of the manuscript. Both authors contributed to manuscript revision, read, and approved the submitted version.

**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.

*Copyright © 2018 Kemper and Lemke. 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) and the copyright owner 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.*

## Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research

Henning Hagmann and Paul T. Brinkkoetter\*

*Department II of Internal Medicine, Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany*

Diseases affecting the glomeruli of the kidney, the renal filtration units, are a leading cause of chronic kidney disease and end-stage renal failure. Despite recent advances in the understanding of glomerular biology, treatment of these disorders has remained extraordinarily challenging in many cases. The use of experimental models has proven invaluable to study renal, and in particular, glomerular biology and disease. Over the past 15 years, studies identified different and very distinct pathogenic mechanisms that result in damage, loss of glomerular visceral epithelial cells (podocytes) and progressive renal disease. However, animal studies and, in particular, mouse studies are often protracted and cumbersome due to the long reproductive cycle and high keeping costs. Transgenic and heterologous expression models have been speeded-up by novel gene editing techniques, yet they still take months. In addition, given the complex cellular biology of the filtration barrier, certain questions may not be directly addressed using mouse models due to the limited accessibility of podocytes for analysis and imaging. In this review, we will describe alternative models to study podocyte biology experimentally. We specifically discuss current podocyte cell culture models, their role in experimental strategies to analyze pathophysiologic mechanisms as well as limitations with regard to transferability of results. We introduce current models in *Caenorhabditis elegans, Drosophila melanogaster*, and *Danio rerio* that allow for analysis of protein interactions, and principle signaling pathways in functional biological structures, and enable high-throughput transgenic expression or compound screens in multicellular organisms.

#### Edited by:

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### Reviewed by:

*Bart Smeets, Radboud UMC Department of Pathology, Netherlands Jan Halbritter, Leipzig University, Germany*

\*Correspondence: *Paul T. Brinkkoetter<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

Received: *14 March 2018* Accepted: *11 June 2018* Published: *13 July 2018*

#### Citation:

*Hagmann H and Brinkkoetter PT (2018) Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research. Front. Pediatr. 6:193. doi: 10.3389/fped.2018.00193* Keywords: model organism, kidney diseases, podocyte, mechanosensation, glomerular filtration barrier

### INTRODUCTION

Chronic kidney disease (CKD) is becoming an increasingly prevalent condition affecting almost 10% of the population in Western societies. The majority of kidney diseases that progress to end stage renal failure start in the glomerulus, the renal filtration unit, as a consequence of a very limited capacity of glomeruli for regeneration and the limited ability of terminally differentiated glomerular podocytes for self-renewal (1). The glomerular filtration barrier consists of three anatomic layers: fenestrated endothelial cells, the glomerular basement membrane and podocytes, post-mitotic epithelial cells located at the outer aspect of the capillary loops (**Figure 1B**). These cells enwrap the glomerular capillaries with their primary and secondary processes and form the outer layer of the filtration apparatus. All three layers contribute substantially to the glomerular filtration barrier and can be affected in human disease. Due to their exposed anatomic localization podocytes are constantly challenged not only by oxygen radicals, cytokines, immune complexes and inflammatory processes but also by mechanical forces. Podocyte damage plays a pivotal role in most, if not all, glomerular diseases that result in glomerulosclerosis (2). As podocyte loss cannot easily be compensated by cell proliferation the cells undergo hypertrophy, autophagy, and/or dedifferentiation depending on the injurious insult (3). Podocyte hypertrophy and the increase in cellular size and the covered area of the GBM represent protective measures to ascertain proper glomerular function. In contrast, dedifferentiation is considered to be maladaptive resulting in albuminuria and persistent podocyte loss. It has to be stressed, that the onset of albuminuria and even nephrotic range proteinuria does not require podocyte depletion. Mere cytoskeletal rearrangements, i.e., foot process effacement, are sufficient to cause massive albuminuria as often seen in patients with minimal change disease (MCD). A condition which lacks evidence of pathology in light microscopy but presents with vast foot-process effacement in electron microscopy. In the event of progressive or severe glomerular disease podocyte loss is the clue and patients develop massive albuminuria in combination with irreversible scarring, i.e., glomerulosclerosis (1, 4–7). Importantly, albuminuria and chronic kidney disease are independently associated with an increased risk for end stage renal failure and cardiovascular disease (8–10).

The finding that independent pathways and pathogenic principles contribute to the identical glomerular phenotype described as focal segmental glomerulosclerosis (FSGS) seems trivial but is of major importance. Podocytes are firmly attached to the underlying glomerular basement membrane and form a unique cell-cell contact to foot processes of neighboring podocytes, a cell junction called slit diaphragm. This specialized cell-cell contact is not only an integral part of the glomerular filtration barrier but also serves as signaling hub to regulate podocyte function (11). Over the past several years, various constituents of the podocyte slit diaphragm cell junction have been identified leading to the concept that the proteins at the slit diaphragm regulate podocyte biology through active signaling. The slit diaphragm bridges the distance between two adjacent foot processes, thus allowing formation of a filtration slit. In severe podocyte damage, the slit diaphragm disappears and podocytes simplify structure and shape due to cytoskeletal alterations, a process called foot process effacement (8, 10). Until recently, the function of the glomerular filtration barrier and the pathogenesis of proteinuria have not been well understood. This has changed with the identification of gene defects in (rare) human genetic diseases known to cause congenital or childhood steroid-resistant nephrotic syndrome and progressive glomerulosclerosis [for review see (9)]. These studies identified distinct deregulated pathways that independently contribute to podocyte injury and, potentially, loss of podocytes. Podocyte depletion has long been known to be the culprit of glomerulosclerosis and progressive loss of renal function (12–14). As multiple different pathogenic mechanisms result in proteinuria and FSGS lesions in kidney biopsies, it is not surprising that several clinical trials including all FSGS patients failed to provide new MCD/FSGS treatment options, e.g. the NIH has spent multimillion dollars on clinical trials that did not yield a single new drug for MCD/FSGS patients (15).

The advent of modern genetics with the development of animal models with cell specific gene manipulation including gene deletions and transgenic gene expression together with systems biology has deepened our understanding of the biology and physiology of the renal filtration barrier in states of health and disease. Despite our tremendous advances in understanding glomerular function and the contribution of the specific anatomic compartments to the renal filtration barrier, essential questions remain to be addressed as treatment of glomerular disorders is still unspecific and primarily based on various immunosuppressive regimens, including glucocorticoids or blockade of the renin-angiotensin-aldosterone system (15).

In the past, the use of experimental models has proven invaluable to study renal, and in particular, glomerular biology and disease. Even after the introduction of novel gene editing techniques, mouse models are time consuming. The relatively long reproductive cycle, high keeping cost, and not least the regulatory standards make these models less flexible. In this review, we will describe alternative models to study podocyte biology experimentally.

### PODOCYTE CELL CULTURE

Podocyte cell culture models were the first models to study podocyte biology and are still widely used (16, 17) as gene and protein expression as well as environmental cues can be easily manipulated in vitro for mechanistic analyses (18, 19). Multiple human, mouse, and rat podocyte cell lines have been generated in the past (16, 17, 20–22). Most groups rely on immortalized mouse or human podocyte cell lines which are cultured under proliferative (33◦C) and growth-restrictive (37◦C) conditions. These studies promoted our understanding of glomerular diseases (19) as well as the cytoskeletal regulation (23), cell cycle control (24), cell death mechanisms (25), signaling pathways (26, 27) as well as protein degradation (28).

However, certain limitations apply and have to be taken into account when translating experimental findings from cultured podocyte cell lines; podocytes in culture are cultivated on petri-dishes as a monolayer in the absence of mesangial and endothelial cells in close proximity. Podocytes in culture do not encounter mechanical stretch nor the flow of primary urine filtrate (29). Hence, it is not surprising that podocyte cell lines do not form secondary processes with slit diaphragms in-between neighboring cells and show only a very limited expression of specific marker proteins including nephrin (30), podocin (31), or transient receptor potential cation channel 6 (26, 32, 33).

In an attempt to further characterize these widely used work horses of podocyte research our group recently applied modern MS/MS technologies and created a comprehensive map at a depth of more than 7,000 proteins expressed in proliferating and differentiated cultured podocytes in vitro (26). To this

end, we examined an immortalized mouse podocyte cell line kindly provided by S. Shankland (Seattle, WA) as well as a conditionally immortalized human podocyte cell line obtained from M. Saleem (Bristol, UK), both are widely used cell-culture models generated by either isolation of primary podocytes from the immortomouse (34) or by retroviral transfection of primary human podocytes with a temperature-sensitive SV40 large T-cell antigen (35) (**Table 1**). The temperature shift from 33 to 37◦C induces in both cell lines a proteostatic shift. Undifferentiated podocytes express high abundance of proteasomal proteins while differentiated podocytes express high abundance of lysosomal proteins. Additional studies using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) and protein degradation assays determined protein dynamics and half-lives and revealed a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal and membrane proteins were particularly stabilized in differentiated podocytes. However, the expression levels of socalled podocyte marker genes or podocytopathy gene products varied significantly as compared to primary cells. Highest levels were detected for Actin-regulating proteins comprising Myosin-9 (MYH9) (36), rho GDP-dissociation inhibitor 1 (ARHGDIA) (37) and alpha-actinin-4 (ACTN4) (5). Out of 15 podocytopathyassociated genes linked to cytoskeletal function 8 were expressed in the cultured cells (MYH9, ARHGDIA, ACTN4, anillin (ANLN), inverted formin-2 (INF2), unconventional myosin IE (MYO1E), synaptopodin (SYNPO), and podocalyxin (PODXL) (22, 38–41). With respect to basement membrane proteins 4 out of 8 were quantified in undifferentiated and differentiated human podocytes [CD151 antigen (CD151), integrin alpha-3 (ITGA3), integrin beta-4 (ITGB4), and laminin subunit beta-2 (LAMB2) (20, 42, 43). In contrast, only one out of six slit diaphragm proteins (CD2-associated protein (CD2AP) (44) could be detected.

*Respective proteins are either marked as expressed ("*+*") or not expressed ("–") in the two examined cell culture conditions (33*◦*C* = *undifferentiated, and 37*◦*C* = *differentiated cells). According to Schroeter et al. (45).*

### MODEL ORGANISMS TO STUDY PODOCYTE BIOLOGY

Caenorhabditis elegans with its short life cycle, completely established cell lineage including a neuronal map (connectome), compact and fully mapped genome, uncomplicated genetic modification by feeding of RNAi-expressing E. coli, and costeffective keeping is a prime model for cell biology (46). In addition and in contrast to other multicellular model organisms, (genetically modified) individuals can be easily frozen, stored for the longer term, and revived with immediate capability to reproduce.

These characteristics make the nematode an ideal model to study signaling pathways and functionality of proteins in a multicellular organism and represent an advantage as compared to mammalian models.

Principle signaling pathways like e.g., the insulin/mTOR signaling cascade are generally well-conserved across species (47). Along this line, the observation that the insulin/mTOR pathway is induced and refers damage in podocytes with mitochondrial dysfunction due to the loss of Prohibitin-2 (PHB-2) was substantiated in C. elegans using worm strains expressing fluorescence labeled DAF-16 (48). After heat-shock DAF-16 is pooled in the nuclear compartment. Redistribution of DAF-16 to the cytoplasm depends on the activity of the insulin pathway. Consistently with data from podocyte-specific PHB-2 knockout mice, phb-2-deficient worms showed accelerated recovery of the DAF-2 (insulin receptor) mediated cytosolic redistribution of DAF-16.

However—with regard to glomerular research—C. elegans does not contain a filtering excretory organ homolog to the mammalian glomerulum. Nevertheless, ortholog genes in analogs structures can be studied in the nematode to understand principle mechanisms of podocyte morphogenesis and podocyte slit-diaphragm function.

The mammalian slit diaphragm is composed of the transmembranous immunoglobulin family proteins nephrin and Neph1. Nephrin and Neph1 are lipid raft associated proteins that refer outside-in signals by tyrosin phosphorylation. Mutations in the nephrin encoding gene NPHS1 or lack of NEPH1 lead to defective assembly of the foot processes and loss of the slit diaphragm which becomes evident as (congenital) nephrotic syndrome (49, 50). The adhesion molecules nephrin and Neph1 are well conserved across species. In C. elegans orthologs of Neph1 and Nephrin are SYG-1 and SYG-2, respectively. SYG-1 and SYG-2 refer cell-cell recognition in synapse development between the hermaphrodite specific neuron (HSN) and specialized epithelial guidepost cells adjacent to the nematode's vulva muscle cells medially in the hermaphrodites' soma. The two HSN (HSNL and HSNR) localize to the lateral aspects in the middle of the nematode and protrude their axonal processes ventrally, where they innervate the vulvar muscle cells and provide the neuronal circuit required for egg laying. Interaction of SYG-1 on the HSN axon with SYG-2 expressed on guidepost cells initiates intracellular signaling processes in HSN to trigger synapse formation and maintenance (51, 52). Mutations in either syg-1 or syg-2 fail to exhibit functional synapses due to aberrant placement of presynaptic sites (53, 54). Interestingly, heterologous expression of mammalian nephrin or Neph 1, −2 or −3 can rescue phenotypes of mutant syg-1 or syg-2 (55, 56). The cytoplasmic tail of SYG-2 is required for subcellular trafficking of SYG-2 itself, whereas the cytoplasmic domain of SYG-1 is required for synapse formation but dispensable in later stages (57). Although elegant ultrastructural analyses in mammalian and avian glomeruli have challenged the concept of heterophilic nephrin and Neph1 interaction at the slit diaphragm, synapse formation at the HSN in C. elegans may represent a suitable model to study signaling mechanisms at the cytoplasmic domains of nephrin and Neph1 by visualization of synaptic vesicles in SNB-1::YFP transgenic worms.

Another example for the utility of C. elegans in glomerular research is based on the homology of mammalian podocin and C. elegans MEC-2. Podocin is an essential constituent of the mammalian slit diaphragm complex, whereas MEC-2 is part of the mechanosensory complex of C. elegans sensing gentle touch. Both stomatin-like proteins share a central stretch of hydrophobic amino acids which refers membrane association while the amino and the carboxy terminal ends face the cytoplasm. The highly conserved PHB domain mediates homophilic interactions and lipid binding via palmitoylation, creating the microenvironment that regulates signaling via the associated ion channel proteins TRPC6 in mammals and the DEG/ENaC channel MEC-4/MEC-10 in C. elegans (58–60). This regulatory role of MEC-2 can not only be assessed by quantification of mechanoreceptor channel currents but also in functional in vivo assays measuring sensitivity to gentle touch in adult hermaphrodites (61). In addition, regular localization of MEC-2 and other components of the mechanosensory complex in a characteristic punctate pattern on the six mechanosensory neurons of C. elegans can be evaluated by staining with MEC-2 specific antibodies or employing MEC-4::YFP transgenic worms (28, 62). Identifying co-localization of the primarily mitochondrial protein Prohibitin 2 (PHB-2) and MEC-4 in mechanosensory punctae of touch receptor neurons in C. elegans as well as partial loss of touch sensitivity in PHB-2 knock down worms helped to establish the role of PHB-2 as a slit-diaphragm protein (63). In a recent paper, the ubiquitin ligase Ubr4 has been shown to control podocin protein stability and conservation of this molecular mechanism could be confirmed for MEC-2 in C. elegans assays, where the loss of the Ubr4 ortholog C44E4.1 (ubr-4) resulted in a more dispersed staining pattern of MEC-2 positive punctae (28).

With regard to glomerular research, studying C. elegans is instrumental as a functional read out for protein interactions, trafficking and protein turnover as well as signaling of conserved pathways in a multicellular organism. This holds true especially for mammalian podocin, nephrin, Neph 1, and their orthologs. A weakness of the nematode as a model of conserved principles in mammalian (patho-)physiology as well as for compound screens is the lack of organs homolog to heart, liver, central nervous systems, and of course filtering organs like the kidney.

Drosophila melanogaster evolved as another high-capacity model organism for glomerular research. Cell type specific gene inactivation and editing make the fly a versatile, adaptable, and expedite model. The reproductive cycle is around 12 days.

Similar to the nematode the Drosophila model allows to study protein interactions in functional biologic structures. In the fly adhesion molecules ortholog to mammalian nephrin and Neph proteins called sticks'n stones/hibris (sns/hbs) and kirre/dumbfounded (duf) are involved in cell-cell recognition, and cellular signaling events to control adhesion, cell shaping, and programmed cell death during eye development in the fly. During embryonal development Drosophila's facet eyes develop from a single layered epithelium and remain undifferentiated during almost the entirety of larval stages. In the final differentiation steps heterophilic interactions of sns/hbs and duf determine cell fate specification and are required for patterning and separation of ommatidea (64, 65).

During the last decade, research explored Drosophila nephrocytes as a novel tool of cell biology. The term was initially coined by Bruntz and Kowalsky, who discovered ammonia carmin absorbing cells around the heart, the digestive organs, and the nervous system in arthropods (66, 67). Nephrocytes are specialized filtrating cells with high endocytic activity, that may have detoxifying and sequestration function. In the adult fly there are two populations of nephrocytes. The Garland nephrocytes, which are assembled along the esophagus, and the pericardial nephrocytes, which palisade the heart tube. Nephrocytes are large cells with a surface laced by invaginations, so called labyrinthine channels, and covered by a continuous basement membrane. Near the apical surface the invaginations are abridged by a slit diaphragm (**Figure 1A**). Loss of the nephrin ortholog sns or the Neph ortholog duf results in loss of slit diaphragm structures, smaller lacunae and thickening of the basement membrane (68). Filtration across the pericardial nephrocyte slit diaphragm is limited to substances smaller than 70 kDa (69). Molecules that get filtered into the labyrinthine channels are taken up by the nephrocyte via endocytosis (70). In this regard the nephrocyte differs significantly from mammalian podocytes as to our current understanding. Filtration across the nephrocyte slit diaphragm as well as endocytosis can be assessed in assays on explanted nephrocytes employing different tracers, like e.g., GFP-, labeled albumin, horseradish peroxidase or dextrans of variable sizes (68, 69, 71, 72).

A novel transgenic fly model expressing secreted atrial natriuretic factor labeled with red fluorescent protein (ANF-RFP) from muscle cells and nephrocyte-specific green fluorescent protein (GFP) combined with the option of nephrocyte specific gene manipulation via a nephyrocyte specific Dot-Gal4 driver has implemented an expedite and reliable screening tool for genes associated with human disease (69). This model was employed to screen Drosophila orthologs of human monogenic nephrotic disease. Reassuringly, most of the pathogenic alterations were conserved in the fly (71, 73). Loss of Coq2 or Rab GTPases e.g., leads to loss of labyrinthine channels and loss of function in nephrocytes (71, 74).

In addition, rescue and overexpression studies with human orthologs are possible in Drosophila knockout models. Both eye and nephrocyte development were evaluated to identify a conserved amino acid motif in mammalian Neph1 to functionally replace Drosophila duf, whereas neither Neph2 nor Neph3 showed similar effects (75).

In general, the fly is an ideal model organism for highthroughput transgenic screens and rescue experiments, when variants of ortholog human genes are expressed in the respective drosophila knock out. The in vivo filtration assay or eye pattern formation serve as reliable and expedite readouts.

Danio rerio, the zebrafish, provides a third model organism of glomerular filtration. In striking contrast to the nonvertebrate Drosophila and C. elegans models, the zebrafish forms vascularized glomeruli in the pronephrons and the mesonephros during development with the mesonephros maintained in adult life. At the pronephros-stage the zebrafish kidney consists of two fused glomeruli with connection to the aorta draining primary urine into the pronephric tubuli followed by the pronephric ducts (76). The pronephros glomeruli already contain fenestrated endothelium, mesangial cells and podocytes that form a functional slit diaphragm (77). In addition, genome conservation of 70% between human and Danio rerio as well as versatile morphlino techniques, CRSPR/Cas-based gene editing and cross-species rescue models make the fish a primary model to study glomerular disease. Loss of zebrafish nephrin and podocin, which are specifically expressed in pronephric podocytes, leads to the loss of slit-diaphragms early in development (78). As in human nephrotic syndrome, pericardial edema, periocular edema, and general edema develops. Likewise disruption of zNeph1 or zNeph2 showed similar phenotypes (55). Besides structural analyses functional assays of glomerular filtration have emerged. Early on, the integrity of the filtration barrier was monitored qualitatively on fixed tissue after injections of large molecular weight dextran, which was detected in tubular epithelial cells in case of disruption of the glomerular filter (78). By now, several assays to quantify glomerular filtration have been established. Amongst these are in vivo fluorescence measurement in the eye or in large vessels in time-laps experiments after injection of fluorescence-labeled 10-, 70-, and/or 500 kDa dextrans (79). Another approach employs transgenic fish expressing eGFP-labeled vitamin D-binding protein (eGFPDBP) of a molecular weight of 78 kDa in the liver, which is repelled from glomerular filtration in healthy fish but leaks into urine in states of glomerular damage and can be quantified in the eye (accumulation in control) and—as excreted protein—in the water surrounding the fish (80).

### ORGANOIDS

Generation of kidney organoids by differentiation of pluripotent stem cells (IPS cells) or re-aggregation of single cell suspensions of embryonic kidney cells in culture provided an important new tool for the study of kidney development and disease (81, 82).

However, the delicate morphology of the glomerular filter as well as the need for specific cellular interactions

and vascularization has hampered the study of glomerular biology on kidney organoids. In addition, recent single cell transcriptomic analysis of organoids has identified incomplete differentiation of all kidney organoid cell types, including podocyte progenitors (83).

In an elegant study, researchers generated organoids mixing murine embryonic kidney cells and implanted these organoids into nephrectomized athymic rats. They describe differentiation of morphologically and functionally intact glomeruli (84). In addition, the authors were able to integrate human amniotic fluid stem cells into chimeric organoids by mixing murine embryonic kidney cells and human amniotic fluid stem cells before in vitro organoid culture. Interestingly, also human amniotic fluid stem cells generated functional podocytes. Similar results were recently confirmed for human pluripotent stem cell derived organoids transplanted under the renal capsule of immunocompromised mice (85).

In conclusion, organoids will become a very important tool also in glomerular research. Again the complexity of glomerular structure and interaction of glomerular cell types is the major challenge to overcome.

### CONCLUSION

Research of the last two decades has boosted our understanding of podocyte cell biology and genetics and provides growing understanding of the composition of the renal filtration barrier and cellular interactions needed to maintain its function. Experimental work in podocyte cell culture models informed on expression, trafficking, interaction and turnover of essential proteins of podocyte function. However, cell culture studies in podocytes are limited due to the fact that podocytes in culture lose their characteristic features. Cultured podocytes lack the intricate foot process morphology, cell polarity is incompletely preserved, and most importantly, intercellular contacts are neither structurally nor functionally close to the slit-diaphragm found in vivo. It is very clear that in vivo models are needed to understand glomerular physiology and to address podocyte diseases experimentally. Besides indispensable rodent models, model organisms like C. elegans, Drosophila melanogaster, and the zebrafish have entered the stage of glomerular research and allow unparalleled functional analyses of inter-cellular interactions and morphogenesis, signaling mechanisms, cell polarity, and filtration in vivo. Kidney organoids may become an additional important tool in the future.

### REFERENCES

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

PB received grant support by the German Research Foundation (BR2955/6-1, KFO329 BR2955/8-1). HH was supported by the EKFS HH (2016-A62). HH and PB also received intramural funds from the Köln Fortune Program, University of Cologne.

### ACKNOWLEDGMENTS

The authors thank S. Koehler for providing Drosophila images.

garland cell nephrocyte. J Am Soc Nephrol. (2017) 28:1521–33. doi: 10.1681/ASN.2016050517

**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.

Copyright © 2018 Hagmann and Brinkkoetter. 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) and the copyright owner(s) 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.

## Using the *Drosophila* Nephrocyte to Model Podocyte Function and Disease

#### *Martin Helmstädter 1,2, Tobias B. Huber 3 and Tobias Hermle1,2\**

*1Renal Division, University Medical Center Freiburg, Freiburg, Germany, 2 Faculty of Medicine, University of Freiburg, Freiburg, Germany, 3 III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany*

Glomerular disorders are a major cause of end-stage renal disease and effective therapies are often lacking. Nephrocytes are considered to be part of the *Drosophila* excretory system and form slit diaphragms across cellular membrane invaginations. Nehphrocytes have been shown to share functional, morphological, and molecular features with podocytes, which form the glomerular filter in vertebrates. Here, we report the progress and the evolving tool-set of this model system. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in *Drosophila*, the nephrocyte has the potential to greatly advance our understanding of the glomerular filtration barrier in health and disease.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Andrew Mallett, Royal Brisbane and Women's Hospital, Australia Michal Malina, Charles University, Czechia Michael Peter Krahn, University Hospital Muenster, Germany*

#### *\*Correspondence:*

*Tobias Hermle<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 20 October 2017 Accepted: 24 November 2017 Published: 07 December 2017*

#### *Citation:*

*Helmstädter M, Huber TB and Hermle T (2017) Using the Drosophila Nephrocyte to Model Podocyte Function and Disease. Front. Pediatr. 5:262. doi: 10.3389/fped.2017.00262*

Keywords: nephrocyte, *Drosophila*, podocyte, glomerular disease, garland cell, endocytosis, renal disease

## INTRODUCTION

Disorders that affect the glomerulus are the predominant cause of end-stage renal disease (ESRD) (1). Beyond renal replacement therapy, potent therapeutic options are not available for most of the disorders from this heterogeneous group. This underscores the unmet need for valid model systems that serve to understand the mechanisms of glomerular diseases.

The most established model system for the glomerulus is currently the mouse. Despite being the undeniable gold standard, this model has its inherent limitations in costs, speed, and considerations of animal welfare. Complementary systems are thus desirable. An excellent alternative can be found in the zebrafish model. But the aforementioned limitations of the mouse model are alleviated only in parts in zebrafish, while additional obstacles like the shortcomings of the morpholino technology need to be considered. Significant insights were also derived from *in vitro* studies, mainly by using cultured podocytes (2). However, here other significant limitations are arising. This is mainly a consequence of the complex glomerular architecture. The glomerular filter is three-layered, including the fenestrated endothelium, the glomerular basement membrane, and the podocytes that form the slit diaphragm. Podocytes are characterized by their most intricate cell shape that is paralleled only by neuronal cells. Cultured podocytes lack crucial features of their *in vivo* counterparts, most notably the formation of slit diaphragms. The *Drosophila* anatomy contains no structure whose analogy to the mammalian kidney appears obvious. Thus, it was unexpected that the *Drosophila* nephrocyte was identified as an invertebrate model that has the potential to meet the needs for a complementary model for glomerular disease (3, 4).

Discovered more than 150 years ago (5) and identified as storage kidneys, the nephrocyte received limited attention until after the discovery that their auto-cellular junctions need to be considered as slit diaphragms that are formed by the orthologs of the mammalian slit diaphragm proteins nephrin (*sns*) and *NEPH1 (kirre)* (3, 4). Loss-of-function of slit diaphragm proteins results in a smooth

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cell surface analogous to podocyte foot process effacement. Nephrocytes thus share molecular, ultrastructural, and functional features with podocytes (3, 4, 6, 7). This makes nephrocytes a unique model system with a functional slit diaphragm in a genetically highly tractable model organism. Now, nearly a decade after the introduction of the nephrocyte to a wider audience in the renal field, the purpose of this review is to examine the progress and the evolving tool-set of this model system for glomerular diseases that still has not reached its peak.

### NEPHROCYTE BIOLOGY

Regarding the history and basic principles of nephrocyte biology, we refer to detailed previous reviews (8–10) in order to focus on the more recent findings.

### Basic Concepts of Nephrocyte As an "Excretory" Organ

The two fundamental functional subunits of renal organs throughout vertebrate biology are the glomeruli that produce an ultrafiltrate and the tubules that process the ultrafiltrate further and finally feed the resulting urine into the disposal system. Together, they serve to eliminate toxins and waste products and maintain water, salt, and pH homeostasis. Following similar principles, *Drosophila* nephrocytes are considered to be part of the fly excretory system. The *Drosophila* renal system has two functional subunits as well: the nephrocytes, regarded as analogous to the glomeruli, and the Malpighian tubules, regarded as analogous to the renal tubular system. There are two distinct nephrocyte populations: the pericardial nephrocytes along the heart tube and the garland cell nephrocytes in a garland-like ring around

Filtration occurs from the blood through the filter into the urinary space. In *Drosophila*, nephrocytes function as individual cells that are clustered. The glomerular filter

is bi-layered, lacking the endothelium. Filtration occurs from the larval blood, the hemolymph, being destined for processing in endosomes.

the esophagus (**Figure 1A**). However, a substantial conceptual difference to the mammalian kidney is that nephrocytes have no connection to the tubular system of *Drosophila* that independently generates, modifies, and finally excretes urine into the intestinal lumen (8).

### Brief History of Nephrocyte Research

Despite the divergence of anatomy and functional concepts, the role of nephrocytes was already discovered in the mid-19th century. The garland cell nephrocytes were first described in 1864 by August Weismann in Freiburg. Studying larvae from *Musca vomitoria*, he noted a string of cells reminiscent of a garland that was floating in the larval body cavity. Accordingly, he termed them "Guirlandenzellen" (German *Girlande* means garland) (11). The function of these cells was an utter mystery to their discoverer but not much later, in 1886, Kowalevsky intended to stain the intestinal mucosa of muscid larvae by feeding them carmine, methylene blue or silver. However, he noted that only nephrocytes took up and stored these tracers (12). He correctly concluded from the tracer experiments that these cells function like a storage kidney to clear the larval blood. This concept was later expanded by Hollande (13). The elaborate ultrastructure of *Drosophila* nephrocytes was described in the sixties of the last century (14, 15) and few years later Crossley (16) showed the size-selectivity of uptake and already noted the analogy of this ultrastructure to podocytes: membrane invaginations called labyrinthine channels are bridged by an auto-cellular slit diaphragm that controls their entry.  Thus, two of the three layers of the glomerular filter in vertebrates are found in *Drosophila* nephrocytes (**Figures 1B** and **2A**).

After the discovery of nephrin (17), Skaer (3), Abmayr and colleagues (4), finally put the pieces together and proved a molecular analogy of podocytes and nephrocytes. They noted that the orthologs are both expressed in nephrocytes and that their absence results in nephrocyte loss of the complex invaginations and a smoothing of the cell surface (3, 4). Interestingly, this disruption of nephrocyte ultrastructure also follows treatment with puromycine (18) and protamine sulfate (7), both of which have the same effect in podocytes. Mammalian NEPH proteins have been shown to be functional within this invertebrate system (19). A most recent and surprising discovery is that nephrocytes potentially exhibit apico-basal polarity (20). Crumbs, a marker of apical polarity, was observed along the membrane of the labyrinthine channels. The presence of integrins on the nephrocyte surface may be suggestive of a basolateral compartment (7). This raises the possibility that despite being spherical cells, nephrocytes might be polarized within their complex ultrastructural morphology. Consequently, analogous to mammalian podocytes, apico-basal polarity may be a potential prerequisite for nephrocyte morphology and function (20). However, at this point it has not been ruled out that the functional role of these proteins in nephrocytes is independent from polarity.

### Endocytosis As an Essential Nephrocyte Function

It is astounding that nephrocytes can exert their function as an "excretory" organ without a connection to the outside world. This feat is performed by endocytosis. The first step is endocytic uptake of material from the blood of the fly that is called hemolymph. Nephrocytes then sort the endocytic cargo for either degradation in the lysosome or recycling back to the hemolymph. Unwanted cargo that cannot be destroyed, will be stored intracellularly. In that way, nephrocytes detoxify the hemolymph and nephrocyte dysfunction reduces survival upon toxin exposure (3). Nephrocytes thus do not contribute to urine formation in any way but nevertheless the high endocytic activtity can be regarded as "excretory" activity (**Figure 1B**). To be optimized for that purpose, they increase their surface area by forming invaginations from the cell surface called labyrinthine channels. Endocytic uptake may occur from the cell surface but the major site of endocytosis is from within the labyrinthine channels (21, 22).  The high endocytic activity attracted researchers to use nephrocytes as a model to study mechanisms of endocytosis (22–26). Endocytic uptake is at least in part dependent on the ortholog of Cubilin (7, 27, 28). In face of this conceptual similarity to the proximal tubular function, it has been proposed that nephrocytes are also a model for the proximal tubule (28). Nephrocytes have been shown to regulate the hemolymph composition through their endocytic activity (29).

### Developmental Aspects

Like podocytes, both subsets of nephrocytes are derived from the mesoderm. Differentiation and maintenance of nephrocytes requires *dKLF15* as a nephrocyte-restricted growth factor (30). *dKLF15* is the ortholog of Krüppel-Like Factor 15 which plays a role in podocyte development (31). The expression of *sns* and *kirre* and the formation of slit diaphragms begin during mid-embryogenesis and are maintained through larval development into adulthood (3, 4, 7, 32). Garland cell nephrocytes undergo a fusion process between embryonic stage E13 and E18, resulting in binucleate cells (4). For unknown reasons, the cell number decreases for both, garland cell and pericardial nephrocytes throughout development. About a quarter of the 25 garland cell nephrocytes and 120 pericardial nephrocytes that are present at the end of embryogenesis are maintained during development into adulthood (4, 7, 8, 33). Adult garland cell nephrocytes exhibit tracer uptake and show regular ultrastructural morphology, supporting these to be functional cells (7).

### Frontiers of Nephrocyte Research

Despite the considerable interest nephrocytes have gained in the recent past, we do not fully understand the significance of nephrocytes within the adult fly organism. Ivy et al. (30) recorded the life-span of adult flies that lack nephrocytes due to the absence of *Klf15* in comparison with wild-type. In contrast to the observations in larval stages, the absence of nephrocytes did not affect survival of adult flies, not even under toxin stress. Further work will be required to determine the functional role of nephrocytes in the organism of adult flies.

A secretory function of nephrocytes that is suggested by the abundance of rough endoplasmic reticulum has been shown for lysozyme (16) but this functional aspect has not been further explored.

Regarding the two subsets of nephrocytes, i.e., pericardial and garland cell nephrocytes, it has become overwhelmingly clear that both cells share critical features. This includes gene expression patterns of the slit diaphragm proteins and other genes like *dKlf15, hand, rudhira*, or *Amnionless*. On the other hand, obvious differences define them as distinct cell lineages, beginning with the number of nuclei. These differences are still not explored in detail. Currently, garland cell and pericardial nephrocytes are best considered to be complementary.

### READOUTS AND STRATEGIES OF NEPHROCYTE RESEARCH

The functional tool-set for nephrocytes is still evolving. As a consequence, there is no defined optimal strategy or established gold standard. However, a general rule emerges that a functional and a morphological assay need to be combined. We will discuss the merits and pitfalls of different strategies to give an overview.

### Tracer Endocytosis As a Readout of Nephrocyte Function

Tracer endocytosis stood at the very beginning of nephrocyte research when they enabled Kowalevski to identify clearance of the larval circulation as their functional role. Until present time, virtually any manuscript regarding nephrocytes employs tracer uptake as a functional readout. This is based on the fact that the "excretory" function of nephrocytes occurs *via* endocytosis. An analogous effect has also been shown for endogenous proteins like *imaginal disk growth factor 2* (34).

A broad array of tracers has been used since Kowalevsky, who introduced AgNO3 that is still in use today. The most commonly used tracers are proteins like GFP-derivates (6), albumin (7), avidin (26), wheat germ agglutinin (22), or Horseradish peroxidase (HRP) (16). Another common approach uses polysaccharids like dextrans (3). Less commonly employed are colloidal substances like Coomassie Brilliant Blue (35). The choice of tracer may follow personal preference but it is important to consider the basic principles for these experiments that became clear through recent findings.

The broad range of possible tracer substances is astonishing. This reflects that the uptake of at least some tracers occurs *via* the scavenger receptor Cubilin (7), whose mammalian ortholog shows affinity for a broad range of ligands as well (36). Aside from the drastic reduction of tracer endocytosis upon silencing of Cubilin/Amnionless, this concept is supported by experiments that suggest that tracer uptake occurs receptor-mediated (7). The observation by Crossley (16) that Lanthanum-dioxide and HRP both enter the labyrinthine channels, while only the latter is taken up, indicates a certain extent of selectivity for the entry mechanism which most likely reflects the receptor affinity.

Being dependent on the scavenger receptor, measurements of nephrocyte tracer endocytosis for most tracers thus do not simply reflect the surface area of nephrocytes. The interpretation of experiments applying tracer endocytosis in nephrocytes should always take possible effects on receptor abundance, function, or specificity into account.

Filtration of tracers across the nephrocyte slit diaphragm is an important concept that directly relates to the functional analogy to podocytes and thus also underlines the significance of such experiments. Crossley (16) was able to show that in nephrocytes, tracers may be excluded size-dependently by the basement membrane and the slit membrane, thus defining a cut-off of about 12 nm. More recently, receptor competition experiments revealed that this size cut-off is comparable with the mammalian glomerulus around 70 kDa (7). Basement membrane thickening occurring upon loss of slit diaphragm proteins (3) mainly affects tracers with a high molecular weight while this effect is not directly related to the nephrocyte ultrastructure or filtration. This became obvious when it was shown using a rapid intervention like protamine treatment that perturbs the ultrastructure without thickening the basement membrane. This treatment has no effect on tracers with a high molecular weight while a tracer that is below the filtration cut-off is strongly reduced (7). In light of these findings, a tracer that is smaller than 70 kDa appears to be preferable. Another potential confounding effect comes with the saturability of tracer endocytosis (7). Choosing an excessive tracer concentration or incubation period thus may even blur the effect of silencing *sns* despite using tracers that are small enough to pass the slit diaphragm (3).

The two major alternative strategies are a pulsed uptake by exposing nephrocytes to tracers *ex vivo* or using an endogenous tracer *in vivo*. The latter strategy was first described by Han and colleagues (6). It employs a transgenic fusion protein of red fluorescent protein with the atrial natriuretic factor from rat. This transgenic tracer is expressed endogenously by muscle cells, secreted into the larval circulation followed by endocytosis and degradation by nephrocytes. The fluorescence intensity of larval pericardial nephrocytes can be observed by transcutaneous imaging. This elegant approach is fast and enabled large-scale genetic screens (6). Studying nephrocytes *in vivo*, this approach appears superior to *ex vivo* interventions. On the other hand, one has to be conscious of certain limitations that nevertheless may favor a decision for an *ex vivo* setting. Imaging fluorescence through the larval cuticle may entail weak and variable signal intensity. True *in vivo* imaging is complicated by larval movements that usually require measures to reduce larval motility. The continuous tracer expression furthermore inherently renders the net result from uptake and degradation as readout. A block in degradation and an increase in uptake thus may have the same outcome. Possible variations in tracer production, distribution, and alternative degradation are difficult to control. The *ex vivo* incubation on the other hand, is able to exclusively reflect rapid uptake under controlled conditions with direct imaging of the nephrocytes. These experiments do not require the presence of a transgene that controls the expression of the endogenous tracer and thus can be immediately applied in any genetic background that allows larval survival. This approach further allows the combination with additional interventions like drug exposure. The dissection of garland cell nephrocytes can be accomplished within few seconds which minimizes the impact of an *ex vivo* setting. An intermediate strategy by dissecting nephrocytes from transgenic animals that express a tracer endogenously may allow to combine some of the advantages of both approaches like speed and precision of direct imaging. Then again, this also retains disadvantages from both sides like the confounders entailed by continuous tracer production and the invasiveness of dissection. In summary, application of either strategy needs to be tailored to the experimental question and the available resources.

Finally, an important consideration for any uptake experiment is that it is hard to interpret without additional morphological studies. As we can learn from loss-of-function of endosomal proteins, reduced tracer uptake may occur while the slit diaphragms are maintained (25, 26) (or potentially also *vice versa* in different settings).

### Electron Microscopy As a Readout of Nephrocyte Morphology

The major advantage of nephrocytes lies in the formation of functional slit diaphragms that are easily accessible. These slit diaphragms can be observed directly by transmission electron microscopy (TEM) providing the most unequivocal evidence. That way the ultrastructural analysis is an important complement for the tracer studies whose interpretation can be more difficult but that still are indispensable as a rapid functional assay. Although the technique is well established after many decades of research, study of nephrocytes still holds challenges that entail the complex ultrastructural architecture of these cells. The labyrinthine channels are nearly parallel furrows that are observed across the whole nephrocyte surface (**Figure 2**). The slit diaphragms seal these invaginations like a ceiling. Tangential sections through slit diaphragms therefore impress as parallel lines (4, 7) (**Figure 2B**). The interpretation of equatorial cross sections is more difficult, as they are dependent on their orientation in regard to the lines formed by the slit diaphragm. A crosssection that cuts through the slit diaphragms at an approximately perpendicular angle renders the classical image (**Figure 2A**). However, if the section cuts through the parallel lines of the slit diaphragms at an angle that is too oblique (**Figure 2C**), the slit diaphragms and labyrinthine channels will be distorted and difficult to identify. For that reason, such a section may even be mistaken for a smoothing of the nephrocyte surface. However, these oblique sections are marked by elongated stretches of electron-density (**Figure 2C**) that clearly distinguish them from a true phenotype. Unfortunately, examples for this error can even be found in the literature.

Analogous to the observations regarding the mammalian slit diaphragm (37), one can assume that fixation and sample preparation may influence the resulting image.

Scanning electron microscopy (SEM) can be most useful to gather information regarding cell shape and number (4, 19), while this may be achieved by using approaches involving light microscopy as well. The assessment of the nephrocyte ultrastructure through this approach is complicated as the labyrinthine channels are concealed by the basement membrane. Specific protocols like protease treatments can overcome this limitation but need to be considered carefully to avoid artefact.

### Immunofluorescence (IF) As Complementary Strategy

Immunofluorescence of slit diaphragm proteins facilitated the discovery of nephrocytes as a model for glomerular diseases (3, 4). Still, this assay is regarded a complementary strategy that was omitted in a number of manuscripts. A significant recent observation concerns highly resolved tangential confocal sections of nephrocytes stained for Sns or Kirre. These stainings reveal a fingerprint-like pattern that correlates with the picture obtained in tangential sections in TEM (**Figure 2A** inset) (7). This not only implies that findings obtained with TEM can be backed up by stainings of the slit diaphragm proteins but also that significant information about ultrastructural aberrations can be obtained before applying TEM. Combined with the opportunity of co-IF, this allows a more comprehensive analysis for a number of questions. IF with 3D reconstruction also revealed that the entire cell body is covered by slit diaphragms (7). In a similar fashion, IF can be regarded as an alternative to SEM.

It remains to be seen, if super resolution imaging using stimulated emission depletion microscopy or stochastic optical reconstruction microscopy will be able to render additional information.

### Readouts of Mortality

Observing survival under the exposure to toxins like silver nitrate in larvae has been described as a possible assay that directly relates to nephrocyte function. That way it may be a suitable complement for tracer strategies that documents the effect on the larval physiology. Another assay that has been introduced more recently is to record adult life-span (38). This appears a logical addition to the spectrum of investigations. However, at this point in time, it is difficult to connect reduced adult survival with nephrocyte functions due to the findings by Hartley and colleagues (30). They did not observe an altered life-span when nephrocytes were even entirely absent entirely due to lack of *dKlf15*. Potential confounders for fly survival can result from expression in other tissues. The commonly used *GAL4*-drivers to control expression of RNAi do not follow anatomic structures but are dependent on gene expression patterns. It is very difficult to rule out that temporary or very limited expression in other tissues contributes to a broad compound readout like survival. At least until the role of nephrocytes in the physiology of the adult animal is better understood, in our opinion it appears prudent to rather make use of assays that are more specifically tailored to nephrocytes.

### Loss-of-Function Strategies

The virtually genome-wide availability of cost-effective, offthe-shelf RNAi libraries is a major advantage of *Drosophila* as a model system. Loss-of-function strategies that employ RNAi are inherently limited by potential off-targets or inefficient knockdown. This may result in contradicting results between different RNAi-transgenes. A common strategy in the *Drosophila* field consists in analyzing at least two independent RNAi stocks to confirm the findings. Further confirmation often remains desirable. This can be achieved by utilizing genetic null alleles. However, lethality frequently impedes this strategy like in the case of *sns*. Nephrocytes are not amenable to the classic mosaic analysis in *Drosophila* as they are non-dividing cells. The CRISPR/Cas9 technology allows to introduce a conditional loss-of-function that is not RNAi dependent (7). This can be accomplished by transgenic *Drosophila* that combine nephrocyte-restricted expression of Cas9 in conjunction with ubiquitous expression of a directed guide RNA. In this approach, individual cells may have divergent phenotypes dependent on individual CRISPR/ Cas-induced mutations. Possible draw backs of this strategy are off-targets, inefficient gene disruption, and persistence of mRNA. Therefore, it is reasonable to either use two independent guide RNAs or combine this with an RNAi approach. For both methods, RNAi and CRISPR/Cas, loss-of-function best is further verified, e.g., by IF using a specific antibody or rescue experiments with an RNAi/CRISPR-resistant transgene.

A number of different *GAL4*-driver lines have been utilized to control the expression of transgenes to induce loss-offunction in nephrocytes. These include *Dorothy*-*GAL4* (6, 39), *sns*-GCN-*GAL4* (4, 40), and *Hand*-*GAL4* (41). While these lines express *GAL4* in both subsets of nephrocytes, the equally common *prospero*-*GAL4* (3, 42) has predominantly been used for garland cell nephrocytes. A garland cell nephrocyterestricted expression has been described for *Aug21*-*GAL4* (43). All commonly used driver lines appear to exhibit robust expression during the larval stage. *G447.2-GAL4* (3) was used to direct expression in embryonic garland cell nephrocytes. *Dorothy*-*GAL4* (7, 44, 45), *Hand-GAL4* (46), and *sns-*GCN*-GAL4* (47) have been employed in nephrocytes dissected from adult animals. A systematic comparison of the available *GAL4* lines regarding onset, continuation, and intensity of transgene expression is currently lacking. The same is true for a characterization of the extra-nephrocytic expression that almost certainly occurs in all the *GAL4*-drivers.

## NEPHROCYTE AS DISEASE MODEL

### Monogenic Forms of Nephrotic Syndrome

*Drosophila* shows a surprising extent of conservation of disease genes and this model organism has proven to be appropriate for the investigation of monogenic diseases. These disorders are characterized by mutation of a single gene as the molecular cause of a disease. The role of nephrocytes as a model for genetic kidney disease has recently been reviewed in more detail (48). Nephrocytes are suitable to study the functional role of candidate genes that were identified in genetic studies and also elucidate the involved functional pathways which is facilitated by the low redundancy of the fly genome. The speed, cost-effectiveness, and reliability of this model make it well equipped to handle the considerable number of candidate variants that may result from contemporary sequencing efforts like whole exome sequencing. Beyond that, nephrocytes can also be applied to assess the significance of individual candidate mutations using transgenic rescue constructs. In the near future, *Drosophila* nephrocytes may further facilitate the development of targeted therapies and personalized medicine.

The most extensive work in *Drosophila* nephrocytes concerns steroid resistant nephrotic syndrome (SRNS). This disorder is characterized by childhood onset of edema, massive proteinuria, and progression to ESRD. SRNS is the second most frequent cause of ESRD within the first two decades of life and monogenic mutations explain a significant fraction of cases that manifest before 25 years of age (49). By now, more than 50 different genes have been implicated as a single gene cause for this disorder (50, 51) and more genes are likely to be discovered. The large number of known SRNS genes prompted systematic analyses that showed that about 60–80% of the studied orthologs of human disease genes resulted in a reduction of tracer uptake (7, 38) including genes that are involved in the slit diaphragm complex, actin regulation, CoQ10-biosynthesis, or interaction with the extracellular matrix. This bears testimony to the surprising extent of evolutionary conservation and underlines that central pathomechanisms of podocytopathies can be studied in nephrocytes. This is most obvious for *COQ2*-nephropathy (52) where findings in *Drosophila* indicate formation of reactive oxygen species as a critical event in the pathogenesis. Successful drug treatment of flies further provides evidence for Vanillic acid as a potential treatment (7). Importantly, these data further support that *COQ2*-nephropathy may be treatable (53) and that treatment strategies can be tested in nephrocytes. Some of these findings were confirmed in a recent manuscript that furthermore implicated autophagy and mitophagy as pathomechanistically relevant (54).

For the identification of several of the SRNS genes, studies in nephrocytes were instrumental by providing functional evidence supporting the genetic findings. This includes the KANK proteins (55), *ARHGDIA* (56), *ADCK4* (57), and most extensively *SGPL1* (58). For the ortholog of *SGPL1*, rescue constructs that reflect the mutations from SRNS patients were shown to be inefficient in contrast to a rescue with wild-type constructs. *Drosophila* nephrocytes also exhibited an altered lipid metabolism reflecting the observations in the SRNS patients.

### Diabetic Nephropathy

Diabetic nephropathy represents the single most significant cause of ESRD (1). Targeted therapies are unavailable and the current regimes are only sufficient to slow down the progress of disease. It is thus noteworthy that nephrocytes were found to be a model for diabetic nephropathy (59). High-glucose diet was shown to induce nephrocyte dysfunction and decrease the amount of Sns, which represents the ortholog of nephrin. The authors identified a transcriptional downregulation by a pathway that includes *Knot*, the ortholog of *EBF2*, to be responsible for the reduction of Sns in nephrocytes. Interestingly, the authors were able to validate their findings in *Drosophila* nephrocytes in mouse models of diabetic nephropathy.

### *APOL1-*Associated Nephropathies

Incidence of ESRD is nearly fourfold in African Americans compared with those of European descent. Much of the excess risk is attributable to two risk alleles of the *APOL1* gene. The study of *APOL1* is hampered by its poor evolutionary conservation as orthologs are lacking in most model organisms. Transgenic expression of the human *APOL1* in nephrocytes has been shown to result in a cellular toxicity that was more pronounced upon expression of the human *APOL1* risk alleles (44, 45). Nephrocyte dysfunction and loss was observed with increasing age of the flies, involving a mechanism connected to endosomal trafficking. More recently, the findings in *Drosophila* were confirmed by transgenic expression of the human gene in mouse (60). Essential features of the human disease were recapitulated in this model. It is intriguing, that the role of a common risk allele was studied successfully in *Drosophila* nephrocytes.

### Endocytosis and Proximal Tubular Diseases

Imerslund–Gräsbeck syndrome is characterized by Cobalamindeficiency and tubular proteinuria while renal function is usually preserved. Mutations of Cubilin (61) and Amnionless (62) have been shown to be causative for this disorder. The orthologs of both genes are expressed in *Drosophila* nephrocytes, exercising an analogous role for nephrocyte function (28). Megalin, that may cause Donnai–Barrow syndrome (63) that also involves tubular proteinuria in humans, does not seem to be required for nephrocyte function (7, 27). A megalin-indepdendent role of Cubilin/Amnionless has also been described in humans for the absorption of vitamin B12 in the terminal ileum (64).

### Organ Crosstalk

*Drosphila* is an appropriate model to study communication between organs. In an effort to identify renal cardiomodulatory factors, Hartley and colleagues induced nephrocyte disruption and observed signs of a cardiomyopathy in the fly. Analyzing the hemolymph composition, they were able to identify *secreted protein acidic and cysteine rich* (SPARC), whose circulating levels are downregulated by nephrocytes. Reducing the gene dosage of this factor independently, through a heterozygous null allele, ameliorated the cardiomyopathy. Interestingly, *SPARC* has been linked to cardiac aging and metabolic syndrome in humans.

### Translational Applications

The genetic tool-kit of Drosophila combined with the costeffectiveness and speed inherent to this model offer great opportunities for a translational approach. Only a glimmer of these opportunities can be recognized in the effect of Vanillic acid on defective CoQ10-biosynthesis (7). Screening platforms have been introduced that already enable large-scale genetic screens. These protocols need to be optimized to facilitate the discovery of treatment options for glomerular diseases.

### OUTLOOK

We have seen significant progress in the last decade, and the nephrocyte currently emerges from the phase of experimental establishment to a driving force of glomerular discovery research. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in *Drosophila*, the nephrocyte as a complementary model system is well equipped to reveal mechanisms of podocyte function and glomerular diseases. Once the enormous potential for translational applications is unlocked, the nephrocyte will play its role in the identification of targeted therapies that are urgently needed in nephrology.

### REFERENCES

### AUTHOR CONTRIBUTIONS

The manuscript was written by TH with help from MH, MH designed the figures with help from TH, TBH critically reviewed and edited the manuscript and gave relevant additional insight. The title was suggested by TBH.

### ACKNOWLEDGMENTS

TH is supported by the Deutsche Forschungsgemeinschaft (DFG, HE 7456/1-1, CRC 1140/associated member) and the Faculty of Medicine of the University of Freiburg. TBH is supported by the DFG (CRC 1140, CRC 992, HU 1016/8-1), by the BMBF (01GM1518C), by the European Research Council—ERC grant 616891, and by the H2020-IMI2 consortium BEAt-DKD. We thank Maria Ericsson (Harvard Medical School Electron Microscopy Facility) for technical assistance.

is mutated in congenital nephrotic syndrome. *Mol Cell* (1998) 1:575–82. doi:10.1016/S1097-2765(00)80057-X

**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.

*Copyright © 2017 Helmstädter, Huber and Hermle. 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.*

## Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases

#### Jochen Gehrig1 , Gunjan Pandey 1,2 and Jens H. Westhoff 2 \*

1 Acquifer is a Division of Ditabis, Digital Biomedical Imaging Systems AG, Pforzheim, Germany, 2 Department of Pediatrics I, University Children's Hospital Heidelberg, Heidelberg, Germany

Genetic disorders account for a wide range of renal diseases emerging during childhood and adolescence. Due to the utilization of modern biochemical and biomedical techniques, the number of identified disease-associated genes is increasing rapidly. Modeling of congenital human disease in animals is key to our understanding of the biological mechanism underlying pathological processes and thus developing novel potential treatment options. The zebrafish (Danio rerio) has been established as a versatile small vertebrate organism that is widely used for studying human inherited diseases. Genetic accessibility in combination with elegant experimental methods in zebrafish permit modeling of human genetic diseases and dissecting the perturbation of underlying cellular networks and physiological processes. Beyond its utility for genetic analysis and pathophysiological and mechanistic studies, zebrafish embryos, and larvae are amenable for phenotypic screening approaches employing high-content and high-throughput experiments using automated microscopy. This includes large-scale chemical screening experiments using genetic models for searching for disease-modulating compounds. Phenotype-based approaches of drug discovery have been successfully performed in diverse zebrafish-based screening applications with various phenotypic readouts. As a result, these can lead to the identification of candidate substances that are further examined in preclinical and clinical trials. In this review, we discuss zebrafish models for inherited kidney disease as well as requirements and considerations for the technical realization of drug screening experiments in zebrafish.

Keywords: zebrafish, drug screening, compound screening, genetic kidney disease, high-throughput, highcontent, automated microscopy

### INTRODUCTION

Modern genetic diagnostics allow the rapid discovery of human disease-associated mutations. Moreover, human genetic disorders can often be mimicked in animal models that can be exploited in large-scale chemical investigations for the search of modifiers of disease-associated phenotypes and potentially therapeutic compounds. The zebrafish (Danio rerio) has become an increasingly accepted vertebrate model organism for biomedical research (1, 2).

Despite being a member of the teleost class of fish species, there is great homology in development as well as cell- and organ-specific structural and physiological properties between zebrafish and humans. Furthermore, even with the evolutionary distance, > 80% of human disease-associated genes have orthologs in the zebrafish genome (3). The embryonic and larval

#### Edited by:

Miriam Schmidts, Radboud University Nijmegen, Netherlands

#### Reviewed by:

Ruxandra Bachmann-Gagescu, Universität Zürich, Switzerland Rachel Lennon, University of Manchester, United Kingdom Rebecca Ann Wingert, University of Notre Dame, United States

### \*Correspondence:

Jens H. Westhoff<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 15 February 2018 Accepted: 04 June 2018 Published: 28 June 2018

#### Citation:

Gehrig J, Pandey G and Westhoff JH (2018) Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases. Front. Pediatr. 6:183. doi: 10.3389/fped.2018.00183

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characteristics of zebrafish include small size, ex utero development, optical transparency, and rapidity of organogenesis. In combination with the high fecundity of adult zebrafish and a relatively simple and cost-effective animal husbandry, this enables large-scale in vivo investigations. The zebrafish genome has been completely sequenced, thus facilitating genetic and genomic analysis and manipulation (3, 4). For instance, reverse genetics allow for precise investigation of associated phenotypes, by e.g., transient gene knockdown using antisense morpholino oligos or by genome-editing technologies like the CRISPR/Cas9 system (5, 6).

Due to the simplicity of the pronephros that can be readily studied in embryonic and larval stages, the zebrafish is an applicable experimental model system for the analysis of renal development and disease (7). The pronephros, as the earliest nephric stage, contains two nephrons sharing numerous genetic, structural, and functional aspects with the mammalian nephron (8). Phenotypic changes upon genetic alterations can be easily analyzed within intact live animals (9). Large-scale mutagenesis screens have identified various mutants affecting kidney development allowing the exploration of genetic and molecular mechanisms underlying pronephros development and function (10, 11). Moreover, reverse genetics approaches enable researchers to specifically alter orthologous genetic elements potentially associated with human disease. To date, major fields of research where such zebrafish models are being employed include glomerular (i.e., podocytopathies) and cystic renal disorders (i.e., ciliopathies).

Phenotype-based screening for drug discovery applications is increasingly employed in biomedical and pharmaceutical research. In contrast to target-based screening, phenotype-based approaches do not require exact knowledge of the therapeutic target (7). In addition, whole-organism in vivo approaches have the advantage that they can unravel toxic and other side-effects of drugs at a very early stage of the study. Over the last years, due to the versatility and power of the model, the zebrafish has emerged as the main vertebrate model system for high-throughput and high-content chemical screening experiments and large-scale phenotypic scoring (12, 13). Clear and scalable readouts for in vivo large-scale experiments can be readily established and a plethora of mutant and transgenic models expressing fluorescent proteins driven by tissue-specific promoters is available. In combination with automation technologies and dedicated sample handling workflows, this has led to various biomedical screening assays in fields such as genetics (14, 15), toxicology (16, 17), immunology and infection biology (18, 19), cardiovascular research (20, 21), drug discovery and safety (12, 13, 22, 23), personalized medicine (24), non-coding-genome analysis (25) as well as behavioral analysis (26, 27). Notably, several compounds that were identified in the zebrafish model have made it to preclinical and clinical trials, including new substance classes and repurposed drugs (12). For instance, in a chemical genetic screen testing 2.480 compounds, prostaglandin E2 (PGE2) was identified as an evolutionarily conserved regulator of hematopoietic stem cell (HSC) number in zebrafish embryos (28). Based on these results, a chemical derivative of PGE2 (Prohema), has been developed with the aim of improving the efficiency of HSC transplants using umbilical cord blood. Prohema has meanwhile advanced to Phase II clinical trials.

### GENETIC KIDNEY DISEASES IN ZEBRAFISH

Genetic kidney diseases can affect all parts of the kidney and its functions. To date, mutations in more than 150 genes have been identified that cause genetic human kidney diseases such as alterations of kidney development or specific glomerular and tubular diseases (29). Nephrogenesis in vertebrates is an intricate process that includes the successive formation of up to three kidneys depending on the species position in the phylogenetic tree: the pronephros, mesonephros, and metanephros (30). There is an increasing complexity with each successive kidney developing, however the structure and function of the basic renal units, the nephrons, remains largely unvaried across vertebrates (31).

In zebrafish, major vertebrate organ systems form within a few days after fertilization (32). The zebrafish pronephros is functional by 48 hpf (hours post fertilization) accomplishing the functions of blood filtration and osmoregulation (11, 33). It consists of two nephrons with a fused glomerulus at the midline (**Figure 1A**) (24). The tubular system consisting of the pronephric proximal and distal tubule and pronephric duct contains segment-specific conserved structural and physiological properties and spatio-temporal gene-expression patterns that are homologous to the human kidney (**Figure 1B**) (8, 35, 36). The zebrafish glomerulus is endowed with podocytes with extended and interdigitating foot processes and fenestrated endothelial cells forming a functional glomerular filtration barrier, analogous to the metanephric glomerulus of higher vertebrates (11).

At present, zebrafish is predominantly used as a genetic model for normal and abnormal kidney development, for hereditary glomerulopathies (i.e., podocytopathies) and for the study of ciliopathy-associated human cystic kidney diseases. These encompass polycystic kidney diseases and diseases of the nephronophthisis/medullary cystic kidney disease complex including more complex ciliopathies such as Joubert Syndrome, Meckel-Gruber Syndrome, and Bardet-Biedl-Syndrome (37). In this review, we focus on glomerulopathies and cystic kidney diseases.

### ZEBRAFISH AS A MODEL FOR HUMAN HEREDITARY GLOMERULOPATHIES

Zebrafish models can recapitulate human genetic glomerulopathies, i.e., a variety of podocytopathies that clinically often manifest by steroid-resistant nephrotic syndrome (SRNS) due to podocyte foot process effacement. SRNS is mostly therapy-resistant and leads to end-stage renal disease (ESRD) within a few years of onset. A growing number of SRNScausing mutations have been identified. For example, mutations in NPHS1, encoding Nephrin, cause congenital nephrotic syndrome of the Finnish type. Morpholino knockdown of nphs1 in zebrafish results in edema and loss of slit-diaphragms with

organization of each nephron into glomerulus (G), neck (N), proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early (DE), corpuscle of Stannius (CS), distal late (DL), and pronephric duct (PD) that fuse

to the cloaca (C). Adapted from Wingert and Davidson (34).

abnormal podocyte foot processes (38, 39). Mutations in WT1 (Wilms' tumor gene 1) have been associated with syndromic disorders such as Denys-Drash syndrome and Frasier syndrome, but also with diffuse mesangial sclerosis and early-onset isolated nephrotic syndrome (40). In zebrafish, knockdown of wt1a results in defects in podocyte development leading to glomerular injury and nephrosis (41). Mutations in NPHS2, Podocin, are the most relevant cause of autosomal-recessive SRNS of childhood. Zebrafish nphs2 morphants display pronephric glomerular hypoplasia with pericardial edema and ultrastructural glomerular damage of the filtration barrier (38, 39). Mutations in PLCE1 (Nephrocystin-3, NPHS3) have been identified in patients with SRNS and disease onset in the first year of life with a rapid progression to ESRD (42, 43). Zebrafish plce1 morphants display an impairment of the kidney filtration barrier as measured by tubular uptake of filtered 500 kDa fluorescent dextran, accompanied by edema, and severe disorganization of slit diaphragms (43). Other rare human mutations that were mimicked in zebrafish include ADCK4 (AarF domain containing kinase 4 gene) (44), KANK1 (kidney ankyrin repeat-containing protein 1), KANK2, KANK4 (45), CRB2 (Crumbs homolog 2) (46), NUP107 (Nuclear Pore Complex Subunit 107) (47), and ARHGDIA (48).

Whereas in many studies of genetic glomerulopathies the zebrafish has been used to model human disease-associated phenotypes, disruption of the glomerular filtration barrier can only be visualized by ultrastructural techniques like superresolution or electron microscopy that are not compatible with large-scale chemical screens. Edema formation in zebrafish embryos can indirectly report glomerular barrier impairments; however, despite being easily observed in brightfield microscopy it is not exclusively linked to renal impairment (49). This restricts its value as a phenotypic readout parameter in chemical kidney screens. Functional assessment of glomerular filtration and barrier integrity can be achieved by monitoring the temporal reporter activity after microinjection of fluorescently labeled inulin (50) or dextrans of different molecular weight (51–53) into the vascular system; however, this method is laborious and incompatible with extensive screening experiments. Additionally, a transgenic zebrafish that expresses GFP (green fluorescent protein)-tagged vitamin D-binding protein (VDBP), which acts as a tracer for proteinuria, has been reported (54) and may serve as an attractive alternative for high-content and high-throughput screening (53).

### ZEBRAFISH AS A MODEL FOR HUMAN CYSTIC KIDNEY DISEASES

Cystic diseases of the kidney are frequent monogenic disorders in humans (55, 56), with primary cilia dysfunction being the unifying cellular mechanism leading to most if not all cystic kidney diseases (56–58). Mutations in a variety of genes encoding the primary cilia/centrosome complex cause ciliopathies often associated with the development of renal cysts, in both human and zebrafish (59–64). However, it must be noted that cilia in the zebrafish pronephros are motile, whereas human renal cilia are thought to be non-motile (65, 66), suggesting a potential contribution of lack of fluid dynamics to cyst formation in the zebrafish model.