# NEPHROTIC SYNDROME IN PEDIATRIC PATIENTS

EDITED BY: Agnieszka Swiatecka-Urban, Robert P. Woroniecki and Frederick Jeffrey Kaskel PUBLISHED IN: Frontiers in Pediatrics

#### *Frontiers Copyright Statement*

*© Copyright 2007-2017 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.*

*The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.*

*Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.*

*Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.*

*As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.*

*All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.*

ISSN 1664-8714 ISBN 978-2-88945-298-9 DOI 10.3389/978-2-88945-298-9

### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

### Dedication to quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
## **NEPHROTIC SYNDROME IN PEDIATRIC PATIENTS**

### Topic Editors:

**Agnieszka Swiatecka-Urban,** University of Pittsburgh, United States **Robert P. Woroniecki,** Stony Brook Children's Hospital, United States **Frederick Jeffrey Kaskel,** Children's Hospital at Montefiore, United States

Electron micrograph images showing glomerular capillary loops of normal (left) and diffusely fused or effaced (right) podocyte foot processes resting on the glomerular basement membrane (GBM). Red blood cells (RBC) are seen inside the open capillary loops. Foot process effacement is an invariable morphological feature of glomerular disease presenting with nephrotic syndrome. 40,000x. Image: Dr. Sarangarajan Ranganathan, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.

Cover image: Human podocytes were cultured in Dr. Swiatecka-Urban's laboratory at Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine. Cover image was obtained by Dr. Donna Beer Stolz from the Imaging Center at the University of Pittsburgh.

Nephrotic syndrome (NS) - characterized by heavy glomerular protein loss (proteinuria), edema, hypoalbuminemia, and hyperlipidemia - has diverse causes and frequently leads to chronic kidney disease. This E-book encompasses articles on a variety of topics in NS, including a historical perspective on understanding and treatment of NS, followed by stateof-the-art reviews of the molecular pathomechanisms, clinical outcomes, as well as current and emerging treatment strategies for NS. We hope that this comprehensive review will help to reduce the gaps between the research and the dayto-day care of patients with NS and inspire new research efforts towards updating and expanding the treatment armamentarium for the future.

**Citation:** Swiatecka-Urban, A., Woroniecki, R. P., Kaskel, F. J., eds. (2017). Nephrotic Syndrome in Pediatric Patients. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-298-9

# Table of Contents

### **Chapter I: Introduction**

Cathie Spino, Jordan S. Jahnke, David T. Selewski, Susan Massengill, Jonathan Troost and Debbie S. Gipson

*78 The Evidence-Based Approach to Adult-Onset Idiopathic Nephrotic Syndrome* Pietro A. A. Canetta and Jai Radhakrishnan

# Editorial: Nephrotic Syndrome in Pediatric Patients

#### *Robert P. Woroniecki 1 \*, Agnieszka Swiatecka-Urban 2,3 and Frederick J. Kaskel 4*

*1Stony Brook Children's Hospital, Stony Brook, NY, United States, 2Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, PA, United States, 3Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 4Division of Pediatric Nephrology, Children's Hospital at Montefiore, Albert Einstein College of Medicine, New York, NY, United States*

Keywords: podocytopathy, APOL1, FSGS, calcineurin inhibitor, outcomes, edema, endocytic trafficking, obesity

### **Editorial on the Research Topic**

### **Nephrotic Syndrome in Pediatric Patients**

Nephrotic syndrome (NS) remains a clinical diagnosis, encompassing proteinuria, dyslipidemia, hypoalbuminemia, and gravity-dependent edema. We came a long way since the initial gross descriptions of NS as dropsy, lipoid nephrosis, and nil disease to the most recent classification of podocytopathies as causes on NS. Recent advances demonstrate that mutations of a number of genes regulating structure and/or function of glomerular filtration barrier are causally associated with histologic diagnosis of focal segmental glomerulosclerosis (FSGS), diffuse mesangial sclerosis, and clinical unresponsiveness to glucocorticosteroids in patients with NS. These findings explain why a subset of children who do not respond to treatment have genetic causes of NS. While the genetic causes represent only a minority of childhood NS, the exact molecular mechanisms underpinning the steroid-responsive NS remain largely unknown. Newer data explain that remission of NS after treatment with glucocorticosteroids, and an overall favorable prognosis are not consistent across all pediatric age groups and ethnic backgrounds.

In this special pediatric nephrology series, we have combined contributions from experts in childhood NS working in a variety of fields, including basic science, clinical medicine, and epidemiology to provide the reader with a comprehensive and the most up-to-date information on different aspects of NS. We were fortunate to enlist a prominent group of 31 authors to contribute a wide range of articles. In total, 10 papers have been included, with 3 original research articles, 6 reviews, and 1 historical perspective (Pal and Kaskel; Ranganathan; Swiatecka-Urban; Ellis; Hjorten et al.; Beins and Dell; Spino et al.; Chanchlani and Parekh; Woroniecki et al.; Canetta and Radhakrishnan).

We introduced the topic by historical perspective authored by Pal and Kaskel. The article walks readers through inspiring stories of tremendous efforts and international collaborations pawing the milestones in understanding of the pathophysiology and treatment of NS. Progress in the field is measured by a huge decrease in mortality related to NS from above 50% to less than 3% in the span of half of a century. As authors note, continued multicenter collaborations are critical to achieve new milestones and progress to personalized approach in the treatment of this rare childhood disease. Ranganathan reviewed the histopathology of congenital NS, minimal change disease, and its variants, and FSGS (Ranganathan). Swiatecka-Urban focused on the role of endocytic trafficking at the mature podocyte slit diaphragm and reviewed the critical role of signaling in maintaining the integrity of slit diaphragm that protects from to glomerular protein loss (proteinuria), manifesting as NS. Ellis described current pathophysiology of edema formation in NS (Ellis). The author reviewed "the under and overfill hypothesis" and current evidence for loss of plasmin and other serine proteases in the urine resulting in upregulation of the epithelial sodium channel (ENaC) causing Na+ and fluid retention (Ellis). Hjorten et al. reviewed the long-term outcomes of NS in

### *Edited by:*

*Michael L. Moritz, Children's Hospital of Pittsburgh, United States*

#### *Reviewed by:*

*Franz Schaefer, Heidelberg University, Germany*

#### *\*Correspondence:*

*Robert P. Woroniecki robert.woroniecki@ stonybrookmedicine.edu*

#### *Specialty section:*

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

*Received: 23 June 2017 Accepted: 18 July 2017 Published: 03 August 2017*

#### *Citation:*

*Woroniecki RP, Swiatecka-Urban A and Kaskel FJ (2017) Editorial: Nephrotic Syndrome in Pediatric Patients. Front. Pediatr. 5:167. doi: 10.3389/fped.2017.00167*

childhood noting the importance of steroid responsiveness as a prognostic indicator. While steroid-resistant NS (SRNS) is associated with a high-risk (HR) of developing end-stage renal disease (ESRD), long-term complications of steroid-sensitive NS are under-recognized (Hjorten et al.). Beins and Dell in their retrospective chart review surmise that although majority of SRNS patients initially respond to calcineurin inhibitor therapy, a significant percentage still progresses to ESRD, despite achieving short-term remission. Spino et al. reviewed current therapeutic options for FSGS and discussed the problem of varied efficacy related to specific therapy, patient characteristics, cost, and common side effects, and noted that this variability of response to treatment is likely caused, at least in part, by the heterogeneity in the etiology of FSGS.

Chanchlani and Parekh further reviewed the emerging role of genetic factors determining the response to treatment within an ethnic group, noting that among African-Americans (AA), the risk variants in *APOL1* are associated with a more than 10-fold increase in risk of FSGS and HR carriers have a twofold greater risk

**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.

The handling editor declared a shared affiliation, though no other collaboration, with one of the authors AS-U and states that the process nevertheless met the standards of a fair and objective review.

of progression to ESRD. Woroniecki et al. presented original data indicating that AA children with HR *APOL1* genotype and FSGS have increased prevalence of obesity and left ventricular hypertrophy (LVH) despite a later age of FSGS onset, while adjusting for socioeconomic status. They noted that treatment of obesity may be an important component of chronic kidney disease and LVH management in this population (Woroniecki et al.). Finally to contrast with children, Canetta and Radhakrishnan reviewed the evidence-based approach to adult-onset NS.

Our overarching goal was to stimulate interest of academic and practicing physicians and scientists in this relatively rare childhood disease with diverse pathophysiology and outcomes. We hope that we will be seeing more studies and research in this field in the near future.

### AUTHOR CONTRIBUTIONS

RW wrote and reviewed the editorial, AS-U and FK contributed to review process and editing.

*Copyright © 2017 Woroniecki, Swiatecka-Urban and Kaskel. 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.*

# History of Nephrotic Syndrome and Evolution of its Treatment

### *Abhijeet Pal\* and Frederick Kaskel*

*Division of Pediatric Nephrology, Children's Hospital at Montefiore, Albert Einstein College of Medicine, New York, NY, USA*

The recognition, evaluation, and early treatment of nephrotic syndrome in infants and children originate from physicians dating back to Hippocrates. It took nearly another 1000 years before the condition was described for its massive edema requiring treatment with herbs and other remedies. A rich history of observations and interpretations followed over the course of centuries until the recognition of the combination of clinical findings of foamy urine and swelling of the body, and measurements of urinary protein and blood analyses showed the phenotypic characteristics of the syndrome that were eventually linked to the early anatomic descriptions from first kidney autopsies and then renal biopsy analyses. Coincident with these findings were a series of treatment modalities involving the use of natural compounds to a host of immunosuppressive agents that are applied today. With the advent of molecular and precision medicine, the field is poised to make major advances in our understanding and effective treatment of nephrotic syndrome and prevent its long-term sequelae.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

### *Reviewed by:*

*Michal Malina, Motol University Hospital, Czech Republic R. Morrison Hurley, University of British Columbia, Canada*

#### *\*Correspondence:*

*Abhijeet Pal<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 26 February 2016 Accepted: 17 May 2016 Published: 30 May 2016*

#### *Citation:*

*Pal A and Kaskel F (2016) History of Nephrotic Syndrome and Evolution of its Treatment. Front. Pediatr. 4:56. doi: 10.3389/fped.2016.00056*

Keywords: nephrotic syndrome, early recognition, evaluation, natural history, treatments

### HISTORY OF NEPHROTIC SYNDROME

Nephrotic syndrome, as we know it today, is a combination of proteinuria, hypoalbuminemia, hyperlipidemia, and edema, a concept that took some time to be developed. Interestingly, the effective treatments became available only recently in the mid 1900s, with the advent of steroids, antibiotics, diuretics, and other immunomodulators. Even today, there is a gap in our understanding of the etiology(s) of nephrotic syndrome of childhood, and better treatments are still required in the more resistant forms. Nephrotic syndrome as such is a combination of clinical and laboratories findings that is seen with a variety of pathologic lesions affecting the glomerulus.

Generalized edema, referred to as dropsy in the earlier literature, has been documented from the times of Hippocrates (1, 2), although the differentiation between various causes (heart vs. liver vs. nutritional disturbances vs. renal abnormalities) was not made. One observation by Hippocrates was: "when bubbles settle on the surface of the urine, it indicates a disease of the kidney and that the disease will be protracted" (1). Cornelus Roelans of Belgium described in 1484 a child with nephrotic syndrome and "whole body swelling." He went on to recommend the treatment as follows: "take the tops of elder plant and daneswort, cook in white wine and wrap the child in hot clothes by applying the poultice in whole or in part, and so cure him" (3).

During the sixteenth to eighteenth centuries, dropsy was considered a disease *per se*, the patient had dropsy, without differentiating between the causes (4). One of the first accurate descriptions of nephrotic syndrome in children was made by Theodore Zwinger of Basel in 1722 (5). He also noted decreased urine output and attributed this to "obstruction and compression of the tubules of the kidney," thus placing the seat of the disease in the kidney, since at that time, it was recognized that in pediatric practice heart and liver disease rarely manifest. His important observation had little influence on the scientific community, and his findings were not quoted by any author in

the following 200 years. Of note, these observations were made even before Morgagni was able to establish the idea that disease might arise from specific organs (6). Morgagni's disciple William Heberden went on to say: "Dropsy is very rarely an original distemper but generally a symptom of some other which is too often incurable" (7).

Later in the eighteenth century, dropsy was divided into those dependent on morbid viscera (liver and heart) and a general form, supposedly inflammatory. With humoralism, a prevalent concept in Europe at that time, bloodletting was a common practice for these so called "inflammatory diseases" and was a commonly prescribed. Around the same time, several observers [namely, Cotugno (8), Cruikshank (9), Wells (10), and Brande (9)] noted coagulability of urine in the patients. It was finally in 1827 that Richard Bright (1789–1858) was able to put together the triad of generalized edema, proteinuria, and kidney disease, as presenting features of this disease (11). John Bostock, a colleague of Bright, also noted that when protein in the urine was highest, it was lowest in the serum (11). Christison confirmed these findings in 1829 (12). Hence by 1830, nephrotic syndrome of profound albuminuria, hypoalbuminemia, and edema, resulting from diseased kidneys was established. Subsequently, on the postmortem analysis, these kidneys revealed a diseased state. Bright described three varieties of postmortem appearance of the kidneys, Chritison seven, Pierre Rayer six in 1840, and Carl Rokitansky no less than eight in 1846, including the "specknierre" or bacon kidney, recognized later as amyloidosis (9).

Over the next few years, the various lipid abnormalities in nephrotic syndrome became the forefront of discussion. Latescent or milky appearance of the serum was known to be present in nephrotic syndrome (noted with bloodletting sessions). Christison showed this to be from ether soluble fat (13). In 1846, Johnson not only described the fatty nature of the kidneys on gross appearance in many such patients but also fat globules and fat casts in the tubules (14). More attention was given to the cellular and tubular components of the kidneys as they were more obvious by the histological techniques available at that time.

Virchow introduced the term "parenchymatous nephritis" for a pathological picture with primary tubular involvement (15). With advances in microscopy, glomerular involvement became clearer along with that of the parenchyma. In acute nephritis, the presence of pale exsanguinated glomeruli was long known. In 1872, Klebs coined the term "glomerulonephritis" to describe the exudative glomerular changes seen under the microscope (16). In 1905, the term "nephrosis" was coined by Müller to describe all "non-inflammatory" diseases of the kidney as a substitute for parenchymatous nephritis, contrasting it with exudative and inflammatory disease, which would retain the name nephritis (17). This concept of "nephritis in contrast to nephrosis" was further popularized by F. Volhard, T. Fahr, and C. Munk.

### TREATMENTS FOR NEPHROTIC SYNDROME

Introduction of the renal biopsy in the mainstream clinical practice of nephrology during the 1950s–1960s added a new dimension to the understanding of the histological findings in nephrotic syndrome. The histological classifications were based on the light microscopic appearance and included membranous glomerulonephritis, proliferative glomerulonephritis, a mixed membranous and proliferative glomerulonephritis, diabetic nephropathy with hyaline nodules, and focal segmental glomerular sclerosis; these are summarized in a case series from 1958 (18). The availability of electron microscopy and immunofluorescent localization of proteins was a welcomed coincidence. These new techniques transformed the ideas on morphology and pathogenesis, and made definite correlations between clinical presentation and pathological picture. The 1961, a Ciba Foundation symposium on the use of the renal biopsy was a landmark in these regards (19).

Antibiotics played an important role in treating the infections that arose from nephrotic syndrome, and the mortality rate reduced drastically from two-third to 35% (20). It is important to point out that prior to availability of steroids multiple desperate treatments were tried. A study from Boston noted the various treatments that were attempted from 1926 to 1948 for nephrotic syndrome. Dietary modification and low salt diet were probably the most effective treatments at that time. There were some weak mercurial diuretics with little if any action. Other drastic measures, such as the induction of measles and vaccinia, were instituted. Many of the children inoculated had some form of post-infection diuresis and decrease in proteinuria (11 out of 14), only to recur. Other supportive measure included blood transfusions, antibiotic therapy, treatment with thyroid extract, decapsulation of the kidney, testosterone, multiple vitamins, horse antiserum, and parathyroid hormone.

Steroid hormones were first isolated and identified in 1936 (21, 22). By 1946, cortisone was first prepared by partial synthesis from bile acids (23). On the other hand, adrenocorticotropic hormone (ACTH) was extracted from pig and sheep pituitary gland by isoelectric precipitation (22, 24) in the 1940s. One preparation of ACTH was commercially available as sterile powder reconstituted with isotonic saline for intramuscular injection. In the early 1950s, intramuscular injections of Cortisone (25) and ACTH (26) were first being used for treatment for nephrotic syndrome in children by Arneil and Wilson from Glasgow. These were relatively short courses, 5 days of daily 100–300 mg of intramuscular cortisone in the first study and 40–80 mg of an ACTH intramuscular injection for 12 days in the second study (25, 26). Later on, prednisone was first synthesized by oxidation of cortisone (27, 28). ACTH and cortisone were quickly replaced by prednisolone and prednisone as they could be administered orally without the need for daily injections (29, 30). With the advent of steroid therapy, mortality from nephrotic syndrome dramatically decreased to 3% (31).

An important series of studies, in the era after renal biopsy and steroids, to better understand the management of childhood nephrotic syndrome were heralded by the International Study of Kidney Disease in Children (ISKDC) established in 1965 with participants from North America, Europe, and Asia. A series of prospective, multicenter cooperative studies by the ISKDC established definitions, clinicopathological correlations, and recommendations for therapy that provided a basis for diagnosis and management of pediatric nephrotic syndrome that persists today. Between January 1967 and June 1974, children with the nephrotic syndrome who were older than 12 weeks and younger than 16 years were enrolled into the clinical survey from the 24 participating clinics (32–34). Of the 521 entered, 76.6% had minimal-change nephrotic syndrome, 7.5% had membranoproliferative glomerulonephritis, and 6.9% had focal segmental glomerulosclerosis (35). All participants had biopsies prior to starting steroid therapy with prednisone. Initial treatment was 60 mg/24 h/m2 (maximum dosage 80 mg/24 h) in divided doses for 4 weeks, followed by 40 mg/24 h/m2 in divided doses, three consecutive days out of seven for 4 weeks. A seminal observation was made that patients with non-minimal lesions had a varied and limited response to steroids (36).

Azathioprine, a purine synthesis inhibitor, was also tested by this group in a randomized placebo-controlled trial (37). Patients that were considered to be early non-responders to steroids (not responding to the initial 8-week therapy) or frequently relapsing were randomized to receive every other day prednisone with azathioprine (test group) or placebo (control group). No significant decrease in proteinuria or number of relapses was noted in the test group vs. the control group (37).

The use of an alkylating agent in steroid-resistant nephrotic syndrome was first initiated with nitrogen mustard as early as 1958 (38). This was extended to other alkylating agents, cyclophosphamide and chlorambucil, in the 1960s and prompted the ISKDC to conduct a randomized control trial to define the role of cyclophosphamide in children with early non-responders and frequently relapsing nephrotic syndrome. Cyclophosphamide was shown to remit proteinuria in early non-responders and decrease the number of relapses of nephrotic syndrome (39). Thus, it proved to be an important agent to decrease steroid use and prevent steroid toxicity, although it caused gonadal failure in post-pubertal males (39). Chorambucil, although noted to be as effective as cyclophosphamide (40), had concerning side effects of acute leukemia and renal carcinoma (41).

The initial 8-week steroid regimens (4 weeks daily and 4 weeks every other day) were then compared with a longer steroid treatment duration in the late 1980s (42). Mounting evidence supported a longer duration of steroids, which helped decrease the number of future relapses and steroid dependence, and the initial 8-week steroid regimen has been recommended to be increased to at least 12 weeks (43, 44).

Levamisole, known for its anthelmintic, was first reported to be used in children with nephrotic syndrome in 1980 (45) and continues to be used as a steroid sparing agent in many countries. Cyclosporine A (CsA), a calcineurin inhibitor, was first isolated from the fungus *Tolypocladium inflatum* found in a soil sample obtained in 1969 from Norway, by Hans Peter Frey (46). It was initially used in humans for renal transplantation in 1978 and has since changed the face of transplantation (47). The first use for CsA was reported in 1986 among adults with difficult to treat nephrotic syndrome (48). By the late 1980s, there were reports of its successful use in children with steroid-resistant or steroiddependent nephrotic syndrome that had not responded well to alkylating agents with the target levels of CsA between 50 and 200 ng/ml (49, 50).

Tacrolimus, another calcineurin inhibitor, was first extracted from *Streptomyces tsukubaenis* in 1987 by a Japanese group (51) and was initially used as a drug in human organ transplantation. Its use for nephrotic syndrome was first started in adults in the early 1990s (52–54) and then reported to be used children by the early 2000s (55–57). It was shown to be similar to CsA with regard to efficacy and renal toxicity but without the cosmetic side effects of hirsutism and gingival hypertrophy (58).

Mycophenolate mofetil (cellcept®) and, more recently, mycophenolate sodium (myfortic®) are prodrug forms of mycophenolic acid, another purine synthesis inhibitor, identified from *Penicillium* species. It was first reported to be used in pediatric nephrotic syndrome in 2000 (59) after being successfully applied in other glomerular diseases (60) and renal transplantation. It has been shown to be useful in steroid-dependent and frequently relapsing steroid-sensitive nephrotic syndrome, as a first-line agent, although there is evidence to indicate that it is probably less effective that calcineurin inhibitors (43, 61–63).

Rituximab, a monoclonal antibody against a B-lymphocyte antigen CD20, came into the armamentarium after case reports describing the incidental finding of a beneficial effect of rituximab on childhood nephrotic syndrome (64). Benz et al. first used rituximab to treat ITP, resistant to steroids and immunoglobulin therapy, in a 16-year-old boy who also suffered from steroid-dependent nephrotic syndrome. As well as effectively resolving the ITP, rituximab also improved the nephrotic syndrome, inducing a relapse-free period for over 12 months on low-dose CsA (65). In the other two cases, rituximab was used to treat post-transplant lymphoproliferative disease (PTLD) in boys who also had recurrence of FSGS in their transplants (66, 67), with beneficial effects on proteinuria. Its efficacy was recently demonstrated as a steroid-sparing agent in childhood onset frequently relapsing and steroid-dependent nephrotic syndrome by a multicenter, double-blind, randomized, and placebo-controlled trial (68) conducted by Iijima et al. Patients in the rituximab-treated group were relapse-free for prolonged period of time despite being weaned off other immunosuppressants such as mycophenolate or cyclosporine. There have been few case reports of its efficacy in refractory primary FSGS; however, controlled clinical trails are needed to define its exact role (69, 70). It has been proven to be effective in combination with plasmapheresis for post-transplant recurrence of FSGS (70, 71). Part of this effect may be mediated by its direct action on the podocyte (72).

### FUTURE TREATMENTS AND CONCLUSION

Interestingly, ACTH is currently remerging as a potential treatment for nephrotic syndrome. In Europe, it is available as a synthetic depot formulation (Synacthen®) and in the US as a highly purified formulation (Acthar® gel) from porcine or bovine sources (73). There are reports on its efficacy in multiple causes of nephrotic syndrome, including idiopathic membranous nephropathy, FSGS, minimal-change disease, and mesangial glomerulonephritis, with a response rate varying from 29 to 100% (73–79). Recently, it has been shown to be particularly beneficial in idiopathic membranous nephropathy. Although the exact mechanism of action for ACTH is not known, it is thought to act directly on the podocytes *via* melanocortin receptors on the podocytes. There is need for a multicenter randomized control trial to assess its use in idiopathic nephrotic syndrome.

With discovery of various gene defects associated with nephrotic syndrome, there is an expanding knowledge of various podocyte signaling pathways that play a role in the pathogenesis of nephrotic syndrome (80). A better understanding of these pathways is needed to develop future targeted therapy. The high risk variant genotype of APOL1 gene (codes of apolipoprotein 1, known for its trypanolytic properties), was found to confer increased risk to kidney disease by gene-wide association studies (GWAS) (81–83). However, its association and role in nephrotic syndrome and podocyte biology are yet to be defined (80). New drugs on the horizon, such as losmapimod (p38 MAPK inhibitor), sparsentan (endothelin receptor type 1A antagonist), and

### REFERENCES

biologics, such as adalimumab (anti-TNF-α) and abatacept (anti-CD80), hold promise in the treatment of steroid-resistant nephrotic syndrome and prevention of renal progression (79).

In summary, the seminal contributions of the ISKDC identified the importance of global collaborations in order to conduct meaningful multicenter trials aimed at effectively treating children with nephrotic syndrome, preventing renal progression and facilitating the opportunity for each child to reach their full potential and quality of life. The availability of advanced genetic and molecular applications to personalized medicine offer unique opportunities along this promising journey.

### AUTHOR CONTRIBUTIONS

The authors contributed to the manuscript. Dr. AP is the first author as he did most of the work in reviewing the literature and formatting the style and content of the submission. Dr. AP is the corresponding author.

technology. *Am J Nephrol* (1997) 17(3–4):347–58. doi:10.1017/CBO97811074 15324.004

gel. *Clin J Am Soc Nephrol* (2013) 8(12):2072–81. doi:10.2215/ CJN.02840313

tissue, primary cells, and immortalized cell lines. *J Am Soc Nephrol* (2015) 26(2):339–48. doi:10.1681/ASN.2013091017

83. Ruchi R, Genovese G, Lee J, Charoonratana VT, Bernhardy AJ, Alper SL, et al. Copy number variation at the APOL1 locus. *PLoS One* (2015) 10(5):e0125410. doi:10.1371/journal.pone.0125410

**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 © 2016 Pal and Kaskel. 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.*

## Pathology of Podocytopathies Causing Nephrotic Syndrome in Children

### *Sarangarajan Ranganathan\**

*Department of Pathology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA*

Nephrotic syndrome (NS) in children includes a diverse group of diseases that range from genetic diseases without any immunological defects to causes that are primarily due to immunological effects. Recent advances in molecular and genomic studies have resulted in a plethora of genetic defects that have been localized to the podocyte, the basic structure that is instrumental in normal filtration process. Although the disease can manifest from birth and into adulthood, the primary focus of this review would be to describe the novel genes and pathology of primary podocyte defects that cause NS in children. This review will restrict itself to the pathology of congenital NS, minimal change disease (MCD), and its variants and focal segmental glomerulosclerosis (FSGS). The two major types of congenital NS are Finnish type characterized by dilated sausage shaped tubules morphologically and diffuse mesangial sclerosis characterized by glomerulosclerosis. MCD has usually normal appearing biopsy features on light microscopy and needs electron microscopy for diagnosis, whereas FSGS in contrast has classic segmental sclerosing lesions identified in different portions of the glomeruli and tubular atrophy. This review summarizes the pathological characteristics of these conditions and also delves into the various genetic defects that have been described as the cause of these primary podocytopathies. Other secondary causes of NS in children, such as membranoproliferative and membranous glomerulonephritis, will not be covered in this review.

Keywords: congenital nephrotic syndrome, Finnish type, diffuse mesangial sclerosis, minimal change disease, focal segmental glomerulosclerosis, C1q nephropathy

### INTRODUCTION

The kidney is the main organ of filtration in the body, and the daily protein loss is only a small portion of the total protein ingested. Nephrotic syndrome (NS) is characterized by heavy proteinuria that exceeds 1.66 g/1.73 m2 /day in children, edema, hypoalbuminemia, and hyperlipidemia (1). Although the causes of NS are many and diverse, it is a frequent cause of renal disease in children with an annual incidence of about 2 to 7 children per 100,000 (1). The International Study of Kidney Disease in Children lists minimal change disease (MCD) as the commonest cause of primary NS in children affecting 77% of the children followed by focal segmental glomerulosclerosis (FSGS) at about 8% followed by membranoproliferative glomerulonephritis (MPGN) and membranous glomerulonephritis (2). The causes of NS also vary to some extent with the age of presentation with the congenital forms more directly associated with specific genetic defects and those appearing in later childhood related more often to secondary causes.

#### *Edited by:*

*Robert P. Woroniecki, State University of New York, USA*

#### *Reviewed by:*

*Francois Cachat, University Hospital of Bern, Switzerland Mohamad Ikram Ilias, Hospital Universiti Sains Malaysia, Malaysia*

#### *\*Correspondence:*

*Sarangarajan Ranganathan<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 16 December 2015 Accepted: 21 March 2016 Published: 31 March 2016*

#### *Citation:*

*Ranganathan S (2016) Pathology of Podocytopathies Causing Nephrotic Syndrome in Children. Front. Pediatr. 4:32. doi: 10.3389/fped.2016.00032*

## PATHOPHYSIOLOGY OF NS

The primary defect in NS is loss of proteins in the kidney. Although lack of tubular reabsorption could lead to proteinuria, NS range proteinuria usually implies permeability defects in the glomerular membrane that results in this excessive protein loss. This leads to albuminuria and hence the associated hypoalbuminemia and edema, the two main manifestations of NS. The hyperlipidemia is usually due to the increased lipoprotein synthesis induced by the hypoalbuminemia, and this may lead to increased platelet aggregation and thrombosis, one of the complications of NS. The loss of other proteins, minerals, and vitamins with the proteinuria may also predispose to malnutrition and infections. The most dramatic advances for understanding the pathophysiology of NS has occurred in the area of podocyte biology and the structure of the slit diaphragm (3–5). The glomerular filtration barrier consists of the fenestrated capillary endothelium, the extracellular basement membrane, and the intercalated podocyte foot processes. NS is associated with the biopsy finding of effacement of podocyte foot processes. Effacement is characterized by flattening of the podocyte, retraction of foot processes, and sometimes microvillous transformation. It has also been understood that MCNS and FSGS can be classified as podocytopathies, in which disruption of slit diaphragm and normal podocyte function can lead to proteinuria and glomerular disease. Details of the structure and mechanisms of podocyte injury and defects are beyond the scope of this article, and readers are directed to several reviews on podocytes (3–5).

### CAUSES OF NEPHROTIC SYNDROME IN CHILDREN

The causes of NS show some variations depending on the age of the child with early onset NS usually representing primary genetic or idiopathic causes, such as congenital NS, due to any of the known genetic defects or MCD and less often FSGS (**Table 1**) (2, 6, 7). As the age increases, the ratio of MCD and FSGS may vary with secondary causes becoming more common in the second decade. Although NS may be the only presentation in children, often the condition is precipitated or can be seen as part of a nephritic syndrome. There is still some controversy on whether MCD and FSGS are related entities with MCD representing the early form of a disease and FSGS the late stage. This is especially so since there is often the finding of an early biopsy with MCD changes with refractory disease and a later biopsy showing features of FSGS. Also, while several genes have now been identified in MCD and FSGS, no distinct genetic defects have been found for these two diseases (7).

### GENETICS OF NS

The podocyte, as mentioned before, is the critical structure and has a critical role in pathogenesis of NS (**Table 2**; **Figure 1**). Podocytes form foot processes and slit diaphragms as part of normal development. This leads to the expression of specific proteins, such as ZO-1, in the slit diaphragm and nephrin, podocin, and CD2AP (adhesion molecule CD2-associated protein) are expressed (5). Nephrin gene mutations cause Finnish-type nephropathy (7, 8).

#### TABLE 1 | Causes of nephrotic syndrome in children.

#### Genetic

Congenital nephrotic syndrome of Finnish type Diffuse mesangial sclerosis (DMS) Isolated DMS Part of Denys–Drash syndrome Epidermolysis bullosa associated Steroid-resistant nephrotic syndrome Familial focal segmental glomerulosclerosis (FSGS)

#### Infectious causes

Congenital infections including syphilis, toxoplasmosis, and HIV Cytomegalovirus HIV-associated nephropathy

#### Idiopathic

Minimal change nephropathy Focal segmental glomerulosclerosis Diffuse mesangial hypercellularity Membranous glomerulonephritis Membranoproliferative GN (MPGN) (NS may predominate or with nephritic syndrome)

#### Others

Lupus nephropathy IgA nephropathy Drugs Malignancies Hemolytic uremic syndrome (HUS)

Other developmental genes involved in podocyte structure and function include Pax-2 and WT1. The WT1 gene mutation results in the Denys–Drash syndrome and Frasier syndrome. Podocin gene defects cause familial autosomal-recessive steroid-resistant NS in early childhood. The individual genetic defects are described further in the respective disease sections. It is critical to understand the structure of the podocyte and the basement membrane with all its collagen to understand the genetic defects and their potential effect. Discovery of novel mutations has led to further understanding of the biology and prognosis of these diseases.

### ROLE OF RENAL BIOPSY IN DIAGNOSIS OF NS

In many instances, there may be no role for pathologist for diagnosis due to the ability to do genetic testing for individual mutations and due to the response to steroid therapy, but increasing incidence of resistance to steroid therapy has increased the role of the pathologist in determining if FSGS is the cause of steroid resistance. Other causes for biopsy are presence of coexisting nephritic syndrome and for determination of other causes of NS. All renal biopsies need triaging for adequacy to perform immunofluorescence and electron microscopy (EM). In view of the broad etiology and associated conditions, the pathologist very often has a critical role in guiding therapy in these patients. Routine histology warrants a PAS and silver stain for assessment of basement membranes and a Masson trichrome stain for assessment of fibrosis and glomerulosclerosis as well as vascular changes. The standard panel for IF includes IgG, IgA, IgM, C3, C4, C1q, and albumin with kappa and lambda where needed. EM is necessary for diagnosis in many instances.

#### TABLE 2 | Genetic causes of nephrotic syndrome.

nephrotic syndrome.

### PATHOLOGY OF COMMON CAUSES OF NS IN CHILDHOOD

### Congenital Nephrotic Syndrome of Finnish Type

It is an autosomal-recessive disorder with an incidence of 1:8200 births in Finland (9). It is also seen in North American and European populations and is a cause of congenital NS with prenatal presentation in many cases. Diagnosis has been made *in utero* by amniocentesis that shows elevated alpha-fetoprotein as early as 16–18 weeks gestation (10). It results in small for gestational age neonate with prematurity, deformities due to contractures and an abnormally enlarged placenta. It results in heavy proteinuria in the neonatal period with selective proteinuria in

proximal tubules, a characteristic feature of congenital NS (H&E 100×). (C) Another image from the opposite kidney showing the same morphological features with no segmental sclerosis evident at this time (H&E 100×).

FIGURE 3 | Histological images for diffuse mesangial sclerosis. (A) An image from another nephrectomy specimen showing the numerous dilated tubules with small occasional glomeruli (H&E 40×). (B) Higher magnification of the glomeruli showing a solidified appearance on light microscopy (H&E 200×). (C) A Jones silver stain highlighting the solidified loops with accentuation of the epithelial cells on the surface. No capillary loops are identified (Jones methanamine silver 200×). (D) A trichrome stain showing the mesangial sclerosis characteristic of this disease. Note again the prominent epithelial cells. Progressive disease leads to glomerular obsolescence and interstitial fibrosis seen in this image (Trichrome 200×).

the early stages and more non-selective proteinuria in the later stages as the disease progresses. Most infants do not survive beyond 1 year of life, usually due to infections and sepsis related to loss of immunoglobulins. The gene for Finnish nephropathy (FN) is *NPHS1* mapped to the long arm of chromosome 19 (19q13.1) that codes for nephrin (7, 11, 12). Several nephrin mutations have now been identified. The typical mutations are a 2-bp deletion in exon 2 (Fin-major) or a nonsense mutation in exon 26 (Fin-minor). Renal biopsy shows normal glomeruli or some with mesangial hypercellularity, hyperlobulated capillary tufts, and some scarring. Microcystic dilatation of proximal and distal tubules is also seen, and there may be associated interstitial fibrosis and inflammation (**Figure 2**). Immunofluorescence is negative, whereas EM shows diffuse foot process effacement with or without villous transformation. Immunohistochemical stain for nephrin, now commercially available will show negative staining of podocyte. This stain helps in differentiating FN from other causes of congenital NS. Development of anti-nephrin antibodies and recurrence in allograft kidney has been reported (13).

### Diffuse Mesangial Sclerosis

This is the second most common cause of congenital NS diagnosed on renal biopsy. Although the presentation is similar to that of FN, diffuse mesangial sclerosis (DMS) can present later up to 4 years of age. It presents with unremitting NS within the first 9 months of life usually. Infants develop hypertension with rapid onset of renal failure. The combination of DMS, Wilms tumor, and male pseudohermaphroditism constitutes Denys–Drash syndrome (14). Other associations include cataract, strabismus, nystagmus, myopia and aniridia, mental retardation, microcephaly,

#### TABLE 3 | Common forms of familial FSGS.

deafness, musculoskeletal abnormalities, and cleft palate. DMS is also part of the Galloway–Mowat syndrome, Pierson syndrome (LAMB2 mutations), and Frasier syndrome (7). It is caused by WT1 gene mutations in exon 8 or 9 (14). Frasier syndrome is usually caused by a splice variant mutation in exon 9 (15). The earliest light microscopic feature is increased mesangial matrix that is global and diffuse. There is a gradient of changes seen from the outer to the inner cortex with the most severe sclerosis being seen in the outer cortex, DMS in the mid zone and milder sclerosis in the inner zone (**Figure 3**). In some cases, prominent focal or global glomerulosclerosis may be seen. Podocytes may be prominent over the tufts. Severe tubulointerstitial damage with cysts and tubular ectasia may be seen. Immunofluorescence shows no immune deposits or non-specific mesangial IgM, C3, and C1q, whereas EM shows variable effacement of foot processes. Glomerular basement membrane lamellations and splitting similar to Alport's syndrome may be seen. In late stages, there is widening of the BM with thickened capillary loops and finally a sclerotic glomerulus.

### Minimal Change Disease

Minimal change disease is more common in boys than girls with a ratio of 2:1. Almost 80% of cases occur in children <6 years of age (median age 2.5 years) (1, 16). It can be idiopathic, secondary, or familial. The most common form is sporadic MCD that is usually steroid sensitive. The genes mutated in MCD include NPHS1 and NPHS 2 (7). Mutations in the dysferlin gene have also been described. More recently, mutations in epithelial membrane protein 2 (EMP2) gene have been shown to cause childhood-onset NS. Crumbs homolog 2 (CRB2) defects have been associated with steroid-resistant NS. NPHS2 mutations have been associated with steroid resistance (17, 18).

Light microscopy usually shows normal glomeruli and tubules with the only change being prominence and swelling of visceral epithelial cells (2, 16). Patchy mild expansion of the mesangium may be seen in some cases (**Figure 4**). Histological finding of even a single glomerulus with segmental sclerosis, hyalinosis, or synechiae is enough to warrant a diagnosis of FSGS. Immunofluorescence is usually negative for any immune deposits, although there may be weak staining for IgM or C3 in a mesangial location in some cases. Albumin is positive within tubules reflecting the albuminuria. EM shows diffuse foot process effacement with prominent villous transformation, directly correlating with the severity of proteinuria and less prominent and patchy following treatment. Although this is the usual pattern, variants with increased mesangial hypercellularity are described. Immature glomeruli may be seen with this mesangial hypercellularity variant. In those patients presenting with acute renal failure, acute tubular injury and interstitial inflammation may be seen. Tubular atrophy is, however, not a feature of MCD. In those cases where IF shows deposits, the most likely ones are IgM (IgM nephropathy) or C1q (C1q nephropathy). In those where IgA is the dominant immunoglobulin, coexistent IgA nephropathy must be considered (19). The differential diagnosis for MCD is FSGS and as mentioned earlier, presence of any of the glomerular changes or foci of tubular atrophy in the absence of typical FSGS lesion, should raise the possibility of FSGS. Foot process effacement is not specific to MCD and can be seen in any cause of severe proteinuria. The debate over C1q being a specific subtype is still open as more recent data seems to suggest that though the initial response to therapy is poorer in this group, the overall long-term outcome is not different from cases of MCD.

The diffuse mesangial hypercellularity variant of MCD defined by presence of more than four mesangial cells per mesangial region affecting at least 80% of the glomeruli is a rarer cause of NS in children (16, 20). These patients can present with hematuria and hypertension unlike classic MCD children. IF shows mainly IgM and C3 in the mesangium, whereas EM shows paramesangial deposits besides the diffuse foot process effacement. This variant is usually associated with increased initial resistance to steroid therapy, but the overall remission rates are similar to MCD.

IgM nephropathy is another unusual variant of MCD characterized by at least 2+ staining intensity for IgM on IF (20). The distribution is mainly mesangial but could be membranous, but EM shows only a few small paramesangial deposits, suggesting that this deposition may be more of protein trapping rather than an immune complex disease. Initial steroid resistance is followed by remission (21).

### Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis is defined by the presence of segmental sclerotic lesions within glomeruli causing NS. It is the cause of NS in about 10–20% of cases in children. There is an association with low birth weight infants, especially for secondary FSGS (22). The causes are manifold and include

FIGURE 5 | Primary focal segmental glomerulosclerosis. (A,B) Photomicrographs showing two glomeruli with a segmental lesion characterized by obliteration of capillary loops with solidification and eosinophilia of the segment due to sclerosis (H&E 200×). (C,D) PAS stained sections showing a focal sclerosing lesion in a segment in (C) and in the perihilar region in (D) close to the vascular pole. Note again the preservation of capillary loops in other segments of the glomeruli (PAS 200×). (E) A trichrome stain showing the perihilar zone of sclerosis as evidenced by the blue staining of that segment. Note also some fibrin deposition in that area (red) (Trichrome 200×). (F) Immunofluorescence showing strong staining for albumin in the tubules, a characteristic feature of nephrotic syndrome in general. Not shown is the associated IgG deposition (DIF 100×). (G,H) Two electron photomicrographs showing collapsed loops in the (H) with effacement of foot processes in both images. Typically, the foot process effacement may be segmental and over the sclerosed segments (EM 3400×).

primary idiopathic FSGS, genetic, and familial cases included under secondary FSGS, and secondary FSGS due to systemic diseases, which are more common in adults but can be seen in older children too (**Table 3**).

### Primary (Idiopathic) FSGS

This is the most common pattern of FSGS in children with a slight male preponderance and increased incidence in the African-American population (23). It presents with NS in 90%

of cases but may present with renal insufficiency, hypertension, and hematuria in a proportion of cases. The histological variants described for FSGS include the classic or FSGS NOS variant, cellular variant, tip lesion, FSGS with mesangial hypercellularity, perihilar FSGS, and collapsing FSGS. Light microscopy for FSGS NOS is characterized by discrete segmental solidification of the glomerular tuft with a predilection for the juxtaglomerular region in the early stages of the disease. They commonly affect the vascular pole or the periphery of the tuft. The capillaries are occluded by acellular matrix material that produces intramembranous hyalinosis lesions highlighted as bright pink with a PAS stain and red with a trichrome stain, with progressive wrinkling of the membranes, development of adhesions and synechiae with the Bowman's capsule with a prominence of visceral epithelial cells over this tuft (**Figure 5**). While focal lesions are the rule in early cases, with progression, glomerular obsolescence results. Tubular atrophy and interstitial fibrosis are evident in the vicinity of the sclerosed glomerulus involving the same nephron unit. Tubules contain protein resorption droplets and lipid droplets (24–26). IF usually shows focal and segmental granular IgM and C3, both within the membranes of the sclerosed segment and mesangium. Albumin will be noted in tubules. EM shows wrinkling and retraction of the glomerular basement membrane with accumulation of hyaline. There is complete effacement of foot processes overlying the sclerotic region with podocyte hypertrophy and focal microvillus transformation. Effacement may be mild to severe in the adjacent non-sclerotic glomeruli but affects more than half the surface membrane.

FIGURE 7 | Collapsing glomerulopathy. (A,B) H&E images showing a low power and higher magnification appearance of a glomerulus with prominent epithelial cells and closed capillary loops better visualized on special stains (H&E 100× and 200×). (C) A PAS stain showing the collapsed cords of basement membranes with exuberant epithelial cell proliferation that seems to "choke" the capillary loops (PAS 400×). (D) A silver stain showing another glomerulus with wrinkling of the basement membranes and prominent epithelial cell proliferation (Jones 400×).

### Cellular FSGS

This variant is characterized on light microscopy by segmental hypercellularity resembling focal proliferative GN with areas of the tuft showing endocapillary proliferation with luminal obliteration of capillaries, increased mesangial cells, and prominent increase in inflammatory cells, including neutrophils, foam cells, and monocytes, with marked podocyte hyperplasia giving the appearance of crescents (pseudocrescents). IF shows some IgM and C3, whereas EM shows no significant deposits but prominent foot process effacement with intact basement membranes.

### Tip Lesion

This is characterized by proliferation of swollen podocytes and endocapillary proliferation with progressive obsolescence located at the tip of the tuft at the origin of the proximal tubule. As the lesion progresses, it results in a segmental scar in that area. There is associated tubular atrophy and interstitial fibrosis develops over time and may not be a feature in the early classic tip lesions. IF again shows IgM and C3, whereas EM resembles the cellular variant.

### Primary FSGS with Mesangial Hypercellularity

In this variant, the histology reveals classic segmental sclerosing lesions associated with podocyte hyperplasia but with the associated finding of mesangial hypercellularity in the non-sclerotic glomeruli. The IF shows a diffuse presence of IgM and C3 in the mesangium of non-sclerotic glomeruli and in the zone of sclerosis. The EM shows extensive foot process effacement with no electron dense deposits.

### Familial FSGS

Familial FSGS probably accounts for about 20% of cases of FSGS and can manifest at any age (27). The incidence increases to twothirds of cases discovered in the first year of life. Several newer mutations have now been described to be associated with FSGS pattern of injury including mutations in the *NPHS2* gene (podocin defect, chromosome 1q25-31), with an autosomal-recessive mode of inheritance, actinin 4 defect (*ACTN4*, autosomal dominant), TRPC6 (transient receptor potential cation channel, subfamily C, member 6), and CD2AP (7, 28, 29). Of these the podocin defect is the most common and can be detected by immunohistochemistry that shows loss of podocin staining. Other mutations and their known associations are shown in **Table 3**.

### Secondary FSGS

Segmental sclerosis can occur commonly in other diseases, not only in adults but also in children. The most common pediatric diseases that can have a component of associated NS in addition to a nephritic picture with histological evidence of FSGS include IgA nephropathy, Hereditary nephritis (Alport's syndrome), and lupus nephritis The histological picture and immunofluorescence usually reveal the underlying disease, for example, mesangial hypercellularity and mesangial IgA in IgA nephropathy, fullhouse pattern in lupus nephritis, and thin basement membrane

C3, IgG, and IgM (DIF 100×). (E,F) Electron micrographs showing open capillary loops with mesangial, paramesangial, and even some subendothelial deposits. Note diffuse foot process effacement (3400× and 7100×).

disease with basement membrane abnormalities on EM in Alport's syndrome (**Figure 6**).

### OTHER HISTOLOGICAL VARIANTS OF FSGS

### Collapsing Glomerulopathy

Collapsing variant of FSGS is characterized by progressive obliteration of capillary lumina due to wrinkling and shrinking of the basement membranes (23, 25, 30). It is relatively uncommon in children and was originally described in patients with HIV, but subsequently also seen in the absence of HIV. Other causes of collapsing glomerulopathy (CG) include infections, such as malaria and visceral leishmaniasis, drugs, such as interferon-alpha, bisphosphonates, and valproic acid, and autoimmune diseases, such as thrombotic microangiopathy and hematologic malignancies (31). They are as a group associated with progressive renal failure and have more severe disease at presentation. In children, rare forms associated with mitochondrial disorders have been reported. The COQ2 mutation is the most common and inherited as an autosomal-recessive condition, encoding for para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ10 synthesis pathway (32). The MYH9 gene, an encoding non-muscle myosin IIA heavy chain gene, has been implicated in HIV associated CG and genetic FSGS in patients of African descent (33). A renal biopsy shows characteristic CG with glomerular epithelial cells showing abnormal mitochondria. It is important to recognize

#### TABLE 4 | Pathological features of podocytopathies in children.

this variant, as it responds to ubiquinone replacement therapy. Histologically, CG is characterized by an implosive collapse of the capillary loops with wrinkling and contraction of the basement membrane with compensatory hypertrophy and hyperplasia of the podocytes, which tend to fill the Bowman's space resembling crescents (**Figure 7**). There is no endothelial proliferation in this condition and no hyaline droplets or lipid is noted. Prominent tubulointerstitial changes are a feature. Typically, no significant deposits are noted in the membrane besides some IgM and C3. EM shows wrinkling with little to no thickening of the basement membranes with marked hypertrophy of the overlying podocytes. There is marked foot process effacement involving even the glomeruli without the collapsing lesions. In general, patients with CG progress rapidly to renal failure and show resistance to steroid therapy.

### C1q Nephropathy

As mentioned before, the existence of C1q nephropathy as a distinct subgroup of NS has been debated. The original description of this entity was by Jennette and Hipp (34). Although originally described as a form of FSGS, the histological spectrum for C1q nephropathy (**Figure 8**) could range from MCD to mild mesangial proliferation to FSGS (35). The clinical manifestations include nephrotic range proteinuria with or without hematuria, with hypertension and renal insufficiency in a subset of patients (36, 37). Serology is usually negative, and serum C3 and C4 levels are normal. Most patients seem to present with steroid-resistant NS. The hallmark is presence of strong (2+ and above) staining for C1q on IF in a mesangial pattern (35, 38). IgG and/or IgM may also be present to some extent as can IgA. EM would show features of either MCD or FSGS with some mesangial electron dense deposits. The deposits may be predominantly seen in a "paramesangial" location. Foot process fusion will also be noted (34, 35). The important differential diagnosis to exclude for this condition is lupus nephritis and IgA nephropathy. These two conditions are distinguished by their distinct immunofluorescence pattern and in the case of lupus nephritis with finding of tubuloreticular inclusions on EM. A membranoproliferative pattern of histology would also not favor a diagnosis of C1q nephropathy.

The exact significance of this entity is still debated with its relationship to outcome being variable. They do not necessarily portend a worse outcome as previously believed with a median renal survival in C1q nephropathy/FSGS being about 81 months (35). They may be, however, associated with relapses, steroidresistant, and steroid-dependent NS (21).

In conclusion, the pathology of podocytopathies have some unique and some overlapping features and are frequently associated with specific genetic mutations. A summary of the pathological findings have been summarized in **Table 4** for the reader.

### AUTHOR CONTRIBUTIONS

The author has reviewed the literature and provided a succinct account of the genetics and pathology of nephrotic syndrome in children.

### REFERENCES

change nephrotic syndrome? *Pediatr Nephrol* (2014) **29**(1):67–74. doi:10.1007/ s00467-013-2551-3

**Conflict of Interest Statement:** The author declares 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 © 2016 Ranganathan. 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.*

## Endocytic Trafficking at the Mature Podocyte Slit Diaphragm

### *Agnieszka Swiatecka-Urban1,2\**

*1Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, PA, USA, 2Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA*

Endocytic trafficking couples cell signaling with the cytoskeletal dynamics by organizing a crosstalk between protein networks in different subcellular compartments. Proteins residing in the plasma membrane are internalized and transported as cargo in endocytic vesicles (i.e., endocytosis). Subsequently, cargo proteins can be delivered to lysosomes for degradation or recycled back to the plasma membrane. The slit diaphragm is a modified tight junction connecting foot processes of the glomerular epithelial cells, podocytes. Signaling at the slit diaphragm plays a critical role in the kidney while its dysfunction leads to glomerular protein loss (proteinuria), manifesting as nephrotic syndrome, a rare condition with an estimated incidence of 2–4 new cases per 100,000 each year. Relatively little is known about the role of endocytic trafficking in podocyte signaling and maintenance of the slit diaphragm integrity. This review will focus on the role of endocytic trafficking at the mature podocyte slit diaphragm.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Michal Malina, NH Hospital, Czechia Max Christoph Liebau, University Hospital of Cologne, Germany*

#### *\*Correspondence:*

*Agnieszka Swiatecka-Urban<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 03 October 2016 Accepted: 03 February 2017 Published: 24 February 2017*

#### *Citation:*

*Swiatecka-Urban A (2017) Endocytic Trafficking at the Mature Podocyte Slit Diaphragm. Front. Pediatr. 5:32. doi: 10.3389/fped.2017.00032*

Keywords: nephrotic syndrome, proteinuria, endocytic trafficking, endocytosis, podocytes, nephrin, podocin, glomerular slit diaphragm

## MECHANISMS OF ENDOCYTIC TRAFFICKING

All living cells process information by trafficking cargo from the plasma membrane in endocytic vesicles (i.e., endocytosis) and by returning much of the internalized membrane to the cell surface by a reciprocal process called recycling. The balance between endocytosis and recycling controls the plasma membrane composition and provides cells with information that has been resolved in time and space. Endocytic trafficking controls the supply of adaptor proteins and cargo, sorts the internalized cargo to specific intracellular compartments, and orchestrates the crosstalk in intracellular vesicles (1–3). As a result, cells turn over the equivalent of the entire plasma membrane one to five times per hour. Although endocytosis and recycling are ubiquitous, specific endocytic motifs and an assortment of protein adaptors guide cargo to diverse trafficking pathways. Defective endocytic trafficking has been associated with human disease, including congenital malformations, cancer, inflammation, and immunodeficiency (1). The mechanisms of endocytic entry of protein cargo, endocytic compartments, and mechanisms of cargo recycling were recently reviewed and will not be discussed here (4).

### ENDOCYTIC TRAFFICKING AT THE MATURE PODOCYTE SLIT DIAPHRAGM

The kidney glomerulus is a filtering apparatus that allows passage of water and solute into the urinary space while retaining the vast majority of plasma proteins within the circulation. The functional

unit of glomerular filtration is formed by the epithelial cells podocytes, glomerular basement membrane (GBM), and fenestrated capillary endothelial cells (5). The mature podocyte consists of the cell body, primary and secondary processes with microtubule and intermediate filament-based cytoskeleton, and the actinbased foot processes. Resting on the GBM, foot processes form interdigitating extensions linked by the slit diaphragm, which provides the only cell–cell contact between mature podocytes (6). Slit diaphragm is critical for (i) providing selective permeability between the blood and urinary space, (ii) separating the apical and basolateral plasma membrane, and (iii) serving as a signaling platform (4). Structurally, a network of proteins residing at the slit diaphragm membranes connects with the actin cytoskeleton through a juxtaposed cytoplasmic protein scaffold (7). Strong evidence demonstrates that the slit diaphragm is a dynamic unit (8, 9), suggesting that endocytic trafficking of membrane proteins plays an integral role in regulating the dynamics (**Figure 1**). Recent data provided genetic, functional, and high-resolution ultra-structural evidence highlighting a model of dynamic and multilayered architecture of the slit diaphragm (10). According to the model, the mammalian intercellular slit has a specific

FIGURE 1 | Schematic of the functional Neph1–nephrin protein complex at the mature mammalian slit diaphragm. Homophilic interactions of the integral membrane proteins nephrin and Neph1 form the mature mammalian slit diaphragm with a ratio of 1:2.5, respectively (10). Nephrin homophilic interactions are located more apically while the Neph1 strands localize more basally at the intercellular slit (10). The number of Ig repeats in the extracellular domain (9 and 5 for nephrin and Neph1, respectively) determines, at least in part, the width of the intercellular slit (10). The PHB domain-containing protein podocin anchors nephrin and Neph1 at the cell membrane. The intracellular domain of nephrin and Neph1 interacts with the cortical actin cytoskeleton *via* the juxtaposed cytoplasmic protein networks of junctional scaffolding proteins, channels, adaptor proteins, including endocytic adaptors, protein kinases, and motor proteins. Fyn-mediated phosphorylation of nephrin and Neph1 is essential for the podocyte architecture and signaling. Growing evidence supports a model where endocytic trafficking of the integral membrane proteins dynamically controls podocyte architecture and signaling. While the endocytic itineraries of nephrin are better understood, very little is known about the Neph1 endocytic mechanisms. Published data demonstrate that the motor protein Myo1c recruits Neph1 to the podocyte membrane and controls the cell membrane turnover of Neph1 in an actin-dependent manner (20, 21).

arrangement of the integral membrane proteins nephrin and Neph1 at a ratio of 1:2.5. The homophilic interactions between extracellular domains of nephrin are formed more apically, while the homophilic interactions of Neph1 localize closer to the basement membrane at the intercellular slit (10). Difference in the length of extracellular domain of nephrin and Neph1 results in a different length of the intercellular strands formed by the homophilic interactions that subsequently determine the width of the slit (10). Nephrin is an immunoglobulin-type cell adhesion molecule critical to the slit diaphragm function (11, 12). Mutations in the *Nphs1* gene encoding nephrin protein lead to congenital nephrotic syndrome characterized by absence or profound impairment of the slit diaphragm and manifesting as severe proteinuria or nephrotic syndrome (13, 14).  Data reviewed below demonstrate current understanding of the protein–protein interactions that regulate nephrin endocytic trafficking. The role of Neph1 is discussed in a separate section.

Nephrin localization at the slit diaphragm is determined by podocin, a lipid raft-resident protein encoded by the *Nphs2* gene (22, 23). The prohibitin homology (PHB) domain—a lipid recognition motif present in podocin may target the podocin– nephrin complex to the lipid raft domains of slit diaphragm membranes (24, 25). Certainly, the *Nphs2* gene mutations that mislocalize podocin also mislocalize nephrin and lead to steroid resistant, hereditary, and sporadic nephrotic syndrome (22, 26). The podocin C-terminal T339VV motif regulates the cell surface localization as well as the lipid raft-independent podocin stability (27). While tagged podocin resides specifically in the late endosomal/lysosomal compartment (27), mislocalized endogenous podocin co-localizes with an early endosomal marker Rab5 in the puromycin nephrosis rat model (28). Despite the above data, a large gap in understanding of the podocin endocytic itineraries still exists. Flotillins are members of the PHB domain-containing protein family. PHB domains contain hairpin-forming, hydrophobic regions that help to insert a protein into the inner leaflet of plasma membrane. The PHB domain and a region upstream of the N-terminus, modified posttranslationally by palmitoylation and myristoylation, mediates binding of flotillins to the inner leaflet of plasma membranes (24). The predicted topology of mouse podocin has the intracellular N- and C-terminal regions, a transmembrane domain, and the PHB domain, which unlike the PHB of flotillins, is not involved in hairpin formation (22). Flotillins form homo- and heterophillic interactions mediated *via* the C-terminal region conserved only within the flotillin family while in podocin both the N- and C-terminal domains are predicted to mediate homooligomerization (22, 24). As of now, nothing is known about the molecular organization of the podocin hairpin domain or whether podocin undergoes palmitoylation and/or myristoylation. The PHB domain of flotillins plays an essential role in endocytic trafficking of several receptors, including those activated by the Src family kinase Fyn (29). Nephrin is phosphorylated by Fyn, and the phosphorylation augments nephrin interaction with podocin and facilitates nephrin endocytosis *via* a clathrin-independent pathway, activating nephrin signaling (15, 30). By contrast, dephosphorylation of the nephrin-Y1193 increases nephrin endocytosis mediated by β-arrestin, decreases nephrin signaling, and impairs the slit diaphragm integrity (31). These data suggest that different endocytic pathways may play opposite roles in modulating nephrin function and that the endocytic itineraries of nephrin are regulated by the phosphorylation/ dephosporylation cycle (see below).

PKC-α, stimulated by the high glucose concentration, induces nephrin endocytosis and leads to proteinuria while depletion of PKC-α stabilizes nephrin in diabetic nephropathy (32, 33). PKC-α phosphorylates two residues in the intracellular domain of nephrin, T1120 and T1125, and facilitates nephrin interaction with β-arrestin in murine podocytes (16). These data suggest that PKC-α promotes β-arrestin-mediated nephrin endocytosis, most likely *via* the clathrin-dependent pathway. Following clathrin-dependent endocytosis, receptors are transported either to the fast or slow recycling route, depending on the stability of interaction between the receptor and β-arrestin. In addition to regulating endocytosis, β-arrestin controls post-endocytic itineraries of several receptors. Nothing is currently known about post-endocytic sorting of nephrin. Understanding the structural basis and kinetics of binding between nephrin and β-arrestin may help to elucidate the fate of internalized nephrin, whether it undergoes lysosomal degradation or recycling to the plasma membrane or both.

CD2AP may control nephrin endocytic trafficking by regulating actin assembly and vesicle sorting. The importance of CD2AP in podocytes is demonstrated by glomerulosclerosis and foot process effacement, leading to renal failure in mice lacking CD2AP (34). CD2AP localizes in the slit diaphragm cytoplasmic region juxtaposed to the lipid raft membranes and co-localizes with the actin-related protein 2 (ARP2) and ARP3 (Arp2/3) and cortactin. CD2AP regulates endosomal sorting and vesicle trafficking by regulating actin assembly (35). Moreover, CD2AP associates with the dynamic actin pool and co-localizes with the endocytic adaptors Rab5, Rab4, the clathrin-dependent endocytic adaptor assembly polypeptide-2 complex (AP-2), and participates in formation of multivesicular bodies (35, 36). CD2AP interacts with podocin and nephrin, anchoring the proteins to the actin cytoskeleton (37, 38). CD2AP and nephrin interact with the p85 regulatory subunit of the phosphoinositide 3-OH kinase (PI3K), leading to its recruitment to the plasma membrane (39). PI3K plays an essential role in endocytic trafficking, including regulation of membrane lipid composition, release of clathrin-coated vesicles, and the Rab5 recruitment (40). The interactions of PI3K with nephrin, CD2AP, and other slit diaphragm proteins suggest that PI3K plays an important role in endocytic trafficking at the slit diaphragm (41).

CIN85/RukL is a closely related homolog of CD2AP (42). In podocytes, CD2AP blocks CIN85/RukL expression by sumoylation while CD2AP depletion increases CIN85/RukL abundance and leads to CIN85/RukL-mediated nephrin ubiquitination and endocytosis (43, 44). CIN85/RukL depletion has the opposite effect and preserves nephrin expression at the slit diaphragm membranes and reduces proteinuria in diabetic mice (45). Studies showed that unlike CD2AP, CIN85 does not contain an actin-binding domain (43, 46). Absence of CD2AP may facilitate nephrin interaction with CIN85, impair nephrin partitioning to lipid rafts, and induce nephrin endocytosis. However, another study has shown that CIN85 interacts directly with actin and together with CD2AP bundles actin filaments and modulates podocyte migration (47). Moreover, CIN85 clusters Src family kinases, providing a scaffold for Arp2/3-mediated actin assembly, and regulates cell polarization and motility. Expression of multiple CIN85 splice variants in different cell types may explain the reported difference of CIN85 function.

Notch activation alters foot process architecture in mature podocytes and induces proteinuria by stimulating the dynamindependent and lipid raft-independent nephrin endocytosis (17). The Wnt/planar cell polarity (PCP) pathway increases clathrin/βarrestin-dependent nephrin endocytosis, and depletion of the PCP protein Vngl2 increases nephrin abundance at the cell surface and disturbs glomerular maturation (19).

The following protein networks have been shown to play a role in endocytic trafficking in podocytes, either directly or by regulating actin dynamics but their role in nephrin trafficking has not been demonstrated. α-Actinin-4 provides structural stability at the slit diaphragm by cross-linking and connecting actin filaments with nephrin and membrane associated guanylate kinase inverted (MAGI-1) (7, 48). The α*-actinin-4* gene mutations are associated with nephrotic syndrome called focal segmental glomerulosclerosis (49). Direct interactions between members of the multi-protein scaffold organized by α-actinin-4, MAGI-1, and the endocytic adaptor megalin indicate a role of the scaffold in endocytic trafficking at the slit diaphragm (50, 51). As a member of the cytoskeleton-associated recycling or transport (CART): an Hrs/actinin-4/BERP/myosin V, α-actinin-4 mediates constitutive recycling of transmembrane receptors *via* the rapid recycling route on actin filaments (52). The role of α-actinin-4 or other members of the CART complex in cargo recycling at the slit diaphragm remains unknown. Endocytic adaptors, synaptojamin 1, and endophilin 1–3 are critical for the foot process formation, while dynamin I and II play a role in the foot process maintenance (18).

### STRUCTURAL BASIS FOR NEPHRIN ENDOCYTIC TRAFFICKING

A protein frequently utilizes different endocytic itineraries to diversify its function (1). Clathrin-dependent endocytosis is one of the most important internalization routes in eukaryotic cells (1). Endocytic adaptors recognize linear internalization signals located in the intracellular C-terminal tail domains of transmemembrane proteins and recruit these proteins as cargo to clathrin-coated pits, which are plasma membrane deformities coated with clathrin and clathrin-dependent endocytic adaptors. Subsequently, additional adaptors cleave off clathrin-coated pits and release them into the cell interior as clathrin-coated vesicles [reviewed in Ref (4)]. Clathrin-independent endocytosis includes a diverse group of internalization mechanisms sharing a requirement for free cholesterol, proteins, and lipids that reside in sphingolipid-rich lipid raft membranes (53).

Little is known about linear endocytic motifs in nephrin or other integral membrane proteins at the slit diaphragm. For example, the nephrin-Y1193 and subsequent amino acid residues D–E–V conform to a canonical, tyrosine-based endocytic signal of the YxxØ type, which is essential for clathrin-mediated endocytosis. Phosphorylation of the tyrosine residue inhibits the interaction of the YxxØ motif with the μ2 subunit of AP-2 and prevents endocytosis. By contrast, dephosphorylation of the tyrosine residue allows the YxxØ interaction with AP-2 and facilitates endocytosis. Thus, the phosphorylation state of the tyrosine residue serves as a regulatory switch controlling protein retention at the plasma membrane or its endocytosis *via* the clathrin-dependent pathway (54). Consistent with this model, Quack et al. demonstrated that β-arrestin mediates clathrin-dependent endocytosis of nephrin dephosphorylated at the conserved Y1193 residue (31). By contrast, phosphorylation of nephrin-Y1193 by Fyn augments nephrin interaction with podocin, prevents nephrin interaction with β-arrestin, attenuates β-arrestin-mediated nephrin endocytosis, and augments nephrin signaling (31). Moreover, the Fynmediated phosphorylation facilitates nephrin endocytosis *via* the clathrin-independent pathway (15, 30). These data demonstrate that the phosphorylation state of nephrin-Y1193 regulates nephrin signaling by directing nephrin to different endocytic pathways, either the clathrin-independent endocytosis to augment signaling or the clathrin-dependent endocytic to attenuate nephrin signaling. Although β-arrestin is known to interact predominantly with the clathrin-dependent adaptors, it was also found to modulate the clathrin-independent pathway by interacting with Arf6, and it may also utilize ubiquitination to inactivate receptor signaling (55, 56). At this time, the role of β-arrestin in nephrin ubuquitination or clathrin-independent internalization is unknown. Presence of several sequences conforming to the tyrosine-based endocytic motifs in nephrin cytoplasmic C-terminal domain suggests that regulation of nephrin endocytic itineraries is even more complex. The complexity is further increased by additional protein–protein interactions between nephrin, Fyn, and the actin cytoskeleton. Nephrin tyrosine phosphorylation is critical for recruitment of actin adaptors, such as p85/PI3K, Cas/Crk, and Nck, facilitating cytoskeletal dynamics in the podocyte foot processes (39, 41, 57–60). Nck facilitates Fyn-mediated nephrin phosphorylation, while Nck depletion leads to decreased nephrin tyrosine phosphorylation and foot process effacement (61, 62). Three conserved, Nck-binding Y–D–x–V motifs in the C-terminal nephrin tail, Y1176DEV, Y1193DEV, and Y1217DQV mediate these effects (63). Replacing the Y1176, Y1193, and Y1217 residues with the non-phosphorylated tyrosine mimic phenylalanine leads to proteinuria associated with foot process effacement, irregular thickening of the GBM, and dilated capillary loops in a mouse model (63).

### THE ESSENTIAL ROLE OF NEPH1 AT THE MAMMALIAN SLIT DIAPHRAGM

Neph1, a transmembrane protein partially homologous to nephrin is another key component of the mammalian slit diaphragm (10, 64). Nephrotic syndrome resulting from mutations in the *neph1* gene in humans as well as severe proteinuria, foot process effacement, and early postnatal death in *neph1* KO-mice demonstrate its importance for the glomerular function (64). Although the extracellular domain of Neph1 can interact with nephrin, recent data show that such heterophilic interactions are rare while the hemophilic Neph1 and nephrin interactions are critical for the mammalian slit diaphragm architecture (10, 65). Similar to nephrin, the Neph1 cytoplasmic domain engages directly with podocin, anchoring the integral membrane proteins at the cell membrane and the scaffolding protein ZO-1 that connects with the cortical actin cytoskeleton (66, 67) (**Figure 1**). Upon signaling engagement, Neph1 and nephrin form a phosphorylation-dependent unit transmitting outside-in signaling through a multi-protein scaffold of junctional proteins to the podocyte actin network (68, 69). While the Fyn-mediated tyrosine phosphorylation of the Neph1 intracellular domain leads to recruitment of protein adaptor Grb2 necessary for the Neph1-mediated actin polymerization, tyrosine phosphorylation of nephrin intracellular domain leads to recruitment of Nck, necessary for nephrin-mediated actin polymerization (58, 69–71). Activation of the Neph1–nephrin functional complex occurs during junction formation and during podocyte injury, both requiring intense signaling and actin cytoskeleton reorganization (69). One study demonstrated that inhibiting Neph1 signaling in podocyte culture preserves podocyte architecture and function in the puromycin aminoglycoside (PAN) injury model, while maintaining high Neph1 levels protects podocytes from injury in *in vivo* zebrafish PAN and adriamycin injury models (72). Another group demonstrated that renal ischemia induces a rapid loss of Neph1 interaction with ZO-1 in an *in vivo* rat ischemic model (73). Induction of injury by ATP depletion in podocyte culture resulted in rapid loss of Neph1 and ZO-1 interaction and redistribution of both proteins from the cell membrane to the cytoplasm while recovery from the injury resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 membrane localization and interaction (73). The structural basis for the Fyn-mediated Neph1–ZO-1 interaction essential for the podocyte function has been recently elucidated. The ZO-1 PDZ1 domain interacts directly with the PDZ-binding region in the C-terminus of Neph1 where the T787HV and L761T residues are critical for protein–protein binding and its stability (67, 74). The functional Neph1–nephrin unit also engages with the Par3– Par6–atypical protein kinase C (aPKC) complex to maintain a three-dimensional foot process architecture and integrity of the slit diaphragm (75). Neph1 may influence localization of other proteins in the foot process. For example, Neph1 interaction with the large-conductance Ca2+-activated (BKCa) channel affects membrane localization of the channel (76). Data reviewed above attest to a dynamic role that Neph1 plays in the slit diaphragm remodeling. Yet, little is known about Neph1 trafficking itineraries. Emerging data demonstrate that Neph1 is recruited to the podocyte membrane by a motor protein Myo1c that interacts with Neph1 intracellular region directly *via* cargo-binding, C-terminal domain in an actin-dependent manner (20, 21). Moreover, Myo1c may control the cell membrane turnover of Neph1 (21). Myo1c also binds nephrin directly. However, by contrast to the effect on Neph1, Myo1c reduces nephrin localization at the podocyte membrane (20). It remain unknown how Myo1c exerts opposite effects on the membrane localization of Neph1 and nephrin.

### SUMMARY

Recent data confirm that endocytic trafficking allows rapid regulation of the signaling strength and duration at the slit diaphragm. Identifying endocytic motifs of the integral membrane proteins and examining the structural basis of the protein–protein interactions that control endocytic trafficking may unravel novel mechanisms and enrich our understanding of the role of this essential biological function at the podocyte slit diaphragm, as

### REFERENCES

well as teach about the mechanisms of nephrotic syndrome and measures to correct it.

### AUTHOR CONTRIBUTIONS

AS-U reviewed the literature and wrote the manuscript.

### FUNDING

This study was supported by the American Heart Association grant 15GRNT25700019 and the National Institutes of Health grant R56HL127202 (to AS-U).

neurons and podocytes. *Am J Physiol Cell Physiol* (2009) 297:C1379–88. doi:10.1152/ajpcell.00354.2009

**Conflict of Interest Statement:** The author declares 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 Swiatecka-Urban. 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.*

# Pathophysiology, evaluation, and Management of edema in Childhood Nephrotic Syndrome

### *Demetrius Ellis\**

*Division of Pediatric Nephrology, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA*

Generalized edema is a major presenting clinical feature of children with nephrotic syndrome (NS) exemplified by such primary conditions as minimal change disease (MCD). In these children with classical NS and marked proteinuria and hypoalbuminemia, the ensuing tendency to hypovolemia triggers compensatory physiological mechanisms, which enhance renal sodium (Na+) and water retention; this is known as the "underfill hypothesis." Edema can also occur in secondary forms of NS and several other glomerulonephritides, in which the degree of proteinuria and hypoalbuminemia, are variable. In contrast to MCD, in these latter conditions, the predominant mechanism of edema formation is "primary" or "pathophysiological," Na+ and water retention; this is known as the "overfill hypothesis." A major clinical challenge in children with these disorders is to distinguish the predominant mechanism of edema formation, identify other potential contributing factors, and prevent the deleterious effects of diuretic regimens in those with unsuspected reduced effective circulatory volume (i.e., underfill). This article reviews the Starling forces that become altered in NS so as to tip the balance of fluid movement in favor of edema formation. An understanding of these pathomechanisms then serves to formulate a more rational approach to prevention, evaluation, and management of such edema.

#### *Edited by:*

*Robert P. Woroniecki, State University of New York, USA*

#### *Reviewed by:*

*Dagmara Borzych-Duzalka, Medical University of Gdansk, Poland Roberto Gordillo, University of Illinois at Chicago, USA*

> *\*Correspondence: Demetrius Ellis<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 07 October 2015 Accepted: 07 December 2015 Published: 11 January 2016*

#### *Citation:*

*Ellis D (2016) Pathophysiology, Evaluation, and Management of Edema in Childhood Nephrotic Syndrome. Front. Pediatr. 3:111. doi: 10.3389/fped.2015.00111*

Keywords: edema, nephrotic syndrome, children/pediatrics, pathophysiology, management

### INTRODUCTION

Edema is an essential clinical feature of the diagnosis of nephrotic syndrome (NS) of various etiologies. It is defined as a palpable swelling resulting from an accumulation of fluid in the interstitial fluid compartment. Massive generalized edema (anasarca) is common, especially in children with primary minimal change disease (MCD) and serves as the main clinical justification for hospital admission for "diuretic management." In such children, the selective loss of large amounts of albumin in the urine leads to hypoalbuminemia and decreased plasma oncotic pressure favoring fluid sequestration in the interstitial fluid compartment, and secondarily triggers renal Na+ and fluid retention so as to preserve intravascular volume and blood pressure, hence preventing an "underfill" state. In contrast to MCD, in NS associated with glomerulonephritis the magnitude of the proteinuria is variable and reduction in GFR is common. Hence, in disorders, such as focal and segmental glomerulosclerosis, acute post-streptococcal glomerulonephritis, Henoch Schoenlein nephritis, lupus nephritis, and several other glomerulonephritides, intravascular fluid volume is typically normal or expanded because of inappropriately stimulated Na+ and fluid retention which together with decreased GFR results in an "overfill" state. The clinical distinction of these two predominant physiological states is critical to the proper management of edema in children with NS. However, the role of hypoalbuminemia has been questioned, and it is apparent that other mechanisms such as water retention and vascular permeability factors, and activation of channels that promote Na+ reabsorption, may also play an important role in edema formation in diverse causes of NS. Thus, multiple mechanisms acting simultaneously may explain the clinical variability in response to measures aimed at removing excessive fluid among individual children with NS. This has led to inconsistent or improper clinical evaluation and management of this relatively common disorder.

While the precise mechanism(s) of edema formation may be controversial and turn-around time of several laboratory tests used to confirm the underlying mechanism(s) may not be sufficiently rapid so as to influence acute management decisions, in most cases, there is adequate information to aid the clinical differentiation of the underlying basic mechanism of edema. This review discusses the pathophysiological alterations that favor edema formation in NS. This background then serves as the basis for developing selection criteria for hospital admission of children with NS who may benefit from edema fluid removal, establishing more standard guidelines for the clinical evaluation of nephrotic edema, and delineation of more uniform management guidelines aimed at providing effective management of the edema. Ultimately, the goal of therapy is to minimize the risk for hypovolemia, acute kidney injury (AKI), and other potentially serious complications ensuing from inappropriate diuretic regimens.

### PATHOPHYSIOLOGY OF EDEMA FORMATION IN NS

Compared to adolescents and adults, neonates and younger children have a greater proportion of total body and interstitial (IS) fluid volume, which can double or triple because of edema related to NS (1). Nephrotic edema is a transudate with low protein concentration (<3 g/dL) and a few or no cells. In contrast to other forms of edema, it is pitting, tends to be generalized and is more prominent in dependent body areas in which capillary hydraulic pressure (PCAP) is high (ankles and feet), or in tissues with low resistance or low interstitial hydraulic pressure (PIF) (eyelids, gastrointestinal tract/abdomen, and scrotum).

It is apparent that while total body water remains constant during health, the fluid within each compartment is not static; there is continuous movement between compartments. The process of *diffusion* across cell membranes by far accounts for most fluid turnover (about 80,000 L/day in a 70 kg adult). By contrast, nephrotic edema represents net movement of water from the intravascular (IV) into the IS fluid compartment through the process of *filtration* across the capillary wall. Such filtration is dependent on the balance or net gradient, between the hydrostatic pressure and the oncotic pressure gradients across the capillary, as initially described by Starling in 1896 (2–4). Starling's equation or "Law" was later modified as it became apparent that the net filtration is also determined by **Lp**, **S**, and **s**, as shown below:

$$\begin{aligned} \text{Net filtration} &= \text{LpS} \times \text{(\Delta hydrostatic pressure} - \Delta \text{ onactive pressure)}\\ &= \mathbf{LpS} \times \text{[(Pcap -- Pf') - s(\pi \text{cap} - \pi \text{if)})]} \end{aligned}$$

**Lp** is the unit permeability (known as porosity or "hydraulic conductivity") of the capillary wall, which in the kidney refers to the glomerular capillary; **S** is the surface area available for fluid movement; **Pcap** and **Pif** are the capillary and interstitial fluid hydraulic pressures; **πcap** and **πif** are the capillary and interstitial fluid oncotic pressures; and **s** represents the reflection coefficient of proteins across capillary walls which ranges from 0 for vessels completely permeable to proteins and approaches a value of 1.0 in protein-free ultrafiltrate. The interstitial fluid oncotic pressure is derived primarily from filtered plasma proteins and to a lesser degree from proteoglycans in the interstitium.

Because of unique anatomy, physiological properties, and Starling forces of glomerular capillaries, the kidney plays a central role in total body fluid and electrolyte homeostasis as depicted elsewhere (5).  Because normal glomerular capillaries are essentially impermeable to protein (so oncotic pressure in the filtrate is zero), this is a true ultrafiltrate with a high reflection coefficient (**s** is about 1.0). Several forces are altered in NS so as to favor net fluid filtration both in glomerular and in peripheral capillaries. These include reduction in **πcap**, increased **Lp**, elevation in **Pcap** depending on the underlying etiology of NS, increased **πif** and lymphatic vessel obstruction which may interfere with removal of filtered fluid thereby enhancing edema formation. Children with MCD tend to have more marked proteinuria and lower πcap than adults, while those with acute post-streptococcal glomerulonephritis tend to have Na+ retention and rise in Pcap, favoring edema formation.

While mathematically and conceptually sound in providing a theoretical framework, direct measurement of Starling forces is complex and particularly challenging to assess in NS in which there are multiple alterations acting in concert to alter all components of Starling's equation to different degrees in any given individual. For example, **Lp** can differ based on histopathological cause of NS, type and extent of elaboration of permeability factors that affect pore size and protein permeability, secretion of neuroendocrine hormones involved in preserving **Pcap**, and the concentration of plasma and interstitial albumin and other proteins that influence **πcap** and **πif**. Thus, in clinical practice, a glimpse of the basic operative mechanisms is often inferred by clinical assessment of hypovolemic symptoms or signs and by measurement of a limited number of blood and urinary conformational biochemical studies. Consequently, from a practical standpoint, two major mechanisms of edema formation are prevalent. First, in relation to edema associated with MCD or other non-inflammatory conditions resulting in massive proteinuria, an increase in transcapillary oncotic pressure gradient is the single most important driver of edema formation. This is because albumin which contributes 6 mmHg of oncotic pressure per g/ dL, or, 24 out of 26 mmHg of normal plasma oncotic pressure, is markedly reduced in such disorders. According to the *underfill hypothesis* reduction in plasma oncotic pressure promotes net movement of fluid out of the intravascular compartment, leading to volume depletion, or underfilling (6–12). This then causes appropriate or compensatory physiological activation of several mechanisms ultimately resulting in secondary Na+ and fluid retention aimed at restoring intravascular volume and blood pressure. This is the most prevalent mechanism in edematous children presenting with NS. This is in contrast to the second mechanism of nephrotic edema, or *overfill hypothesis*, exemplified by acute post-streptococcal glomerulonephritis and other inflammatory proteinuric disorders in which the pathophysiological process activates several mediators sub-serving primary Na+ retention, resulting in intravascular volume expansion, increased capillary hydraulic pressure and edema. A schema depicting the two major mechanisms of nephrotic edema is presented in **Figure 1**.

### CONTROVERSY OF MECHANISMS OF NEPHROTIC EDEMA

### Primary vs. Secondary Na**+** Retention

While previous schemes of nephrotic edema depicted Na+ retention through distinct secondary (in underfill) and primary (in overfill) mechanisms, there is now compelling evidence that primary Na+ retention may occur by a mechanism common to all individuals with "nephrotic urine," independent of underlying histology or blood volume status [see below, Ref. (13)]. Several studies implicate loss of plasmin and other serine proteases in the urine in the up-regulation of the epithelial sodium channel (ENaC) causing Na+ and fluid retention (see below). This is also highlighted in **Figure 1**.

### Blood Volume Status

Based on clinical observations and theoretical grounds, the long held prevailing opinion has been that children and adults presenting with NS edema, are in a state of intravascular volume depletion. Although a few studies support the presence of hypovolemia in adults with MCD compared to other histological forms of NS (14), and in some but not all children with MCD (15, 16), other investigations indicate that most adults and some children with NS of any cause have an adequate or expanded effective blood volume and do not exhibit orthostatic hypotension or other hypovolemic symptoms and signs (16–18).

To gain greater insight into the pathophysiology of edema in children with NS, Vande Walle et al. (19) studied children in early relapse of NS and found that they grouped into three presentations: (a) incipient proteinuria without edema, Na+ retention, and slightly elevated circulating aldosterone, increased renal plasma

FIGURE 1 | Pathophysiology of edema formation in NS. In disorders with massive proteinuria and marked hypoalbuminemia but minimal or absent renal inflammatory infiltrate, as in most children with minimal-change disease (MCD), the reduction in capillary colloid oncotic pressure (πcap) favors net fluid exit from the vascular to the interstitial fluid compartment thereby reducing effective circulatory blood volume, denoted as "underfill." This then triggers secondary, or compensatory, Na+ retention and hemodynamic alterations aimed at achieving blood pressure homeostasis. By contrast, various glomerulonephritides and inflammatory renal disorders, such as acute post-streptococcal glomerulonephritis, may be associated with variable degrees of proteinuria and with pathologic release of mediators, which promote primary renal Na+ and water retention, as well as vasoconstrictive hormones that are released despite an intact or even expanded intravascular volume. These factors together with reduction in glomerular ultrafiltration coefficient (Kf, or LpS in the Starling equation), lead to reduced glomerular filtration rate (GFR) and combine to further limit Na+ excretion, resulting in an "overfill" state and rise in capillary hydraulic pressure (Pcap). In turn, this causes net fluid accumulation in the interstitial fluid compartment. Note that nephrotic urine may be a common pathway for primary Na+ retention in both undefill and overfill disorders (refer to text and Tables 1–3 for the mediators sub-serving each of these main mechanisms of edema formation).

flow, and normal norepinephrine (NE) levels; (b) symptoms of hypovolemia, edema, Na+ retention, elevated plasma renin, aldosterone and NE levels, low atrial natriuretic peptide, and reduced GFR; and (c) edema, no hypovolemia, no active Na+ retention, normal plasma hormones, and normal GFR.

These data are very important because they indicate that there is both clinical and biochemical heterogeneity in children with NS. Distinguishing these fundamental mechanisms aids, the clinical prediction of safety and benefit of diuretic therapy: diuretics are well tolerated in children with primary renal Na+ retention but, if underfilling is the predominant mechanism, diuretic use can exacerbate hypovolemia and tissue hypoperfusion.

### Role of Hypoalbuminemia in Nephrotic Edema

**Figure 1** highlights the role of hypoalbuminemia in children with nephrotic edema. However, with the exception of marked hypoalbuminemia (<2.0 g/dL) and plasma oncotic pressures below 8–10 mmHg, several clinical and experimental observations call into question the central role of hypoalbuminemia in the pathogenesis of nephrotic edema (8). For example, studies in analbuminemic rats show no significant change in transcapillary oncotic pressure gradient or tendency to edema compared to controls (20). Also, 81% of individuals with congenital analbuminemia (dysfunction of the albumin gene resulting in marked hypoalbuminemia) have little or no edema (21). It appears that in this disorder, **πcap** is partially compensated over time by other plasma proteins and may explain the parallel decrease in **πif**.

Other observations in experimental animals as well as in humans undergoing repeated plasmapheresis to intentionally produce modest reductions in serum albumin concentrations show that the transcapillary oncotic gradient is preserved; only with greater degree or more rapid achievement of hypoproteinemia does blood volume fall and edema develops (22, 23). This is because as plasma oncotic pressure falls, a parallel reduction in tissue oncotic pressure also occurs. Moreover, administration of albumin concentrates does not appreciably mobilize edema fluid in many nephrotic subjects, although Na+ excretion can increase transiently in some (24–26). Finally, patients with MCD who are treated with corticosteroids often undergo a diuresis and natriuresis well before the serum albumin concentration starts to rise; this finding suggests that correction of the hypoalbuminemia might not be essential in steroid-induced natriuresis (27). However, several of the experimental conditions investigating the role of albumin *per se* in the pathomechanism of nephrotic edema may not be appropriate models for this condition in humans in whom vascular permeability factors and cytokines may, in fact, play an important role in preventing the counterbalance of oncotic pressure gradient evident in congenital analbuminemia or in experimental plasmapheresis.

Despite the controversy surrounding the "central role" of hypoalbuminemia in the development of edema in NS, nearly all children with NS and edema have hypoalbuminemia and in the author's opinion this controversy has little baring on the clinical evaluation and management of edema.

## Factors That Protect Against Edema Formation in NS

Because normally there is a small net pressure gradient favoring net filtration across capillaries, it might be expected that only a minor change in these hemodynamic forces would lead to edema. However, experimental and clinical observations indicate that there must be at least a 15 mmHg increase in the net pressure gradient favoring filtration before edema can be detected. With lesser reduction of this gradient, edema is unlikely to occur because of three compensatory factors (27, 28). First, experimental evidence indicates that there is increased lymphatic flow which, by bulk flow, will remove albumin as well, and help remove some of the excess filtrate (29, 30). Second, fluid entry into the interstitium will eventually raise the interstitial hydraulic pressure, thereby oppose filtration and interstitial fluid accumulation (28). Third, fluid accumulation in the interstitium simultaneously reduces interstitial oncotic pressure in subcutaneous tissue which in humans it is normally 12–15 mmHg (7). Thus, a gradual fall in plasma oncotic pressure in NS is associated with a parallel decline in interstitial oncotic pressure and rise in interstitial hydraulic pressure (7, 27), which minimizes the change in the transcapillary pressure gradients favoring net fluid movement out of the vascular space and results in relative preservation of plasma volume.

As a result of these compensatory physiological responses, there is usually little change in the transcapillary oncotic pressure gradient in children with NS, and therefore little tendency to plasma volume depletion, unless the hypoalbuminemia is severe. Similarly, as long as children with NS are not overdiuresed, plasma volume is typically preserved during diuretic therapy for edema removal.

### VOLUME REGULATORY HORMONES SUB-SERVING UNDERFILL AND OVERFILL MECHANISMS OF EDEMA FORMATION IN NS

In children who are hypovolemic or "underfilled," there is interplay of several volume regulatory hormones and nephron channels listed in **Table 1**, which tend to attenuate the effect of vascular underfilling mainly through modulation of renal Na+ and fluid retention. Activation of the RAAS is an important mechanism in the majority of children presenting with nephrotic edema. It is notable that several of the hormones shown in **Table 1**, including AT II and anti-diuretic hormone (ADH), have dual Na+ or water retaining, and vasoconstriction properties, and are therefore very suitable in preserving systemic blood pressure in children with NS and reduced circulatory volume. Similarly, NE release in response to stimulation of low pressure cardiopulmonary receptors mediates neuronal control of renal tubular Na+ reabsorption (31). Edema may thus be viewed as a byproduct of these adaptive processes.

In addition, there may be several relatively unique pathological perturbations that promote edema formation in NS. Thus, children with acute post-streptococcal glomerulonephritis manifesting edema and hypertension often have increased

#### TABLE 1 | Contribution of volume and blood pressure regulatory hormones and channels which mediate or participate in renal Na+ and edema formation in NS.

*RAAS, renin angiotensin aldosterone system; AT II, angiotensin II; Na*+*/K*+ *ATPase, sodium potassium adenosine tri-phosphatase; DCT, distal convoluted tubule; CD, collecting duct; PCT, proximal convoluted tubule; CCT, cortical collecting tubule.*

circulating ANP and reduced ADH, plasma renin activity, aldosterone, and NE concentrations. Experimental models of unilateral NS or glomerulonephritis show increased Na+ reabsorption in the collecting tubules (26, 32), which is also the site of action of ANP and the related renal hormone urodilatin. Urodilatin is an ANP analog or isoform secreted by distal convoluted tubule (DCT) and collecting duct (CD) epithelium and exerts a paracrine function similar to ANP in promoting natriuresis and diuresis. In both experimental and human NS, a state of relative resistance to ANP and urodilatin has been observed (6, 33–35). This defect is due at least in part to urinary plasmin loss in individuals with NS which then stimulates phosphodiesterase activity, leading to more rapid degradation of the second messenger of ANP, cyclic guanosine monophosphate (cGMP), in the collecting tubules. Infusion of a phosphodiesterase inhibitor largely reverses this defect and restores the natriuretic response to volume expansion (33, 35).

Also, in many individuals with inflammatory forms of glomerulonephritis and NS there is activation of several tubular channels and transporters that promote Na+ reabsorption and edema formation. These include:

1. Increased activity of the Na+-hydrogen exchanger (NHE3) that mediates a large portion of proximal Na+ reabsorption (36, 37). However, as demonstrated in experimental unilateral NS, overall Na+ retention is influenced to a larger degree by mechanisms that promote Na+ reabsorption in the distal tubule (26).

### Clinical Evaluation of the Predominant Mechanism of Edema formation in NS

Timely clinical assessment of hemodynamic aspects, including circulatory volume, is the key to determining management approaches to reduce edema in children with NS. This will help avoid exacerbation of the most common complication encountered in hospitalized children with NS and "underfill," AKI (43), or hypertensive and pulmonary complications in those with "overfill." **Tables 2** and **3** summarize the typical clinical and laboratory features, which serve to distinguish children with NS with underfill or overfill physiology.

It should be noted that because Na+ retention occurs both in underfill and overfill states (**Figure 1**), it is not possible to use the FENa+ to clinically differentiate primary from secondary Na+ retention in NS. Also, measurement of several hormonal markers shown in **Tables 2** and **3** are not readily measured in hospital laboratories and may not be clinically useful in confirming the underlying operative mechanism or influence point-of-care decisions. However, an increase in RAAS and circulating aldosterone effect can be inferred on the basis of more readily measured values such as an increased transtubular potassium gradient (TTKG) index or, UK+/UK+ + UNa+, which is observed in hypovolemic children and not when blood volume is preserved. Similarly, a TTKG index below 60% along with FENa+ above 0.5%, and normal or suppressed plasma aldosterone concentrations, highly implicate a primary Na+ retention mechanism leading to increased intravascular volume (i.e., overfill) (10, 19). This also suggests a primary role of aldosterone in the intrinsic activation of Na+/K+ ATPase in the cortical CD. Such children may benefit from diuretic use. By contrast, diuretic use in children with NS and secondary Na+ retention triggered by hypovolemia or circulatory

#### TABLE 2 | Mechanism of edema formation in nephrotic syndrome: "underfilling."

#### Clinical characteristics

Neuromuscular weakness, pallor, cool extremities, tachycardia, and other signs and symptoms of orthostatic hypotension, abdominal pain secondary to gut edema, abdominal compartment syndrome, or thrombosis of vena cave or renal veins

#### Laboratory findings

Reduced urine volume FENa+ < 0.2% UK+/UK+ + Na+ > 60% (increased TTKG index) Reduced urinary Na+ and high potassium concentration Very low serum albumin (≤2 g/dL) Low serum creatinine level GFR > 75 mL/min/1.73 m2 Hemoconcentration High circulating PRA, aldosterone, vasopressin, and norepinephrine Low ANP concentration

*FENa*+*, fractional excretion of sodium; UK*+ *and UNa*+*, urinary potassium and sodium concentrations; TTKG, transtubular potassium gradient; GFR, glomerular filtration rate; PRA, plasma renin activity; ANP, atrial natriuretic peptide.*

#### TABLE 3 | Mechanism of edema formation in nephrotic syndrome: "overfilling."

#### Clinical findings

Normal or elevated BP without tachycardia or orthostatic symptoms, and no signs to indicate distal extremity hypoperfusion

#### Laboratory findings

FENa+ > 0.5% while on no salt restricted diet UK+/UK+ + UNa+ < 60% (decreased TTKG index) Hematuria and cellular casts Serum albumin >2 g/dL Elevated serum creatinine and BUN GFR < 50 mL/min/1.73 m2 Decreased vasopressin Low circulating PRA and norepinephrine Low or normal plasma aldosterone High ANP

*FENa*+*, fractional excretion of sodium; UK*+ *and UNa*+*, urinary potassium and sodium concentrations; TTKG, transtubular potassium gradient; GFR, glomerular filtration rate; PRA, plasma renin activity; ANP, atrial natriuretic peptide.*

insufficiency may have serious deleterious consequences (see below).

### MANAGEMENT OF NEPHROTIC EDEMA

Before reviewing the management of edema in children with NS it is worth noting the change in the incidence of known clinical complications of NS that may relate to edema or its improper medical management. In a recent study involving hospital discharges of 4,701 children admitted with NS, Rheault et al. found that the frequency of infectious and thromboembolic complications has not changed much over the past 10 years; however, the incidence of AKI had increased from 3.3 to 8.5% (158%) over the period of 2001 and 2009 (43). The increasing use of nephrotoxic medications such as calcineurin inhibitors and

#### TABLE 4 | General aspects of management of edema in children with NS.

#### Avoid NSAID use

Avoid placement of deep lines to prevent thromboembolic events Reduce dietary salt No fluid restriction unless brisk diuresis is achieved Insure adequate nutrition Monitor urine output, renal function, electrolytes, serum albumin, body weight, and vital signs Elevate extremities or use compression stockings when ambulating; water immersion is helpful but impractical Avoid ACE inhibitors as remission can occur in many children with corticosteroid monotherapy

angiotensin converting enzyme/angiotensin receptor blockers to co-manage steroid dependent and steroid resistant NS may be partly responsible for this trend. However, aggressive diuresis in children not recognized as having intravascular volume depletion (underfilling) may enable progression from incipient AKI to established AKI. Furthermore, prevention of AKI is of great importance because it may be a precursor to future development of chronic renal injury and hypertension. Inappropriate diuresis may also promote a thrombotic tendency in this disorder (44–47). Consequently, children with "underfill" physiology may benefit first by circulatory volume expansion using salt-poor albumin infusions, and delayed start of diuretics until after restoration of tissue perfusion is achieved.

### NON-PHARMACOLOGICAL MEASURES

Apart from managing the underlying condition leading to NS according to established guidelines (48–51), **Table 4** summarizes other basic aspects of care. Because Na+ and fluid retention is a fundamental feature of all causes of NS and because treatment regimens that include corticosteroids tend to enhance this effect, all children presenting with edema are counseled on dietary Na+ restriction (35 mg Na+/kg/day, or approximately1.5 mEq/kg/day) and are monitored for clinical signs of hypovolemia (52). Fluid restriction is usually self-limited in children who adhere well to Na+ restriction, and it is not recommended in children managed in the outpatient setting.

Attention to nutrition is very important particularly in conditions associated with massive proteinuria, such as Finnish type NS. Given the T1/2 of albumin of 21 days, when provided with adequate calories and amino acids, the liver can produce 200 mg albumin/kg/day to replace albumin catabolism or urinary loss. This normal synthetic function can double when the oncotic pressure in hepatic sinusoids falls as in the setting of NS. However, many children with NS are "picky eaters" and may have intestinal edema and abdominal pain, resulting in poor appetite as well as protein-losing enteropathy that may further compromise nutrition. In addition, the degree of proteinuria may be underestimated because of the large influence of the proximal tubule in albumin catabolism in NS. Thus, recycled amino acids are not used to replace albumin exclusively. Provision of supplemental calories, egg white protein, and nutritional supplements, such as Boost or Pediassure, may be helpful if clinically indicated.

Once a brisk diuresis is achieved, fluid intake may be limited to 2/3 of maintenance, or 1/2 or less of urine output, so as to produce the intended negative Na+ and fluid balance. Close monitoring of vital signs, fluid input and output, and of electrolytes is required in order to assure safety. Diuretic therapy should be temporarily discontinued if there is an unexplained decrease in urine output, elevation in serum creatinine or clinical manifestations of hypovolemia (e.g., weakness, orthostatic hypotension, and/or cool extremities) (52, 53).

#### TABLE 5 | Albumin infusion in the management of edema in NS.

#### Dosing of salt-poor albumin (25% SPA, or, 25 g/100 mL)

0.5 g/kg infused over 1-h, 2–3 times daily. Slower infusion rates may enhance equilibration of albumin between the intravascular and interstitial fluid compartments, thereby undermining fluid mobilization and removal. Larger dosages may be more effective but may cause acute volume expansion and pulmonary congestion

#### Indications

Tense ascites with abdominal compartment syndrome limiting diaphragmatic excursion, lymphatic flow, and venous return

Severe pleural effusions compromising breathing

Oliguria with incipient acute kidney injury (AKI)

Marked eyelid edema compromising vision

Severe scrotal or labial edema, risking skin breakdown

#### Precautions

Expensive

Low supply

Obtained from multiple blood donors risking viral transmission, tissue allosensitization, etc.

Pulmonary edema

#### TABLE 6 | Diuretics used to manage edema in children.

### PHARMACOLOGICAL MANAGEMENT OF NEPHROTIC EDEMA

### Albumin Infusion

In hospitalized children with nephrotic edema, excessive fluid can usually be removed, relatively safely, without exacerbating volume depletion (refer to **Table 5**). This is often accomplished through the combined administration of salt-poor, or, 25% albumin (SPA) to facilitate reabsorption of IS fluid, thereby supporting plasma volume and diuretics to enhance fluid removal. The more available 5% albumin solution can increase blood volume but does not raise oncotic pressure, while it delivers fivefold higher Na+ for each gram of albumin infused. By expanding plasma volume, albumin infusion suppresses vasopressin release induced by hypovolemia, thereby increasing water diuresis and improvement in hyponatremia. Albumin infusion is associated with more profound diuresis, at least in a subpopulation of pediatric patients with NS (54–57), particularly those with reduced effective arterial blood volume. For example, in one series of children with nephrotic edema and fractional excretion of Na+ (FENa+) of <0.2% suggesting reduced effective arterial blood volume were treated with diuretics plus albumin infusion (52). Their diuresis rivaled that obtained by diuretic monotherapy in children with FENa+ > 0.5% suggesting circulatory volume sufficiency or expansion (58).

**Table 5** lists specific indications for albumin infusion recommended by the author. Caretakers should also carefully weigh the precautions or potential drawbacks to such therapy, and consider avoiding pharmacotherapy for edema particularly if prompt remission of NS is anticipated. Notably, cosmetic effects related to edema are not an indication for diuresis in children with NS.

### Non-Protein Colloid Alternatives

The clinical utility of non-albumin colloids, such as (hyperoncotic 12% dextran solution given at 1.2–1.8 g/kg body weight daily as a single dose or on 3–5 consecutive days), was previously investigated in children with nephrotic edema (59). The precise mechanism of action has not been clarified. However, despite being effective in achieving a brisk diuresis at a lower cost than albumin, dextran use has not gained clinical favor because of safety concerns including increased blood pressure, headache, gastrointestinal discomfort, pain upon tissue infiltration, and bleeding diathesis with epistaxis. The clinical response to other non-protein colloid alternatives such as gelatin and hydroxyethyl starch to induce diuresis in nephrotic edema has not been investigated.

### Angiotensin Inhibition

Nearly, all children with chronic proteinuric disorders receive an ACE inhibitor or an angiotensin II receptor blocker as adjunctive synergistic drugs aimed at averting progressive renal injury caused by the underlying renal disorder. Such agents also exert a significant anti-proteinuric action and rise in serum albumin concentration which enhances the response to diuretics. However, the author recommends not using such agents in children with marked proteinuria because they tend to exaggerate hyponatremia and increase the risk of AKI because of lowering of systemic and intra-glomerular pressure. Although these agents serve as anti-proteinuric agents regardless of the underlying etiology of chronic proteinuria they are of little benefit in the acute management edema or if prompt remission of proteinuria is anticipated after staring steroids, as in the case of childhood MCD.

### DIURETIC MANAGEMENT OF NEPHROTIC EDEMA

Recognizing that in the majority of children nephrotic edema is the result of appropriate compensatory physiological mechanisms aimed at restoring diminished circulatory volume and tissue perfusion, use of diuretics to remove edema fluid should be undertaken with great caution so as not to interfere with adaptive responses (refer to **Table 6**).

With the possible exception of children with inflammatory glomerulonephritis, nephrotic edema and coexisting hypertension or clear evidence of "overfilling," the author rarely recommends diuretic use in the outpatient setting. This is because of the potentially catastrophic risks of diuretic use in NS, such as thrombosis and thromboembolism, AKI, and electrolyte imbalance. By contrast, concurrent use of diuretics and salt-poor albumin or diuretic monotherapy is often utilized to manage edema in the inpatient setting.

Many children with NS respond well to loop diuretics, although there is generally lesser natriuresis than when such diuretics are utilized to manage edema associated with other medical disorders (60, 61). Experimental studies in drug-induced NS suggest that the loop of Henle may be relatively resistant to loop diuretics (62). Several factors are thought to play an important role in inducing this relative diuretic resistance: