From Podocyte Biology to Novel Cures for Glomerular Disease

Elena Torban1, Fabian Braun2, Nicola Wanner2, Tomoko Takano1, Paul R. Goodyer3, Rachel Lennon4, Pierre Ronco5, Andrey V. Cybulsky1 and Tobias B. Huber2

1Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada2III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany3Department of Pediatrics, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada4Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, UK5Service de Néphrologie et Dialyses, Hôpital Tenon, Paris, France.

All authors contributed equally

$To whom correspondence should be addressed:elena.torban@mcgill.ca or t.huber@uke.de

Running Title: 12thInternational Podocyte Conference

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Abstract

The podocyte is a key component of the glomerular filtration barrier. Podocyte dysfunction is

central to the underlying pathophysiology of many common glomerular diseases, including

diabetic nephropathy, glomerulonephritis and genetic forms of nephrotic syndrome.

Collectively, these conditions affect millions of people worldwide, and account for the majority

of kidney diseases requiring dialysis and transplantation. The 12th International Podocyte

Conference was held in Montreal, Canada from May 30 to June 2, 2018. The primary aim of

this conference was to bring together nephrologists, clinicianscientists, basic scientists and

their trainees from all over the world to present their research and to establish networks with

the common goal of developing new therapies for glomerular diseases based on the latest

advances in podocyte biology. This review briefly highlights recent advances made in

understanding podocyte structure and metabolism, experimental systems in which to study

podocytes and glomerular disease, disease mediators, genetic and immune origins of

glomerulopathies, and the development of novel therapeutic agents to protect podocyte and

glomerular injury.

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Introduction

The theme of the 12th International Podocyte Conference was “from podocyte biology to

novel cures for glomerular disease”. The meeting began with sessions on podocyte

development, podocyte ultrastructure, and interactions of podocytes with neighboring cells

and the glomerular basement membrane (GBM). Subsequent sessions covered mechanisms

of nephrotic syndrome, mediators of podocyte injury, metabolic origins of podocyte disease,

genetics, experimental model systems, and therapeutics of glomerulonephritis.

Podocyte origin in development and disease

Our understanding of the molecular and morphological processes underlying glomerular

development has improved significantly in recent years. Ultrastructural analysis with cutting-

edge electron microscopy approaches has provided insight into the maturation of primitive

podocytes and their mature protrusions, as well as the intricate network of foot processes

and the slit diaphragm (Fig. 1)1. Jordan Kreidberg and colleagues developed new methods

for the analysis of the genetic programs guiding these cellular changes.Using chromatin

immunoprecipitation (ChIP) sequencing they identified a number of genes that are targets of

the Wilms Tumor 1 (WT1) transcription factor2. In experimental nephrotic syndrome, WT1

binding sites in some genes were lost, while new sites were acquired, implying that WT1-

regulated genes may becausative of nephrotic diseases in humans. The tight regulation of

the podocyte transcriptome also relies on othermechanisms. Jacqueline Ho presented

compelling data on the role of microRNAs in podocyte development and disease.

Herresearch has revealed specific microRNA clusters involved in the development of

nephron progenitor cells. Mutations in the MIR17HG cluster (miR-17-92 in mice) were

detected as the first miRNA mutations to cause renal developmental defects in Feingold

syndrome, characterized by impaired progenitor cell proliferation and a reduced number of

developing nephrons3. Conversely, miRNAs might represent a valuable therapeutic option,

as overexpression of different miRNA speciesis associated with modulatory effectson cyst

growth in experimental models of polycystic kidney diseaseand diabetic nephropathy4.Nicola

Wanner discussed epigenetic control of nephron number in kidney development, a major

determinant of long-term renal function. Nutritional restriction of kidney growth is associated

with DNA hypomethylation. DNA methyltransferases Dnmt1 and Dnmt3a are highly

expressed in the developing kidney. By analogy to nutritional growth restriction, deletion of

Dnmt1 in nephron progenitor cells ledto a reduction in nephron number and renal hypoplasia

at birth. DNA hypomethylation resulted in downregulation of genes crucial for initiation of

nephrogenesis, thus representing a key regulatory event of prenatal renal programming,

linking maternal nutritional factors during gestation and reduced nephron number5. To further

elucidate the relevance of progenitors cellsin glomerular development, Stuart Shankland and

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colleagues used elegant fate tracking experiments to identify cells of renin lineage thatexhibit

pluripotency and potential to replenish podocytes6,7. Ryuichi Nishinakamura provided an

overview of new models of glomerular development, such as kidney organoids8,9 and

podocytes differentiated from induced pluripotent stem cells10,11. These novel exciting

techniques will sharpen our knowledge in the future. The latter offers the potential to model

the glomerular filtration barrier on a chip12.

Podocyte architecture: Focus on the cytoskeleton, slit diaphragm and their

regulation in health and disease

Very few cells in higher vertebrates develop a cell body that matches the intricacy of the

podocyte with primary and secondary foot processes forming both a filtration barrier and a

signaling hub represented by the slit diaphragm (Fig. 2). A significant contributor to this

shape and function is the actin cytoskeleton. Rho GTPases, with 22 members, have long

been known for their involvement in actin cytoskeletal remodeling, cell motility, and podocyte

disease. Tomoko Takano and hergroup deepened our understanding of actin dynamics by

identifying the Rho GDP dissociation inhibitor, ARHGDIA, as a gene mutated in congenital

nephrotic syndrome13. At the cellular level, these mutations promote Rac1

hyperactivation14,which in turn causes podocyte detachment15. The roles of numerous Rho

GTPase regulatory proteins are yet to be elucidated in podocytes and their roles are actively

being investigated. Barbara Ballermann and colleagues identified chloride intracellular

channel protein 5a (Clic5a) as an upstream regulator of the actin connecting protein ezrin.

Clic5a is expressed in the podocyte apical domain, as is podocalyxin, and controls the

activation of phosphatidylinositol-5 kinaseand activation-specific phosphorylation of ezrin.The

interaction of podocalyxin with the actin cytoskeleton via activated ezrin appears to be vital

for intact podocyte structure. Knockout of Clic5a in mice leads to glomerular abnormalities16,

microaneurysms and hypertension17.

Besides the complex control of the podocyte cytoskeleton, the podocyte connection to

the extracellular matrix (ECM), specifically the GBM, plays an essential role in glomerular

health. Roy Zent’s group investigated the role of integrins in kidney biology. Deletion of the

β1-integrin linking kinase, ILK, in the ureteric bud of miceduring kidney development led to

reduced branching morphogenesis and severe interstitial fibrosis at 8-10 weeks age18,19.

Similar effects were seen in mice harboring a mutation in the binding site Y783A of talin, a

focal adhesion protein connecting the cytoplasmic tail of integrins to the cytoskeleton, while a

complete loss of talin led to renal agenesis and neonatal death20.

The crucial slit diaphragm protein nephrin and its phosphorylation have been the

focus of research by Nina Jones and colleagues. Disruption of three nephrin tyrosine

phosphorylation sites (Y3F mice), which serve as adaptor sites for the proteins Nck1 and 4

Nck2, led to proteinuria21. Likewise, homozygous Y3F/Y3F mice showed a prolonged

recovery in the nephrotoxic serum nephritis model, since nephrin clustering and Nck binding

were impaired, resulting in decreased nephrin endocytosis and turnover. Nephrin turnover is

in part regulated by ShcA, a phosphotyrosine adaptor protein. ShcA associates with multiple

phosphorylation sites on nephrin, promotes phosphorylation and reduces nephrin signaling.

Overexpression of ShcA, which is found in several proteinuric kidney diseases, may also

reduce nephrin signaling, pointing towards a common pathway involved in the generation or

maintenance of proteinuria22. There is mounting evidence to suggest that in many

glomerulopathies, actin dynamics and cell adhesion are abnormal and lead to subsequent

dysregulation of intercellular signaling. Thus, there have been extensive efforts to find

therapeutic druggable targets to inhibit and potentially reverse pathogenic changes. A

promising candidate is the small molecule Bis-T-23, as presented by Sanja Sever and

colleagues. Bis-T-23 facilitates the oligomerization of dynamin, a GTPase. Bis-T-23 was

found to successfully restore actin polymerization in injured podocytes inseveral renal

disease models by reversing defects in post-translational protein modification, such as O-

GlcNAcylation23,24.

Podocytes and friends

The podocyte cannot be viewed in isolation when investigating its functional role in health

and disease. It has become clear that there is intricate communication between podocytes

and other glomerular cells and structures (Fig. 3). Rachel Lennon and colleagues defined the

composition of the glomerular ECM using proteomic approaches25 and these data can now

be compared to ECM from other tissues using the powerful Matrisome Project resource

(http://matrisomeproject.mit.edu). The glomerular matrisome was compared to cell-derived

ECM from podocyte and endothelial cell co-cultures and whilst there was a significant

overlap, key GBM components including collagen IV3α4α5 have low abundance in vitro26.

To improve understanding of in vivoECM composition, they report a protocol to implant

kidney organoids differentiated in vitro fromhuman pluripotent cellsinto immunodeficient mice

to generate perfused glomerular structures with regions of mature GBM andevidence of

filtration function27.

Novel imaging techniques have sharpened our understanding of the morphology of

ECM proteins. Serial block face-scanning electron microscopy further delineated changes in

GBM morphology during the development of Alport syndrome, a hereditary glomerulopathy

related to mutations in GBM collagen IV. Changes included sub-podocyte expansions of the

GBM and podocyte protrusions invading the GBM28. Beyond that, super resolution

immunofluorescence microscopy opens up entirely new possibilities to investigate the

extracellular environment of the podocyte29. David Unnersjö-Jess employed similar

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techniques in combination with hydrogel-based optical clearing tostudy the morphology and

composition of the slit diaphragm in more detail30,31.

Concerning cellular crosstalk, Pierre-Louis Tharaux addressed the pathogenic signals

that trigger parietal epithelial cell recruitment after podocyte loss, which leads to cell

adhesions and sclerosis in the glomerular tuft, i.e., focal segmental glomerulosclerosis

(FSGS). In mice infused with angiotensin II, endothelial-specific deletion of the endothelial

PAS domain-containing protein 1 (Epas1) gene accentuated albuminuria, recruitment of

pathogenic parietal glomerular epithelial cells and sclerotic lesions. Furthermore, he showed

compelling data on the relevance of the tetraspanin CD9 in parietal epithelial cells for

crescent formation or the generation of sclerotic lesions in glomerulopathies. Besides this,

cells of the macula densa are central in the physiologicalremodeling of the glomerulus via

Wnt signaling and secreted paracrine factors that act on podocyte precursor cells, including

cells of the renin lineage32.

Dylan Burger and his group have investigated the role of urinary microparticles in

glomerular diseases. Specifically, they identified microparticles that are released from

cultured podocytes in response to high glucose exposure or mechanical stretch.

Microparticles of podocyte origin were present in the urine of diabetic rats and humans33.

Furthermore, Burgerpresented data suggesting that podocyte microparticles induce

profibrotic signaling in tubular cells34.

Immune etiology of nephrotic syndrome

Immune cells and immune-epithelial interactions have increasingly become the focus of

investigation into pathogenesis of proteinuric glomerular disease (Fig. 4). Leonardo Riella

introduced the concept of fetomaternal tolerance relying on regulatory T-cells and negative

signals through programmed death-ligand 1 (PDL1) costimulation. Using PDL1 blockade, he

and colleagues identified a shift towards interleukin-17 (IL-17) producing Th17 cells with a

decrease in regulatory T-cells leading to a higher abortion rate. This effect could be

abrogated by IL-17 neutralization35. Riella and his group are involved in the multicenter

TANGO study36 aiming at the discovery of prognostic factors for post-transplant recurrence of

glomerular diseases with a particular focus on immune factors. Furthermore, they are

investigating how changes in the microbiome (by use of dietary modifications) influence

subsequent immunological effects on proteinuria innephrotic syndrome in children. Manuela

Colucci presented work from her group on the underlying immune-mediated effects in

nephrotic syndrome. By examining the effects of rituximab on B- and T-cells in pediatric

patients with frequently relapsing or steroid-dependent nephrotic syndrome, they showed

that the only denominator of relapse was the reconstitution of memory B-cells independent of

the immunosuppressive regime37. The importance of immune-mediated effects on nephrotic

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syndrome was further delineated by a genome-wide association study (GWAS) in children

with steroid-sensitive nephrotic syndrome (SSNS) that identified three single nucleotide

polymorphisms in HLA-DQB1, HLA-DRB1 and BTNL2; all of these genetic loci are involved

in the immune response and associated with independent risk alleles for SSNS38. Similarly,

Samia Khan and colleagues identified three genetic variations in the integrin subunit alpha M

gene (encoding CD11b) that are associated with systemic lupus erythematosus (SLE).

Further research showed that reduction of Toll-like receptor-dependent proinflammatory

signals and suppression of interferon-1 signaling was CD11b-dependent, indicating that its

activation may be a potential therapy in SLE39.

The soluble urokinase-type plasminogen activator receptor (suPAR) has been

intensively investigated for its properties as a soluble immune mediator of proteinuric kidney

disease40,41. More recent evidence points towards suPAR being independently associated

with chronic kidney disease (CKD)42. Jochen Reiser elaborated on recent studies depicting

suPARas a possible predictor of mortality in type 2 diabetes43, of estimated glomerular

filtration rate decline in cardiovascular disease44, as well as the progression of CKD in

children45 with possible larger implications as a prognostic factor. The direct effects of suPAR

on glomerular cells and whether its depletion from patient blood can lead to a robust

amelioration of proteinuric kidney diseases arethe subject of current studies.

David Salant provided an overview of experimental models of podocyte disease. He

noted that models to study alterations in GBM structure in glomerulopathies are lacking.

Recent studies in membranous nephropathy46,47 have resulted in the identification of novel

nephritogenic antigens and their mediation of podocyte injury, as well as signaling pathways

activated by slit diaphragm molecules, including the interaction of nephrin with ephrin-B148.

The direct cytotoxic effects of immune cells in glomerular injury is supported by an

experimental model of rapidly progressive glomerulonephritis. How the microenvironment

plays a specific role in the control of immune-epithelial interactions was elegantly presented

by Detlef Schlöndorff and colleagues49. They injected enhanced green fluorescent protein

(EGFP)-specific CD8+ T-cells from just EGFP death inducing (Jedi) mice into mice with a

podocyte-specific EGFP transgene. In healthy conditions, podocytes were not accessible to

cytotoxic T-cells. However, after the disruption of Bowman's capsule through the induction of

nephrotoxic nephritis, the disease phenotype was aggravated by the injection of Jedi cells:

mice had higher blood urea nitrogen and urine albumin levels and more severe histological

lesions including massive depletion of EGF-positive podocytes. EGFP-specific CD8+ T cells

were observed near breaches in Bowman’s capsule, suggesting that CD8+ T cells can

interact with podocytes only after disruption of Bowman’s capsule, as observed in kidney

biopsies from patients with crescentic glomerulonephritis where glomerular infiltration of

CD8+ T cells was observed primarily at the site of cellular crescents with rupture of

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Bowman’s capsule. Detlef Schlöndorff is currently addressing the next questions on the

implication of parietal epithelial cells and the role of matrix metalloproteases in this process.

This beautiful work is emblematic of Detlef Schlöndorffs visionary, synthetic

conception of science. In a brilliant editorial published in Kidney International in 2014, he

started with "Many of us may have childhood memories of taking apart a mechanical clock

and then trying to put it back together. The taking apart step proved relatively easy; the

putting together proved to be more of a challenge”50. Is this not the same for the glomerulus

clock? As Detlef wrote 10 years ago about mesangial cells, no cell is an island51. The "putting

together" is beautifully illustrated by Detlef Schlöndorffs presentation at this Podocyte

meeting where the pathogenesis of glomerulonephritis can only be understood through

unraveling the complex cell-cell interactions between CD8+ T cells, podocytes and parietal

cells that play their part in opening the barrier towards the podocyte. Detlef Schlöndorffs

elegant studies using cutting-edge technologies to bring definitive answers to important

questions, have opened up new lines of investigation for other researchers and new

perspectives for more precision medicine. It is remarkable that Detlef Schlöndorff has been

following this line of research based on cell-cell interactions since the 80's where his group

described important aspects of mesangial cell biology and the role of chemokines,

chemokine receptors and Toll-like receptors in various inflammatory diseases with a constant

translational perspective. Because we are podocyte lovers, we should follow the advice of

this great mentor to integrate the mesangial, endothelial and podocyte periods of glomerular

research into a holistic period. That would benefit the whole renal community and more

importantly the patients.

Advances in therapeutics for glomerular nephropathies

In addition to a wide variety of molecular and clinical studies on disease mechanisms, further

research has been conducted into novel therapeutic approaches (Fig. 5). Nada Alachkar

presented the results of two studies conducted by her group on potential therapies for FSGS.

In recurrent or de novo FSGS resistant to rituximab or therapeutic plasma exchange,

adrenocorticotropic hormone gel administration led to a decrease in proteinuria, though to

varying degrees51. A second prospective study concluded that preemptive rituximab

administration or plasma exchange did not prevent the recurrence of FSGS after

transplantation while remaining a viable therapeutic option after recurrence52. Moin Saleem

presented work that further delineated the effects of circulating factors in FSGS. Previously,

his group demonstrated that vasodilator-stimulated phosphoprotein (VASP) becomes

phosphorylated (activated) upon exposure to plasma exchange material from patients with

recurrent FSGS53. This activation was shown to be dependent on protease-activated

receptor-1 (PAR1) and leads to podocyte hypermotility in vitro. Current research focuses on

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the function of a constitutively active PAR1 in vivo, the activity of PAR1 in human FSGS, and

how specific human podocin mutations may impair PAR1 trafficking and interactions with the

cytoskeleton thereby leading to FSGS, as well as potential for pharmacologic rescue. Alessia

Fornoni presented a prospective therapeutic approach to Alport syndrome. She

demonstrated that mice with deletion of discoidin domain receptor 1 (DDR1) exhibited a

slower progression in COL4A3 knockout Alport mice. DDR1 is a receptor tyrosine kinase

expressed on the membranes of podocytes, and excessive de novo production of the

collagen IVα1α1α2 network in Alport mice can activate DDR1 and cause podocyte lipid

accumulation and lipotoxicity. Pharmacological modulation of DDR1 or lipid accumulation

could be a novel therapeutic approach.

Rac1 activation results in podocyte damage and Rac1 activating mutations result in

sporadic FSGS54-56. Anna Greka and colleagues sought downstream targets of Rac1

amenable to therapeutic intervention. They identified Rac1-induced activity of the ion channel

TRPC5 and subsequent cytoskeletal remodeling to be a druggable target ofthe small

molecule AC1903. AC1903 successfully blocked TRPC5 channel activity, attenuating

proteinuria in both a rat genetic FSGS model and hypertensive proteinuric kidney disease57.

Using another approach, which involved screening over 5,000 FDA-approved drugs, two

compounds, BRAFV600E inhibitor GDC-0879 and adenylate cyclase agonist forskolin, were

identified to promote podocyte survival, revealing the exciting possibility of repurposing

established therapeutics for glomerular diseases58. These compound screenings and

targeted approaches can now be complemented by unbiased “omics” analyses using patient

material, as was exemplified by the identification of a compartment- and cell type-specific

dysregulation of hypoxia-associated gene transcripts through weighted correlation network

analysis of over 200 renal biopsies with varying CKD stages59. Such studies harbor the

potential of adapting compound screens to promising pathways in future studies.

Complement-mediated diseases in the glomerulus

In addition to direct antibody or T-cell-epithelial interactions, humoral immune factors, such

as the complement system, are important contributors to many glomerulopathies (Fig. 6). Successful treatments such as anti-C5 antibody infusion have outlined the potential of

complement-based interventions. Craig Langman reviewed the genetics of complement-

regulatory proteins in C3 glomerulopathy (C3G) and atypical hemolytic syndrome (aHUS).

He indicated that podocytes express several complement receptors, suggesting that aHUS

can affect podocytes directly. Langman highlighted the potential role for complement

therapeutics in C3G, albeit cautioning that more research was required60. The investigation of

rare genetic variants in aHUS and C3G has in part been hampered by small patient cohorts.

Marina Noris and colleagues were able to detect 371 novel rare genetic variants for aHUS

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and 82 for C3G through the analysis of 13 complement genes in >3,500 patients. The

resulting database has expanded our understanding of the two disease entities, with a clear

non-random distribution of variants over the affected proteins61. Beyond these glomerular

pathologies with important involvement of the complement cascade, Pierre Ronco62

presented compelling evidence for the involvement of complement in human membranous

nephropathy (MN). There is evidence for the activation of the lectin and alternative

complement pathways in MN. Analyzing MN patients with unusual progression, Ronco and

his collaborators identified antibodies to factor H, a regulatory component of the complement

system63. Steven Sacks and colleagues showed the potential of complement inhibition in

ischemia-reperfusion injury and organ transplant tolerance. In particular, they showed an

abnormal L-fucose pattern that is identified by a C-type lectin, collectin-11, subsequently

triggering the complement cascade via the lectin pathway. Inhibition of collectin-11 led to

strong protection from ischemia-reperfusion damage, pointing towards new potential

mechanisms of reducing ischemia-reperfusion injury after transplantation64.

Metabolic origin of glomerular disease

The glomerulus represents a demanding microenvironment requiring high metabolic activity

in its cellular components (Fig. 7). The group of Tobias Huber has made significant

advances in our understanding of metabolic control in podocyte health and disease. The

mammalian target of rapamycin (mTOR) was identified as a primary regulator of podocyte

autophagy during aging and an important signal in guiding compensatory hypertrophy.

mTOR dysfunction may contribute to podocyte loss65-67. Their focus has now shifted to

mechanisms of energy consumption and oxidative phosphorylation in the podocyte with new

data pointing towards podocyte-specific mechanisms in glucose metabolism. An excess of

reactive oxygen species production through NADPH oxidases (NOX) poses a severe, but

potentially druggable threat to the podocyte, as shown by Chris Kennedy's group. Both

expression of NOX5 in mice and overexpression of NOX4 in a diabetic mouse model

resulted in albuminuria and podocyte damage, while inhibition of the latter through knockout

or pharmacological intervention ameliorated the disease phenotype in diabetes68-71.

Alexander Staruschenko presented compelling data implicating calcium channels TRPC5

and TRPC6 in this process70. Besides energy metabolism, Andrey Cybulsky and colleagues

delineated the pathological impact of endoplasmic reticulum (ER) stress and proteotoxicity

on podocytes. It has been appreciated that mutations in α-actinin-4 result in a genetic form of

FSGS72. One reason for the occurrence of podocyte damage is proteotoxicity and aggregate

formation. These effects can be ameliorated by administering the chemical chaperone 4-

phenyl butyric acid to mice with FSGS associated with expression of mutant α-actinin-4. The

chemical chaperone reduces enhanced protein misfoldingand ER stress73.The role for ER 10

stress was also demonstrated by a podocyte-specific knockout of inositol-requiring enzyme-

1α (IRE1α; an ER transmembrane protein and transducer of ER stress), which resulted in

albuminuria and foot-process effacement in aging mice, and was at least in part related to

impaired autophagy74. Knockout of IRE1α also exacerbated injury in anti-GBM nephritis.

Endocytosis and recycling of proteins in podocytes (i.e., nephrin) is deregulated in disease

states, such as diabetes75. Catherine Meyer-Schwesinger and her colleagues have further

investigated the role of podocyte proteostasis by examining the two main protein degradation

mechanisms, the ubiquitin-proteasome system (UPS) and autophagy. Both systems vary in

their activity in different glomerular diseases. In fact, increases in specific UPS proteins can

differentiate between minimal change disease and FSGS76. Impaired autophagy

throughknockout of the autophagy protein 5 (ATG5) gene in podocytes leads to proteinuria

and renal insufficiency67. Tampering with the UPS likewise results in proteinuria and

exacerbation of injury in experimental models of nephritis, underlining the importance of

tightly regulated protein metabolism in sustaining podocyte function77,78.

Genetics of glomerular disease

The study of genetics in glomerular diseases has had a major impact on the understanding

of podocyte biology (Fig. 8). The field is rapidly expanding with the identification of more and

more pathogenic genetic variants resulting in a disease phenotype. COL4A3/4/5 mutations

were previously thought to be associated exclusively with Alport syndrome. Moumita Barua

elaborated on the importance of exome sequencing in patient families exhibiting proteinuric

kidney disease and FSGS by showing that specific COL4A3/4/5 mutations are present in a

significant proportion of these families79. The potential wider importance of Alport gene

mutations was highlighted by Jose Florez who reported recent findings from a large genome

wide association study (GWAS) in diabetic kidney disease. One significant locus was a

variant in COL4A380. Spearheading the search for new genes in steroid-resistant nephrotic

syndrome (SRNS), Friedhelm Hildebrandt and his group have continued to broaden our

understanding of genetic causes of kidney disease, demonstrating that close to a third of

familial SRNS cases are monogenic diseases81. New mutations have been discovered in

DNA-damage response complexes82, sphingosine metabolism83, regulation of small

GTPases84 and nucleoporins85. A technique that has been changing the landscape of genetic

research over the last year has been single-cell RNA sequencing (scRNAseq). Katalin

Suzták’s group recently published the first atlas of scRNAseq of the murine kidney86. The

dataset represents a valuable resource for the correlation of past, current and future GWAS

datasets, as single nucleotide polymorphisms can be ascribed to the cells with the highest

expression of the host gene. Daniel Bichet elaborated on how a monogenic disease can

increase the understanding of podocyte biology usingthe example of Fabry disease. Previous

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studies underlined the impact of dysregulated autophagy, profibrotic signaling and deranged

lipid metabolism on podocyte health87-89.

Jeff Miner and colleagues investigated metabolic and genetic modifiers in Alport

syndrome, and discovered that albumin and its filtration through the damaged GBM is a

significant contributor to the disease phenotype90. Furthermore, others showed that silencing

of the microRNA-21 by specific oligonucleotides led to decreased fibrogenesis as a result of

enhanced peroxisome proliferator-activated receptor-α/retinoic X receptor activity and

improved mitochondrial function, possibly opening an opportunity for therapy91. Similarly,

inducible expression of a COL4A3 transgene in mice, which leads to secretion and assembly

of collagen IV α3α4α5 heterotrimers by podocytes, ameliorated the Alport phenotype by

restoring GBM integrity92. Miner’s group also uncovered genetic modifiers ofthe disease

course, such asa human mutation in LAMB2 93.

Model systems to study podocytes

Podocyte research relies heavily on model systems. A dedicated session, therefore,

highlighted the advances in establishing novel models for glomerular disease and their

analysis (Fig. 9). Nicole Endlich and colleagues introduced super-resolution microscopy and

the measurement of slit diaphragm length per glomerular capillary surface as a novel tool to

robustly evaluate foot process effacement94.The architecture of the Drosophila nephrocyte

resembles the structure of the podocyte foot process and slit diaphragm in striking ways.

Accordingly, this Drosophila model has emerged as a valuable research tool for the

investigation of podocyte biology. Zhe Han focused on two studies in nephrocytes that

revealed the importance of small GTPases Rab 5, 7 and 11 95, as well as coenzyme Q1096 in

nephrocyte health, pointing to similar functions of these molecules in mammalian podocyte

biology. Vineet Gupta’s group established a novel high-throughput assay to simultaneously

screen for the effects of multiple pharmacological compounds on blocking drug-induced

podocyte toxicity (seen as changes in the integrity of actin cytoskeleton and focal adhesions)

using podocytes grown in a multiwell dish. Gupta and co-workers were able to identify 1% of

more than 2,000 FDA-approved compounds to be protective in podocytes; one of these

compounds, pyrintegrin, showed similar protective effects in vivo97. Since then, the procedure

has been fully automated, enabling enhanced screening of more compounds. Weining Lu

introduced a follow-up study of the slit guidance ligand 2 (SLIT2)/roundabout guidance

receptor 2 (ROBO2)/SLIT-ROBO Rho GTPase activating protein 1/non-muscle myosin IIA

heavy chain axis playing a role in podocyte adhesion. He proposed that interference with

ROBO2 signaling may be a potential therapeutic option in glomerular diseases98.

The investigation of immune-epithelial interactions poses specific problems when

studying the process in rodent models, as the current inbred strains only partially resemble

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the human immune system. Eunsil Hahm and her group examined the potential of

humanized mice by injecting peripheral blood mononuclear cells (PBMC) into

immunodeficient mice. Indeed, Hahm showed that, for example, PBMCs from patients with

recurrent FSGS could engraft in the new host body and trigger immune responses such as

suPAR release and foot process effacement. Importantly, these responses were abrogated

upon transplantation of a PBMC population depleted of CD34 cells41. She presented

additional data on how the humanized mouse model represents a valuable tool to delineate

immune processes mediated by mononuclear cells in patients.

Controversies and Discussions

Some of the presentations at the IPC and the associated discussions raised

controversies.

For example, there is strong indirect evidence that supports an extrarenal cause for

idiopathic FSGS. It has been proposed that a circulating factor toxic to podocytes is

produced by the cells of dysregulated immune system; however, after a number of

studies, the factor’s identity and the specific cell lineage producing it remain elusive.

This may not be surprising; although we typically refer to FSGS as a single type of

podocyte glomerulopathy, FSGS represents a histological pattern, most likely with

distinct etiologies. Indeed, at the IPC, it was highlighted that mutations in the

COL4A3/4/5 genes may be a substantial and underappreciated cause of FSGS.

Likewise, so-called “idiopathic” FSGS may have heterogenous pathophysiology.

Serum levels of suPAR appear to be strong predictors of declining renal function and

cardiovascular disease42, but it remains controversial whether suPAR is the driver of

chronic kidney disease and, especially, if it functions as the circulating podocyte-toxic

factor40,99,100. Clinical trials testing the effects of suPAR absorption therapy may be

able to resolve this debate. The lack of clear appreciation of the heterogeneity in

FSGS may also be responsible for conflicting results of FSGS treatments presented

at the IPC. It remains unclear whether drugs used to treat steroid-responsive

nephrotic syndrome (rituximab, glucocorticoids, calcineurin inhibitors and others)

have any role in treating FSGS that recurs after kidney transplantation. Putative

downstream mediators of podocyte-toxic factor(s) were presented at the IPC,

including PAR1, β3-integrin, dynamin and other molecules. Participants of the IPC

debated whether TRPC5 or TRPC6 may drive podocyte injury57,101,102. Studies of

TRPC isoform-specific inhibitors in patient-derived podocytes may be a way to

resolve this controversy.13

Discussions at the IPC identified several obstacles to progress in understanding

podocyte biology and disease. Genetic studies have focused on defining pathogenic

monogenic mutations responsible for specific podocytopathies. Mutations in

complement regulatory proteins are believed to contribute to the pathogenesis of

aHUS and C3 glomerulopathy. However, it was noted at the IPC that such mutations

may be more widespread (e.g. in membranous nephropathy), which raises the need

for examining mutations across several diseases to draw proper conclusions on

genotypes and phenotypes. The majority of the research efforts have been centered

on podocytes themselves and there is a paucity of information on other cells that

impact on podocyte function. For example, contribution of endothelium or parietal

cells to specific forms of glomerulopathies is unclear, and detailed phenotypes of

patients’ immune cells are not known. Such knowledge is essential to improve and

refine therapeutic approaches, e.g. anti-complement therapy in aHUS.

It is recognized that metabolism in cultured cells tends to be glycolytic while

mitochondria provide energy in vivo. Surprisingly, data presented at the IPC

supported a major role for glycolysis in podocytes in vivo, although it remains to be

determined if mitochondrial ATP production is dispensable in health and in

glomerulopathies. Potential effects of the environment on the function of immune

cells were noted at the IPC, but at present, such effects are considered infrequently

in experimental studies on glomerular disease. Likewise, standardized and

reproducible experimental models to study podocyte injury are lacking. In this regard,

humanized rodent models and “mini” organoids from patient-derived cells will likely

provide a new direction, although further fine-tuning of these emerging models is

required to determine the extent to which they recapitulate the functions of podocytes

or immune cells in human health and disease (Fig. 10).

Conclusion

The 2018 12thInternational Podocyte Conference in Montreal highlighted the most recent

discoveries in podocyte biology and mechanisms of proteinuric disease. It brought together

various stakeholders, including clinicians, scientists and trainees from academia and the

pharmaceutical industry, and created a sense of excitement regarding an increasingly prolific

pace of discovery in the field. The conference brought to light developments in transcriptional

and epigenetic control of podocyte gene expression. There were new insights into the

dynamics of the slit diaphragm, actin cytoskeleton and regulators, including RhoGTPases.

The importance of crosstalk of podocytes with other glomerular cells and GBM was 14

emphasized. The roles of immune mediators, complement, protein folding, and regulation of

proteostasis in podocyte diseases were highlighted. Finally, novel techniques in imaging and

ssRNAseq were presented. Several regulators and pathways may constitute druggable

targets for podocyte diseases, and various promising therapeutic approaches were

discussed (Fig. 11). These presentations provide hope that in the future, podocyte diseases

will be preventable or attenuated in many patients by use of such mechanism-based

therapies.

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Figures and Figure legends

Figure 1. Podocyte origin in development and disease. Nephron development starts with the aggregation of the nephron progenitor cells, initiating the transition from renal vesicle to mature nephron. Podocytes are for the first time detectable in the comma-shaped stage (blue) and later form their characteristic foot processes. In this process, transcription factor WT1, DNA methylation and microRNA 17 have been shown to play a role. Me, methylation.

Figure 2. Podocyte architecture: Focus on the cytoskeleton, slit diaphragm and their regulation in health and disease. Rho GTPase pathways, slit diaphragm signaling and actin interactions, as well as focal adhesions have been shown to be crucial for podocyte foot process architecture.

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Figure 3. Podocytes and friends. Podocytes and endothelial cells both contribute to the glomerular basement membrane (GBM). Podocytes, parietal epithelial cells (PECs) and cells from the Macula densa communicate via signaling molecules. Urinary microparticles of podocyte origin can lead to downstream signaling in the tubule.

Figure 4. Immune etiology of nephrotic syndrome. Podocytes are affected by circulating factors (such as suPAR), the microbiome or diet, and different immune subpopulations such as regulatory T cells (Tregs) and Th17 cells. The occurrence of memory B cells can aggravate immune complex depositions. CD8 T cells are able to access podocytes when the Bowman’s capsule is ruptured.

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Figure 5. Advances in therapeutics for glomerular nephropathies. Several approved and potential therapeutic treatments improve proteinuria, podocyte foot process effacement and glomerular nephropathies.

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Figure 6. Complement-mediated diseases in the glomerulus. The complement cascade contributes to many glomerulopathies such as membraneous nephropathy, C3 glomerulopathy, Atypical Hemolytic Uremic Syndrome (aHUS) and ischemia reperfusion transplant tolerance.

Figure 7. Metabolic origin of glomerular disease. Crucial metabolic processes in the podocyte involve mTOR pathway, autophagy, ubiquitin/proteasome system (UPS), mitochondria and endoplasmic reticulum (ER). oxPhos, oxidative phosphorylation. Ub, ubiquitin. NOX, NADPH oxidase.

Figure 8. Genetics of glomerular disease. Novel mutations causing monogenetic nephrotic diseases have been found. The effect of miRNA on podocyte biology is under investigation. Genome wide association studies (GWAS) detect correlations between single nucleotide polymorphisms (SNP) and disease. Single cell RNA-sequencing (scRNA-seq) elucidates gene expression in single glomerular cells.

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Figure 9. Model systems to study podocytes. Novel methods and models are being employed to uncover podocyte function. Super-resolution microscopy has greatly improved resolution of slit diaphragm stainings. High-throughput screening assays of FDA-approved drug libraries uncover podocyte protective compounds. Drosophila melanogaster nephrocytes function as simplified podocyte models. Humanized mouse models are a valuable tool to delineate immune processes mediated by mononuclear cells in patients.

Figure 10. Scanning electron microscopic picture of podocytes with pseudocoloring of foot processes (original picture from Martin Helmstädter and Tobias B. Huber).

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Figure 11. Podocyte diseases, cell-cell interactions, cellular targets, methods and model. CKD, chronic kidney disease. DKD, diabetic kidney disease. MN, membranous nephropathy. RPGN, rapid progressive glomerulonephropathy. FSGS, focal segmental glomerular sclerosis. ECM, extracellular matrix. GBM, Glomerular basement membrane. PECs, parietal epithelial cells. TFs, transcription factors. DNAm, DNA methylation. iPSCs, induced pluripotent stem cells. scRNA-seq, single cell RNA sequencing.

Figure 12. Prof. Detlef Schlöndorff…..

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