From Podocyte Biology to Novel Cures for Glomerular Disease Elena Torban 1 , Fabian Braun 2 , Nicola Wanner 2 , Tomoko Takano 1 , Paul R. Goodyer 3 , Rachel Lennon 4 , Pierre Ronco 5 , Andrey V. Cybulsky 1 and Tobias B. Huber 2 1 Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada 2 III. Department of Medicine, University Medical Center Hamburg- Eppendorf, Hamburg, Germany 3 Department of Pediatrics, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada 4 Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, UK 5 Service de Néphrologie et Dialyses, Hôpital Tenon, Paris, France. All authors contributed equally $ To whom correspondence should be addressed: elena . torban @ mcgill .ca or [email protected]Running Title: 12 th International Podocyte Conference 1
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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.
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).