Drug discovery in focal and segmental glomerulosclerosis
Authors: Nick Pullen, Alessia Fornoni
Journal: Kidney International (2016)

Despite the high medical burden experienced by patients with focal segmental glomerulosclerosis, the etiology of the condition remains largely unknown. Focal segmental glomerulosclerosis is highly heterogeneous in clinical and morphologic manifestations. While this presents challenges for the development of new treatments, research investments over the last 2 decades have yielded a surfeit of potential avenues for therapeutic intervention. The development of many of those ideas and concepts into new therapies, however, has been very disappointing. Here, we describe some of the factors that have potentially contributed to the poor translational performance from this research investment, including the confidence we ascribe to a target, the conduct of experimental studies, and the availability of selective reagents to test hypotheses. We will discuss the significance of genetic and systems traits as well as other methods for reducing bias. We will analyze the limitations of a successful drug development. We will use specific examples hoping that these will guide a consensus for investment and drive greater translational quality. We hope that this substrate will serve to exemplify the tremendous opportunity for intervention as well as facilitate greater collaborative effort between industry, academia, and private foundations in promoting appropriate validation of these targets. Only then will we have achieved our goal for curative therapies for this devastating disease. Despite the high medical burden experienced by patients with focal segmental glomerulosclerosis, the etiology of the condition remains largely unknown. Focal segmental glomerulosclerosis is highly heterogeneous in clinical and morphologic manifestations. While this presents challenges for the development of new treatments, research investments over the last 2 decades have yielded a surfeit of potential avenues for therapeutic intervention. The development of many of those ideas and concepts into new therapies, however, has been very disappointing. Here, we describe some of the factors that have potentially contributed to the poor translational performance from this research investment, including the confidence we ascribe to a target, the conduct of experimental studies, and the availability of selective reagents to test hypotheses. We will discuss the significance of genetic and systems traits as well as other methods for reducing bias. We will analyze the limitations of a successful drug development. We will use specific examples hoping that these will guide a consensus for investment and drive greater translational quality. We hope that this substrate will serve to exemplify the tremendous opportunity for intervention as well as facilitate greater collaborative effort between industry, academia, and private foundations in promoting appropriate validation of these targets. Only then will we have achieved our goal for curative therapies for this devastating disease. Focal segmental glomerulosclerosis (FSGS) is a common cause of primary nephrotic syndrome, with a peak age of onset between 10 and 45 years and with about 2000 individuals reaching end-stage kidney disease each year.1US Renal Data System. 2011 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States. 2011. Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases.Google Scholar As the prevalence and incidence cannot be precisely estimated due to lack of population-based studies, the implementation of longitudinal cohorts of patients affected by nephrotic syndrome as well as the development of national and international registries will be key to understand disease pathogenesis, estimate disease impact, stratify patients, develop alternative clinically meaningful outcome measures, and design feasible clinical trials. This is even more important if we consider that the clinical disease course of patients with biopsy-proven FSGS is highly heterogeneous, as well as the recurrence rates after transplant, the response to treatment, and the morphologic pattern, as reviewed elsewhere.2D'Agati V.D. Kaskel F.J. Falk R.J. Focal segmental glomerulosclerosis.N Engl J Med. 2011; 365: 2398-2411Crossref PubMed Scopus (229) Google Scholar Successful drug development in the field of FSGS has also been hampered by the fact that the etiology of FSGS remains largely unknown (with 80% of cases being idiopathic) and little consensus as well as confidence on key interventional nodes has been reached, as we will discuss. While FSGS, and more broadly chronic kidney disease, represents a silent killer, and affected vulnerable populations such as underrepresented minorities and children are often characterized by a worse clinical outcome, the investment in experimental studies has been somewhat limited in contrast to other disciplines. First, this clearly reflects the influence of the public opinion in the community, which overall is more sensitive to specific areas of research characterized by high morbidity and mortality, such as cancer and heart disease. Interestingly, however, the risk of dying from chronic kidney disease is equal or superior to that of dying from certain forms of cancer,3Murray C.J. Lopez A.D. Measuring the global burden of disease.N Engl J Med. 2013; 369: 448-457Crossref PubMed Scopus (318) Google Scholar yet patients are surely more afraid to be informed they have cancer than chronic kidney disease. While a cultural shift and education are needed to generate awareness about this, there is perhaps also a need to make the discipline more glamorous and attractive to researchers. Second, dialysis and transplantation remain a lucrative therapeutic alternative to preventive or curative strategies in kidney diseases, thus decreasing the attractiveness of drug development. Finally, the lack of alternative outcome measures considered acceptable by the regulatory agencies,4Coresh J. Turin T.C. Matsushita K. et al.Decline in estimated glomerular filtration rate and subsequent risk of end-stage renal disease and mortality.JAMA. 2014; 311: 2518-2531Crossref PubMed Scopus (94) Google Scholar, 5Inker L.A. Levey A.S. Pandya K. et al.Early change in proteinuria as a surrogate end point for kidney disease progression: an individual patient meta-analysis.Am J Kidney Dis. 2014; 64: 74-85Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Thompson A. Cattran D.C. Blank M. et al.Complete and partial remission as surrogate end points in membranous nephropathy.J Am Soc Nephrol. 2015; 26: 2930-2937Crossref Scopus (1) Google Scholar as well as methods for better stratifying patients on their risk profile has discouraged industry from investing in drug development and requires further studies. Alternative biomarkers are being explored, for instance the measurement of podocyturia and urinary podocyte mRNA, which may be especially relevant in FSGS where damage to the podocyte appears to be a key determinant in the disease pathogenesis. These alternatives may help stratify patients and predict disease progression or sensitivity to drug intervention,7Wickman L. Afshinnia F. Wang S.Q. et al.Urine podocyte mRNAs, proteinuria, and progression in human glomerular diseases.J Am Soc Nephrol. 2013; 24: 2081-2095Crossref PubMed Scopus (28) Google Scholar and certainly more investment as well as qualification is warranted. The limitations discussed in the prior paragraph are among the factors that have led to the limited success in the development of new effective drugs for kidney diseases. In fact nephrology, among other subspecialties in internal medicine, has performed very poorly when it comes to conducting randomized clinical trials.8Palmer S.C. Sciancalepore M. Strippoli G.F. Trial quality in nephrology: how are we measuring up?.Am J Kidney Dis. 2011; 58: 335-337Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Table 1 summarizes ongoing clinical trials in FSGS or treatment-resistant nephrotic syndrome. There is a relative paucity of innovative and tailored approaches in development. This may be due to insufficient understanding of the disease pathophysiology, or the confidence in translation of experimental efficacy studies in animals to studies in humans or the cost of clinical development. With this in mind, the National Institutes of Health (NIH) established in 2012 a National Center for Advancing Translational Science (NCATS). Through 2 initiatives, Discovering New Therapeutic Uses for Existing Molecules (New Therapeutic Uses) (http://www.ncats.nih.gov/ntu) and Pfizer's Centers for Therapeutic Innovation for NIH Researchers (http://www.ncats.nih.gov/cti), NCATS has facilitated the engagement of multidisciplinary physician-scientists capable of conducting clinical studies and pharma to support the translation of new ideas. While the first initiative enables access to a diverse portfolio of clinically safe but developmentally curtailed agents and aims to repurpose an asset for an alternative indication, to date no nephrology programs have been sponsored. This is somewhat surprising given the ingenuity and talent of the nephrology community for exploring therapeutic repositioning of existing agents, as we will describe below. This contrasts with the appetite of most pharma and biotechnology companies for de novo discovery and development of highly differentiated as well as novel approaches. As academicians often lack the expertise in drug discovery and pharma has limited access to patient samples and clinical databases supporting their development plan, this makes approaches like the Centers for Therapeutic Innovation very attractive for both parties. Indeed, both initiatives place partnership between industry, academia, potentially private foundations, and government funds at the center, helping to fast-track programs with high probability of success. It will also allow for the early implementation of precision medicine initiatives and for their translatability into the arena of clinical care. The role of private foundations such as NephCure Kidney International engaging patients and families will be key for the success of any development, primarily when rare diseases such as FSGS are being targeted. Furthermore, foundations that are willing to serve as a partnering mediator between the academic investigators and the investors will undoubtedly facilitate the development of new therapeutic strategies. Access to patient registries, either developed by private foundations or supported by the NIH via consortia, that track a longitudinal cohort of affected individuals, as successfully implemented via national and international study networks such as NEPTUNE and CureGN, will be tremendously powerful.Table 1A snapshot of ongoing clinical trials in FSGS captured from ClincalTrials.govIdentifier(NCT)IndicationStudy designAssetMechanismSponsorStageComments02382874FSGSSingle-arm open-label pilot safety study (n = 5)Allogenic AD-MSC transplantNot knownRoyan InstituteINo substantial supporting preclinical data02235857FSGSOpen-label POM study (n = 35)Primary = change in UACR at 10 wkLiposorber LA-15Not knownKaneka Pharma America LLCIbNo substantial supporting preclinical data02000440FSGSSingle-arm open-label POM study (n = 24)Primary = 50% reduction in UACR at 24 wkLosmapimodp38 kinase inhibitionGlaxoSmithKlineIbPathway appears to be altered in FSGS. Some supporting preclinical data96Pengal R. Guess A.J. Agrawal S. et al.Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.Am J Physiol Renal Physiol. 2011; 301: F509-F519Crossref PubMed Scopus (9) Google Scholar, 119Koshikawa M. Mukoyama M. Mori K. et al.Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome.J Am Soc Nephrol. 2005; 16: 2690-2701Crossref PubMed Scopus (102) Google Scholar02585804FSGSSingle-arm open-label POM study (n = 12)Primary = change in inulin GFR at 8 wkDapagliflozinSGLT2 inhibitionAstraZenecaIbRationale is based on observed effects of SGLT2 inhibitors on renal hemodynamics in other diabetic populations (e.g., renal hemodynamic effect of sodium-glucose cotransporter 2 inhibition in patients with type 1 diabetes mellitus)120Cherney D.Z. Perkins B.A. Soleymanlou N. et al.Renal hemodynamic effect of sodium-glucose cotransporter 2 inhibition in patients with type 1 diabetes mellitus.Circulation. 2014; 129: 587-597Crossref PubMed Scopus (159) Google Scholar01164098FSGS (post-transplant)Randomized open-label study (n = 60)Primary = prevention of proteinuria recurrence at 4 wkRituximabCD20 B cell depletionSMPDL3bGenentech/University MiamiIISupported by clinical and preclinical observations38Fornoni A. Sageshima J. Wei C. et al.Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis.Sci Transl Med. 2011; 3: 85ra46Crossref PubMed Scopus (86) Google Scholar01573533FSGS(treatment resistant)Open-label study in treatmentPrimary = remission & change in UACR at 12 moRituximabCD20 B cell depletionSMPDL3bGenentechMayoIISupported by clinical observations02394106Nephrotic syndrome (treatment resistant)Double-blind, 2-parallel-arm, placebo-controlled randomized study (n = 50)Primary = remission and change in UACR at 6 moOfatumumabCD20 B cell depletionIstituto Giannina GasliniIISupported by clinical observations01613118FSGSMultidose randomized placebo-controlled efficacy study (n = 100)Primary = change in UACR at 8 wkRE-021/sparsentanETRA/ARBRetrophinIIETRAs appear to improve UACR and fibrosis in preclinical models. As a class, though, ETRAs have been associated with liver and CV AEs02257697Nephrotic syndromeRandomized controlled open-label study (n = 238)Primary = remission at 52 wkMizoribineIMPDHPodocyte target not knownAsahi Kasei PharmaIIIPreclinical supporting data40Lee H.W. Khan S.Q. Faridi M.H. et al.A podocyte-based automated screening assay identifies protective small molecules.J Am Soc Nephrol. 2015; 26: 2741-2752Crossref Scopus (2) Google ScholarAD-MSC, adipose tissue–derived mesenchymal stem cell; ARB, angiotensin receptor blockade; CV AEs, cardiovascular adverse events; ETRA, endothelin receptor antagonists; FSGS, focal segmental glomerulosclerosis; GFR, glomerular filtration rate; IMPDH, inosine-5′-monophosphate dehydrogenase; NCT, National Clinical Trial; POM, proof of mechanism; UACR, urinary albumin-to-creatinine ratio. Open table in a new tab AD-MSC, adipose tissue–derived mesenchymal stem cell; ARB, angiotensin receptor blockade; CV AEs, cardiovascular adverse events; ETRA, endothelin receptor antagonists; FSGS, focal segmental glomerulosclerosis; GFR, glomerular filtration rate; IMPDH, inosine-5′-monophosphate dehydrogenase; NCT, National Clinical Trial; POM, proof of mechanism; UACR, urinary albumin-to-creatinine ratio. The glomerulus is a highly specialized structure that ensures the selective ultrafiltration of plasma so that essential proteins are retained in the blood.9Somlo S. Mundel P. Getting a foothold in nephrotic syndrome.Nat Genet. 2000; 24: 333-335Crossref PubMed Scopus (194) Google Scholar Podocytes, the primary cellular targets in FSGS, are specialized glomerular epithelial cells whose numerous foot processes support the formation of the slit diaphragm.10Ruotsalainen V. Ljungberg P. Wartiovaara J. et al.Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci U S A. 1999; 96: 7962-7967Crossref PubMed Scopus (456) Google Scholar, 11Tryggvason K. Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar Podocytes contribute to the glomerular filtration barrier through a tight regulation of actin cytoskeleton remodeling.12Kerjaschki D. Caught flat-footed: podocyte damage and the molecular bases of focal glomerulosclerosis.J Clin Invest. 2001; 108: 1583-1587Crossref PubMed Scopus (221) Google Scholar, 13Asanuma K. Mundel P. The role of podocytes in glomerular pathobiology.Clin Exp Nephrol. 2003; 7: 255-259Crossref PubMed Scopus (144) Google Scholar A large variety of new experimental tools have allowed for the identification of specific drug targets that are predominantly expressed by podocytes in health and disease.14Boerries M. Grahammer F. Eiselein S. et al.Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.Kidney Int. 2013; 83: 1052-1064Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Grgic I. Hofmeister A.F. Genovese G. et al.Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo.Kidney Int. 2014; 86: 1116-1129Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar In fact, while nephrin was the only known component of the glomerular filtration barrier when first discovered in 1998, a large body of new podocyte-specific proteins and potential targets have now been identified.16Mathieson P.W. The podocyte as a target for therapies—new and old.Nat Rev Nephrol. 2012; 8: 52-56Crossref Scopus (47) Google Scholar, 17Arif E. Nihalani D. Podocytes as a therapeutic target.Ann Clin Exp Hypertens. 2013; 1: 1003-1013Google Scholar However, target validation has remained a problem, and new strategies to increase the probability of success in drug development remain a matter of intense debate. Therefore, while we do not intend to discuss each of the new targets under experimental and clinical investigation for FSGS, we will provide some examples of how effective validation of new targets may lead to successful drug development. Some guidance criteria for strategic decisions, target selection, and experimental assessment are summarized in Figure 1 and discussed below. The discovery of mendelian inherited genes, such as mutations in nephrin, podocin, etc., that predispose individuals to FSGS signposted the significance of the podocyte in proteinuric disease. Their study has provided insight into potential mechanistic causes of proteinuric disease and opened avenues for patient stratification. Many of these genes have been studied in the context of transgenic and knockout systems, providing potentially important disease-relevant models for efficacy studies. It has been increasingly recognized that the study of disease-causing as well as protective alleles can yield to a rich source of important therapeutic targets. Recent industry analyses of clinical success and failure have indicated that portfolios that are rich in assets derived from genetic validation have a greater chance of success than those derived without.18Cook D. Brown D. Alexander R. et al.Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework.Nat Rev Drug Discov. 2014; 13: 419-431Crossref PubMed Scopus (140) Google Scholar, 19Nelson M.R. Tipney H. Painter J.L. et al.The support of human genetic evidence for approved drug indications.Nat Genet. 2015; 47: 856-860Crossref PubMed Scopus (38) Google Scholar, 20Plenge R.M. Scolnick E.M. Altshuler D. Validating therapeutic targets through human genetics.Nat Rev Drug Discov. 2013; 12: 581-594Crossref PubMed Scopus (116) Google Scholar Many of the genes that have been shown to contribute to proteinuric disease are not easily or obviously targetable. Remarkably little investment has been made in developing approaches against known causal genes. If we take TRPC6 as an example, multiple gain-of function mutations have been mapped to FSGS.21Reiser J. Polu K.R. Moller C.C. et al.TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function.Nat Genet. 2005; 37: 739-744Crossref PubMed Scopus (451) Google Scholar, 22Winn M.P. Conlon P.J. Lynn K.L. et al.A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis.Science. 2005; 308: 1801-1804Crossref PubMed Scopus (584) Google Scholar TRPC6 is a nonselective cation channel that sits at the slit diaphragm. It is a member of a class of channels that are believed to be chemically tractable drug targets. As hyperactivation of the channel is believed to promote podocyte motility, foot process effacement, and apoptosis, antagonism of the channel might be desirable in a disease setting. Indeed, there is supporting evidence of increased expression of the channel in other glomerular diseases, suggesting that this is a pathway that might have a key disease role.23Moller C.C. Wei C. Altintas M.M. et al.Induction of TRPC6 channel in acquired forms of proteinuric kidney disease.J Am Soc Nephrol. 2007; 18: 29-36Crossref PubMed Scopus (165) Google Scholar However, for several potential reasons, this target has not received much attention. Firstly, targeted knockdown or knockout alone in mice does not appear to confer a significant renal phenotype,24Hauser P.V. Pippin J.W. Kaiser C. et al.Novel siRNA delivery system to target podocytes in vivo.PLoS One. 2010; 5: e9463Crossref PubMed Scopus (0) Google Scholar, 25Wang L. Jirka G. Rosenberg P.B. et al.Gq signaling causes glomerular injury by activating TRPC6.J Clin Invest. 2015; 125: 1913-1926Crossref Scopus (1) Google Scholar which suggests potential compensation by other TRP channels or pathways.26Schaldecker T. Kim S. Tarabanis C. et al.Inhibition of the TRPC5 ion channel protects the kidney filter.J Clin Invest. 2013; 123: 5298-5309Crossref PubMed Scopus (32) Google Scholar To this latter point, TRPC5 has also emerged as a potential podocyte target based on some preclinical observations,26Schaldecker T. Kim S. Tarabanis C. et al.Inhibition of the TRPC5 ion channel protects the kidney filter.J Clin Invest. 2013; 123: 5298-5309Crossref PubMed Scopus (32) Google Scholar and we await evidence that TRPC5 activity is altered in human FSGS in order to underwrite the significance of these findings compared with what is known about TRPC6. As TRPC6 appears to be activated in an angiotensin II–dependent fashion, if channel blockade is not superior to angiotensin-converting enzyme inhibition or angiotensin receptor blockade treatment alone, then a TRPC6 antagonist might not have the additional clinical horsepower. TRPC6 is also expressed in many other cell types, and this raises potential safety concerns, especially cardiovascular safety. Finally, the quality and availability of selective TRPC6 tools have limited the validation of this approach; even ML204, which was used to assess the potential contribution of TRPC5 in podocytopathy, has significant polypharmacology at the concentrations tested that might limit interpretation of the preclinical findings.26Schaldecker T. Kim S. Tarabanis C. et al.Inhibition of the TRPC5 ion channel protects the kidney filter.J Clin Invest. 2013; 123: 5298-5309Crossref PubMed Scopus (32) Google Scholar, 27Miller M. Shi J. Zhu Y. et al.Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.J Biol Chem. 2011; 286: 33436-33446Crossref PubMed Scopus (39) Google Scholar The barriers for initiating any de novo drug discovery program are considerable. Increasingly researchers are trying to apply the knowledge of the underlying systems biology, transcriptional, and -omic networks with the genetic causes to repurpose known drugs for therapeutic use.28Martini S. Nair V. Keller B.J. et al.Integrative biology identifies shared transcriptional networks in CKD.J Am Soc Nephrol. 2014; 25: 2559-2572Crossref PubMed Scopus (13) Google Scholar, 29Kretzler M. Sedor J.R. Introduction: precision medicine for glomerular disease: the road forward.Semin Nephrol. 2015; 35: 209-211Abstract Full Text Full Text PDF PubMed Google Scholar This has been most elegantly demonstrated in patients with steroid-resistant nephrotic syndrome and mutations in ADCK4.30Ashraf S. Gee H.Y. Woerner S. et al.ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption.J Clin Invest. 2013; 123: 5179-5189Crossref PubMed Scopus (55) Google Scholar ADCK4 appears to localize to mitochondria, to interact with COQ6. Comparisons with related proteins and COQ mutant phenotypes suggested that ADCK4 promotes CoQ10 biosynthesis and CoQ10 deficiency might be driving the phenotype of individuals harboring ADCK4 mutations. Supporting in vitro studies and preliminary human data strongly suggest that for this small group of individuals with ADCK4 mutations a restoration of renal function can be achieved with CoQ10 replacement. This early success suggests that more examples will emerge from the application of integrated functional genomics and the investigational pharmacopeia. It is postulated that a hallmark of proteinuric disease is the sustained loss of podocytes from the glomerular tuft, either by injury or by impaired function. As these cells have a poor capacity for self-renewal,31Hodgin J.B. Bitzer M. Wickman L. et al.Glomerular aging and focal global glomerulosclerosis: a podometric perspective.J Am Soc Nephrol. 2015; 26: 3162-3178Crossref PubMed Google Scholar, 32Peired A. Lazzeri E. Lasagni L. et al.Glomerular regeneration: when can the kidney regenerate from injury and what turns failure into success?.Nephron Exp Nephrol. 2014; 126: 70Crossref Scopus (1) Google Scholar the effect of the loss of functional podocyte mass on glomerular function is potentially irreversible and leads to progressive scarring and ultimately end-stage kidney disease. Elegant experimental studies performed by the Wiggins group33Fukuda A. Wickman L.T. Venkatareddy M.P. et al.Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease.Kidney Int. 2012; 81: 40-55Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar have shown that selective depletion of 30% to 40% of glomerular podocytes is sufficient to induce an overt proteinuria and progressive glomerulosclerosis that is phenotypically reminiscent of the clinical situation. These observations, coupled with the high phenotypic penetrance of mutations in key proteins necessary for podocyte function in individuals that present with primary FSGS, have brought the podocyte to center stage in the visualization and development of therapeutic approaches that seek to either preserve podocyte mass or restore function. The goal of either an unbiased phenotypic screening approach or hypothesis-driven assessment of any potential target is to conduct studies that generate data that meaningfully signpost a pharmacologic effect and offer insight into differentiation compared with standard of care and a directionally compelling rationale for efficacy in the patient. Several critical elements are needed for the development and execution of a podocyte screening funnel: (i) disease-relevant, human-based in vitro assays and functional end points; and (ii) orthologous reagents and in vivo systems that can be used to evaluate a compound’s efficacy and concentration–dose response. Of the >100 podocyte targets and pathways that have been identified from the phenotypic evaluation of transgenic animals, human genetics, and systems biology approaches as potential causal contributors to nephrotic syndrome, the task of replicating the observation of each one and evaluating their therapeutic potential would be both time-consuming and cost-prohibitive in the absence of sensitive and reproducible as well as predictive semi-high-throughput assays. Podocytes are terminally differentiated cells, and screening requires potentially large volumes of them. As primary cultures derived from sieved glomeruli are limited by tissue availability (human) or dedifferentiate, generating heterogeneous populations in culture during expansion, many groups have therefore taken advantage of conditionally immortalized human and rodent lines.34Saleem M.A. O'Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Crossref PubMed Google Scholar, 35Ni L. Saleem M. Mathieson P.W. Podocyte culture: tricks of the trade.Nephrology. 2012; 17: 525-531Crossref PubMed Scopus (0) Google Scholar However, these cells lack the distinctive morphology of the interdigitated foot process. Whether this is due to the clonal selection, the performance of these cells in the absence of an endothelium and glomerular basement matrix, or the conventional 2-dimensional structure and lack of microfluidics, or just simply because these cells do not express a full repertoire of podocyte proteins, especially those that form the structure of the slit diaphragm, is not clear. New cell lines and systems for deriving podocytes are being developed36Da Sacco S. Lemley K.V. Sedrakyan S. et al.A novel source of cultured podocytes.PLoS One. 2013; 8: e81812Crossref PubMed Google Scholar, 37Song B. Smink A.M. Jones C.V. et al.The directed differentiation of human iPS cells into kidney podocytes.PLoS One. 2012; 7: e46453Crossref PubMed Scopus (54) Google Scholar and may offer promise with respect to some of these deficiencies, but as we will discuss, we are still far from a system that offers all of the desired features of biomimicry. Should we achieve the perfect conditions to culture podocytes, standardized protocols will need to be developed to assess the correlation between in vitro readouts of podocyte function and in vivo disease observation. Notwithstanding these limitations, some groups have been successful in deriving assays that might mimic some of the mechanistic processes associated with proteinuric disease38Fornoni A. Sageshima J. Wei C. et al.Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis.Sci Transl Med. 2011; 3: 85ra46Crossref PubMed Scopus (86) Google Scholar, 39Merscher-Gomez S. Guzman J. Pedigo C.E.