Transplanted organoids empower human preclinical assessment of drug candidate for the clinic.

Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.

Discovery of a Potent and Selective TRPC5 Inhibitor, Efficacious in a Focal Segmental Glomerulosclerosis Model.

The nonselective Ca2+-permeable transient receptor potential (TRP) channels play important roles in diverse cellular processes, including actin remodeling and cell migration. TRP channel subfamily C, member 5 (TRPC5) helps regulate a tight balance of cytoskeletal dynamics in podocytes and is suggested to be involved in the pathogenesis of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). As such, protection of podocytes by inhibition of TRPC5 mediated Ca2+ signaling may provide a novel therapeutic approach for the treatment of proteinuric kidney diseases. Herein, we describe the identification of a novel TRPC5 inhibitor, GFB-8438, by systematic optimization of a high-throughput screening hit, pyridazinone 1. GFB-8438 protects mouse podocytes from injury induced by protamine sulfate (PS) in vitro. It is also efficacious in a hypertensive deoxycorticosterone acetate (DOCA)-salt rat model of FSGS, significantly reducing both total protein and albumin concentrations in urine.

Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury.

BACKGROUND: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, ß-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. METHODS: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. RESULTS: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. CONCLUSIONS: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Inducible podocyte-specific deletion of CTCF drives progressive kidney disease and bone abnormalities.

Progressive chronic kidney diseases (CKDs) are on the rise worldwide. However, the sequence of events resulting in CKD progression remain poorly understood. Animal models of CKD exploring these issues are confounded by systemic toxicities or surgical interventions to acutely induce kidney injury. Here we report the generation of a CKD mouse model through the inducible podocyte-specific ablation of an essential endogenous molecule, the chromatin structure regulator CCCTC-binding factor (CTCF), which leads to rapid podocyte loss (iCTCFpod-/-). As a consequence, iCTCFpod-/- mice develop severe progressive albuminuria, hyperlipidemia, hypoalbuminemia, and impairment of renal function, and die within 8-10 weeks. CKD progression in iCTCFpod-/- mice leads to high serum phosphate and elevations in fibroblast growth factor 23 (FGF23) and parathyroid hormone that rapidly cause bone mineralization defects, increased bone resorption, and bone loss. Dissection of the timeline leading to glomerular pathology in this CKD model led to the surprising observation that podocyte ablation and the resulting glomerular filter destruction is sufficient to drive progressive CKD and osteodystrophy in the absence of interstitial fibrosis. This work introduces an animal model with significant advantages for the study of CKD progression, and it highlights the need for podocyte-protective strategies for future kidney therapeutics.

GDC-0879, a BRAFV600E Inhibitor, Protects Kidney Podocytes from Death.

Progressive kidney diseases affect approximately 500 million people worldwide. Podocytes are terminally differentiated cells of the kidney filter, the loss of which leads to disease progression and kidney failure. To date, there are no therapies to promote podocyte survival. Drug repurposing may therefore help accelerate the development of cures in an area of tremendous unmet need. In a newly developed high-throughput screening assay of podocyte viability, we identified the BRAFV600E inhibitor GDC-0879 and the adenylate cyclase agonist forskolin as podocyte-survival-promoting compounds. GDC-0879 protects podocytes from injury through paradoxical activation of the MEK/ERK pathway. Forskolin promotes podocyte survival by attenuating protein biosynthesis. Importantly, GDC-0879 and forskolin are shown to promote podocyte survival against an array of cellular stressors. This work reveals new therapeutic targets for much needed podocyte-protective therapies and provides insights into the use of GDC-0879-like molecules for the treatment of progressive kidney diseases.

Podocytes and the quest for precision medicines for kidney diseases.

In this review, I describe a 30-year journey in the quest for precision medicines for patients with kidney diseases. In 1987, when I started my reseach career, most scientists studying glomerular disease biology were focused on mesangial cells. The crucial role of the podocyte in many kidney diseases characterized by proteinuria, including focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, had not yet been recognized. We were not aware of genetic causes or drivers of kidney diseases nor of molecular markers and cell culture systems for mechanistic studies of podocyte biology. Tools for generating podocyte-specific knockout mice did not exist, and the key role of the podocyte actin cytoskeleton in the pathogenesis of proteinuria had not yet been identified. Clinically, treatment options for proteinuric kidney diseases were empiric, non-specific, and restricted to steroids and cyclosporine, without an understanding of their underlying mechanism of action. Since then, we have come a long way: a host of genetic causes for FSGS affecting podocytes has been identified, and with the advent of next generation sequencing approaches, the number of genetic causes continues to increase. Thinking "outside the box," empowered me to turn my attention to podocytes, develop the first differentiated podocyte cell culture system, and pioneer studies on the critical role of the podocyte actin cytoskeleton. Now, with the advent of iPSCs, we can build on these efforts by generating human podocytes and kidney organoids from patient cells, which, in combination with CRISPR-Cas9 gene editing and big data analyses, represent important next generation tools for bringing urgently needed precision medicines to patients with kidney disease. These new directions in kidney research should also increase the feasibility of much needed clinical trials in the kidney space. From Heidelberg to Boston, it has been an amazing scientific adventure. I will close with my thoughts about the path forward in making precision medicines for kidney diseases a reality.

Glomerulosclerosis Induced by Deficiency of Membrane-Associated Guanylate Kinase Inverted 2 in Kidney Podocytes.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a component of the slit diaphragm (SD) of glomerular podocytes. Here, we investigated the podocyte-specific function of MAGI-2 using newly generated podocyte-specific MAGI-2-knockout (MAGI-2-KO) mice. Compared with podocytes from wild-type mice, podocytes from MAGI-2-KO mice exhibited SD disruption, morphologic abnormalities of foot processes, and podocyte apoptosis leading to podocyte loss. These pathologic changes manifested as massive albuminuria by 8 weeks of age and glomerulosclerosis and significantly higher plasma creatinine levels at 12 weeks of age; all MAGI-2-KO mice died by 20 weeks of age. Loss of MAGI-2 in podocytes associated with decreased expression and nuclear translocation of dendrin, which is also a component of the SD complex. Dendrin translocates from the SD to the nucleus of injured podocytes, promoting apoptosis. Our coimmunoprecipitation and in vitro reconstitution studies showed that dendrin is phosphorylated by Fyn and dephosphorylated by PTP1B, and that Fyn-induced phosphorylation prevents Nedd4-2-mediated ubiquitination of dendrin. Under physiologic conditions in vivo, phosphorylated dendrin localized at the SDs; in the absence of MAGI-2, dephosphorylated dendrin accumulated in the nucleus. Furthermore, induction of experimental GN in rats led to the downregulation of MAGI-2 expression and the nuclear accumulation of dendrin in podocytes. In summary, MAGI-2 and Fyn protect dendrin from Nedd4-2-mediated ubiquitination and from nuclear translocation, thereby maintaining the physiologic homeostasis of podocytes, and the lack of MAGI-2 in podocytes results in FSGS.

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

Personalized Comments on Challenges and Opportunities in Kidney Disease Therapeutics: The Glom-NExT Symposium.

In the face of ever-increasing incidence and prevalence of kidney disease worldwide, the unmet need for new treatments is unprecedented. Precision medicine is defined as the use of modern technologies to identify mechanisms of diseases in individual patients, and thus deploy treatment using tailored, targeted approaches, in the hopes of avoiding unnecessary toxicities and complications. Is there a place for kidney disease therapeutics in this space? If so, what is required to make significant progress toward precision nephrology? To answer these critical questions, we present a series of personalized comments corresponding to the responses offered to these very questions during the Inaugural Glom-NExT Symposium held at Harvard Medical School on October 23, 2014, a national meeting focused exclusively on kidney disease therapeutics.

Rewiring neuronal microcircuits of the brain via spine head protrusions--a role for synaptopodin and intracellular calcium stores.

Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient.

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53.

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Dendrin ablation prolongs life span by delaying kidney failure.

Podocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin.

Developing therapeutic 'arrows' with the precision of William Tell: the time has come for targeted therapies in kidney disease.

PURPOSE OF REVIEW: A core mission for modern medicine is the development of precision therapeutics. Cancer therapies have been at the leading edge of this effort, while nephrology has lagged on the path to precision medicine. Breaking the stalemate, recent work revealed CD80 (B7-1) as a candidate for targeted therapy in the treatment of resistant nephrotic syndrome. This review aims to summarize the current state of our understanding of podocyte CD80 biology, its therapeutic implications and the challenges that lie ahead in essential future validation studies. RECENT FINDINGS: The CD80 targeting agent abatacept (CTLA4-Ig), approved to treat rheumatoid arthritis, was shown to induce remission of nephrotic range proteinuria in four patients with recurrence of disease posttransplant and one patient with primary, treatment resistant nephrotic syndrome. The concept of 'CD80-positive' proteinuric kidney disease due to podocyte CD80 staining in patient kidney biopsies was introduced as a molecular biomarker to define disease and guide treatment. The mechanism of action of CTLA4-Ig in podocytes was shown to centre on ß1 integrin activation in a T-cell independent fashion. Subsequent work revealed a putative role for podocyte CD80 in diabetic kidney disease. SUMMARY: These studies have direct implications for patient care, and intense interest has focused on validating these findings in upcoming clinical trials.

MAGI-2 scaffold protein is critical for kidney barrier function.

MAGUK Inverted 2 (MAGI-2) is a PTEN-interacting scaffold protein implicated in cancer on the basis of rare, recurrent genomic translocations and deletions in various tumors. In the renal glomerulus, MAGI-2 is exclusively expressed in podocytes, specialized cells forming part of the glomerular filter, where it interacts with the slit diaphragm protein nephrin. To further explore MAGI-2 function, we generated Magi-2-KO mice through homologous recombination by targeting an exon common to all three alternative splice variants. Magi-2 null mice presented with progressive proteinuria as early as 2 wk postnatally, which coincided with loss of nephrin expression in the glomeruli. Magi-2-null kidneys revealed diffuse podocyte foot process effacement and focal podocyte hypertrophy by 3 wk of age, as well as progressive podocyte loss. By 5.5 wk, coinciding with a near-complete loss of podocytes, Magi-2-null mice developed diffuse glomerular extracapillary epithelial cell proliferations, and died of renal failure by 3 mo of age. As confirmed by immunohistochemical analysis, the proliferative cell populations in glomerular lesions were exclusively composed of activated parietal epithelial cells (PECs). Our results reveal that MAGI-2 is required for the integrity of the kidney filter and podocyte survival. Moreover, we demonstrate that PECs can be activated to form glomerular lesions resembling a noninflammatory glomerulopathy with extensive extracapillary proliferation, sometimes resembling crescents, following rapid and severe podocyte loss.

Role of podocyte B7-1 in diabetic nephropathy.

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.

Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2.

Type 2 diabetes is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are susceptible to saturated FFAs, which induce endoplasmic reticulum (ER) stress and podocyte death. Genome-wide association studies indicate that expression of acetyl-CoA carboxylase (ACC) 2, a key enzyme of fatty acid oxidation (FAO), is associated with proteinuria in type 2 diabetes. Here, we show that stimulation of FAO by aminoimidazole-4-carboxamide-1ß-D-ribofuranoside (AICAR) or by adiponectin, activators of the low-energy sensor AMP-activated protein kinase (AMPK), protects from palmitic acid-induced podocyte death. Conversely, inhibition of carnitine palmitoyltransferase (CPT-1), the rate-limiting enzyme of FAO and downstream target of AMPK, augments palmitic acid toxicity and impedes the protective AICAR effect. Etomoxir blocked the AICAR-induced FAO measured with tritium-labeled palmitic acid. The beneficial effect of AICAR was associated with a reduction of ER stress, and it was markedly reduced in ACC-1/-2 double-silenced podocytes. In conclusion, the stimulation of FAO by modulating the AMPK-ACC-CPT-1 pathway may be part of a protective mechanism against saturated FFAs that drive podocyte death. Further studies are needed to investigate the potentially novel therapeutic implications of these findings.

Extensive podocyte loss triggers a rapid parietal epithelial cell response.

Damage to podocytes is a central pathomechanism of proteinuric kidney disease. However, it is not fully understood how podocyte injury evolves to progressive glomerulopathies such as FSGS or collapsing glomerulopathy. In particular, the role of parietal epithelial cells remains controversial. Here, we show that adriamycin induces DNA damage and podocyte lysis in mice without evidence of autophagy, endoplasmic reticulum stress, or necroptosis. After extensive podocyte loss, activated parietal cells mediated tuft re-epithelialization by two distinct mechanisms. In the majority of glomeruli, vacuolized parietal epithelial cells attached to denuded glomerular basement membrane and, occasionally, disengaged from the parietal basement membrane. Less frequently, parietal epithelial cells covered the denuded visceral basement membrane via formation of proliferative pseudocrescents. Notably, "visceralized" parietal epithelial cells did not express vascular endothelial growth factor but upregulated hypoxia-inducible factor 1 expression. The presence of visceralized parietal epithelial cells in sclerosing and collapsing lesions in a kidney biopsy from a patient with diabetes underscores the human relevance of our findings. In conclusion, repopulation of the glomerular tuft by parietal cells may represent a compensatory response to extensive podocyte loss. Our results suggest, however, that visceralized parietal epithelial cells cannot induce revascularization of the hyalinized tuft, resulting in hypoxic cell death and irreversible destruction of the glomerulus.

Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes.

AIMS/HYPOTHESIS: In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes. METHODS: Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection of streptozotocin. Mesenteric arteries were isolated to study ex vivo vascular reactivity with a wire myograph and kidneys were processed for histological examination. Glycation was determined by mass spectrometry and immunohistochemistry. Markers for inflammation, endothelium dysfunction and renal dysfunction were measured with ELISA-based techniques. RESULTS: Diabetes-induced formation of AGEs in mesenteric arteries and endothelial dysfunction were reduced by Glo1 overexpression. Despite the absence of advanced nephrotic lesions, early markers of renal dysfunction (i.e. increased glomerular volume, decreased podocyte number and diabetes-induced elevation of urinary markers albumin, osteopontin, kidney-inflammation-molecule-1 and nephrin) were attenuated by Glo1 overexpression. In line with this, downregulation of Glo1 in cultured endothelial cells resulted in increased expression of inflammation and endothelium dysfunction markers. In fully differentiated cultured podocytes incubation with MGO resulted in apoptosis. CONCLUSIONS/INTERPRETATION: This study shows that effective regulation of the GLO-I enzyme is important in the prevention of vascular intracellular glycation, endothelial dysfunction and early renal impairment in experimental diabetes. Modulating the GLO-I pathway therefore may provide a novel approach to prevent vascular complications in diabetes.