ARF6, a component of intercellular bridges, is essential for spermatogenesis in mice.

Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.

The 14th International Podocyte Conference 2023: from podocyte biology to glomerular medicine.

The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting on May 23, providing young scientists with a platform to present and discuss their research findings. Throughout the main conference, 29 speakers across 9 sessions shared their insights on podocyte biology, glomerular medicine, novel technologic advancements, and translational approaches. Additionally, the event featured 3 keynote lectures addressing engineered chimeric antigen receptor T cell- and mRNA-based therapies and the use of biobanks for enhanced disease comprehension. Furthermore, 4 brief oral abstract sessions allowed scientists to present their findings to a broad audience. The program also included a panel discussion addressing the challenges of conducting human research within the American Black community. Remarkably, after a 5-year hiatus from in-person conferences, the 14th International Podocyte Conference successfully convened scientists from around the globe, fostering the presentation and discussion of crucial research findings, as summarized in this review. Furthermore, to ensure continuous and sustainable education, research, translation, and trial medicine related to podocyte and glomerular diseases for the benefit of patients, the International Society of Glomerular Disease was officially launched during the conference.

Rationale and design of the Nephrotic Syndrome Study Network (NEPTUNE) Match in glomerular diseases: designing the right trial for the right patient, today.

Glomerular diseases are classified using a descriptive taxonomy that is not reflective of the heterogeneous underlying molecular drivers. This limits not only diagnostic and therapeutic patient management, but also impacts clinical trials evaluating targeted interventions. The Nephrotic Syndrome Study Network (NEPTUNE) is poised to address these challenges. The study has enrolled >850 pediatric and adult patients with proteinuric glomerular diseases who have contributed to deep clinical, histologic, genetic, and molecular profiles linked to long-term outcomes. The NEPTUNE Knowledge Network, comprising combined, multiscalar data sets, captures each participant's molecular disease processes at the time of kidney biopsy. In this editorial, we describe the design and implementation of NEPTUNE Match, which bridges a basic science discovery pipeline with targeted clinical trials. Noninvasive biomarkers have been developed for real-time pathway analyses. A Molecular Nephrology Board reviews the pathway maps together with clinical, laboratory, and histopathologic data assembled for each patient to compile a Match report that estimates the fit between the specific molecular disease pathway(s) identified in an individual patient and proposed clinical trials. The NEPTUNE Match report is communicated using established protocols to the patient and the attending nephrologist for use in their selection of available clinical trials. NEPTUNE Match represents the first application of precision medicine in nephrology with the aim of developing targeted therapies and providing the right medication for each patient with primary glomerular disease.

Association of COVID-19 Versus COVID-19 Vaccination With Kidney Function and Disease Activity in Primary Glomerular Disease: A Report of the Cure Glomerulonephropathy Study.

RATIONALE & OBJECTIVE: Patients with glomerular disease (GN) may be at increased risk of severe COVID-19, yet concerns over vaccines causing disease relapse may lead to vaccine hesitancy. We examined the associations of COVID-19 with longitudinal kidney function and proteinuria and compared these with similar associations with COVID-19 vaccination. STUDY DESIGN: Observational cohort study from July 1, 2021, to January 1, 2023. SETTING & PARTICIPANTS: A prospective observational study network of 71 centers from North America and Europe (CureGN) with children and adults with primary minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy. EXPOSURE: COVID-19 and COVID-19 vaccination. OUTCOME: Repeated measure of estimated glomerular filtration rate (eGFR); recurrent time-to-event outcome of GN disease worsening as defined by doubling of the urinary protein-creatinine ratio (UPCR) to at least 1.5g/g or increase in dipstick urine protein by 2 ordinal levels to 3+(300mg/dL) or above. ANALYTICAL APPROACH: Interrupted time series analysis for eGFR. Prognostic matched sequential stratification recurrent event analysis for GN disease worsening. RESULTS: Among 2,055 participants, 722 (35%) reported COVID-19 infection; of these, 92 (13%) were hospitalized, and 3 died (<1%). The eGFR slope before COVID-19 infection was-1.40mL/min/1.73m2 (± 0.29 SD); within 6 months after COVID-19 infection, the eGFR slope was-4.26mL/min/1.73m2 (± 3.02 SD), which was not significantly different (P=0.34). COVID-19 was associated with increased risk of worsening GN disease activity (HR, 1.35 [95% CI, 1.01-1.80]). Vaccination was not associated with a change in eGFR (-1.34mL/min/1.73m2±0.15 SD vs-2.16mL/min/1.73m2±1.74 SD; P=0.6) or subsequent GN disease worsening (HR 1.02 [95% CI, 0.79-1.33]) in this cohort. LIMITATIONS: Infrequent or short follow-up. CONCLUSIONS: Among patients with primary GN, COVID-19 infection was severe for 1 in 8 cases and was associated with subsequent worsening of GN disease activity, as defined by proteinuria. By contrast, vaccination against COVID-19 was not associated with change in disease activity or kidney function decline. These results support COVID-19 vaccination for patients with GN. PLAIN-LANGUAGE SUMMARY: In this cohort study of 2,055 patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy, COVID-19 resulted in hospitalization or death for 1 in 8 cases and was associated with a 35% increase in risk for worsening proteinuria. By contrast, vaccination did not appear to adversely affect kidney function or proteinuria. Our data support vaccination for COVID-19 in patients with glomerular disease.

Precision nephrology identified tumor necrosis factor activation variability in minimal change disease and focal segmental glomerulosclerosis.

The diagnosis of nephrotic syndrome relies on clinical presentation and descriptive patterns of injury on kidney biopsies, but not specific to underlying pathobiology. Consequently, there are variable rates of progression and response to therapy within diagnoses. Here, an unbiased transcriptomic-driven approach was used to identify molecular pathways which are shared by subgroups of patients with either minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). Kidney tissue transcriptomic profile-based clustering identified three patient subgroups with shared molecular signatures across independent, North American, European, and African cohorts. One subgroup had significantly greater disease progression (Hazard Ratio 5.2) which persisted after adjusting for diagnosis and clinical measures (Hazard Ratio 3.8). Inclusion in this subgroup was retained even when clustering was limited to those with less than 25% interstitial fibrosis. The molecular profile of this subgroup was largely consistent with tumor necrosis factor (TNF) pathway activation. Two TNF pathway urine markers were identified, tissue inhibitor of metalloproteinases-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), that could be used to predict an individual's TNF pathway activation score. Kidney organoids and single-nucleus RNA-sequencing of participant kidney biopsies, validated TNF-dependent increases in pathway activation score, transcript and protein levels of TIMP-1 and MCP-1, in resident kidney cells. Thus, molecular profiling identified a subgroup of patients with either MCD or FSGS who shared kidney TNF pathway activation and poor outcomes. A clinical trial testing targeted therapies in patients selected using urinary markers of TNF pathway activation is ongoing.

Kidney Biopsy Features Most Predictive of Clinical Outcomes in the Spectrum of Minimal Change Disease and Focal Segmental Glomerulosclerosis.

BACKGROUND: Heterogeneity in disease course and treatment response among patients with MCD/FSGS necessitates a granular evaluation of kidney tissue features. This study aimed to identify histologic and ultrastructural descriptors of structural changes most predictive of clinical outcomes in the Nephrotic Syndrome Study Network (NEPTUNE). METHODS: Forty-eight histologic (37 glomerular, 9 tubulointerstitial, 2 vascular) and 20 ultrastructural descriptors were quantified by applying the NEPTUNE Digital Pathology Scoring System to NEPTUNE kidney biopsies. Outcomes included time from biopsy to disease progression, first complete remission of proteinuria, and treatment response. Relative importance of pathology and clinical predictors was obtained from random forest models, and predictive discrimination was assessed. RESULTS: Among 224 participants (34% Black, 24% Hispanic), model performance was excellent, with predictive discrimination of 0.9 for disease progression, 0.85 for complete remission, and 0.81 for treatment response. The most predictive descriptors of outcomes included both conventional-e.g., global sclerosis or segmental sclerosis and interstitial fibrosis/tubular atrophy-and novel features, including adhesion, interstitial foam cells, deflation, periglomerular fibrosis, mononuclear white blood cells, endothelial cell abnormalities, microvillous transformation, and acute tubular injury. CONCLUSIONS: The most predictive descriptors of clinical outcomes among MCD/FSGS patients reflected structural changes in multiple renal compartments. Reporting these descriptors should be standardized to guide prognostication of proteinuric glomerular diseases.

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair.

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

Quantification of Glomerular Structural Lesions: Associations With Clinical Outcomes and Transcriptomic Profiles in Nephrotic Syndrome.

RATIONALE & OBJECTIVE: The current classification system for focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) does not fully capture the complex structural changes in kidney biopsies nor the clinical and molecular heterogeneity of these diseases. STUDY DESIGN: Prospective observational cohort study. SETTING & PARTICIPANTS: 221 MCD and FSGS patients enrolled in the Nephrotic Syndrome Study Network (NEPTUNE). EXPOSURE: The NEPTUNE Digital Pathology Scoring System (NDPSS) was applied to generate scores for 37 glomerular descriptors. OUTCOME: Time from biopsy to complete proteinuria remission, time from biopsy to kidney disease progression (40% estimated glomerular filtration rate [eGFR] decline or kidney failure), and eGFR over time. ANALYTICAL APPROACH: Cluster analysis was used to group patients with similar morphologic characteristics. Glomerular descriptors and patient clusters were assessed for associations with outcomes using adjusted Cox models and linear mixed models. Messenger RNA from glomerular tissue was used to assess differentially expressed genes between clusters and identify genes associated with individual descriptors driving cluster membership. RESULTS: Three clusters were identified: X (n = 56), Y (n = 68), and Z (n = 97). Clusters Y and Z had higher probabilities of proteinuria remission (HRs of 1.95 [95% CI, 0.99-3.85] and 3.29 [95% CI, 1.52-7.13], respectively), lower hazards of disease progression (HRs of 0.22 [95% CI, 0.08-0.57] and 0.11 [95% CI, 0.03-0.45], respectively), and lower loss of eGFR over time compared with X. Cluster X had 1,920 genes that were differentially expressed compared with Y+Z; these reflected activation of pathways of immune response and inflammation. Six descriptors driving the clusters individually correlated with clinical outcomes and gene expression. LIMITATIONS: Low prevalence of some descriptors and biopsy at a single time point. CONCLUSIONS: The NDPSS allows for categorization of FSGS/MCD patients into clinically and biologically relevant subgroups, and uncovers histologic parameters associated with clinical outcomes and molecular signatures not included in current classification systems.

APOL1 genotype-associated morphologic changes among patients with focal segmental glomerulosclerosis.

BACKGROUND: The G1 and G2 alleles of apolipoprotein L1 (APOL1) are common in the Black population and associated with increased risk of focal segmental glomerulosclerosis (FSGS). The molecular mechanisms linking APOL1 risk variants with FSGS are not clearly understood, and APOL1's natural absence in laboratory animals makes studying its pathobiology challenging. METHODS: In a cohort of 90 Black patients with either FSGS or minimal change disease (MCD) enrolled in the Nephrotic Syndrome Study Network (58% pediatric onset), we used kidney biopsy traits as an intermediate outcome to help illuminate tissue-based consequences of APOL1 risk variants and expression. We tested associations between APOL1 risk alleles or glomerular APOL1 mRNA expression and 83 light- or electron-microscopy traits measuring structural and cellular kidney changes. RESULTS: Under both recessive and dominant models in the FSGS patient subgroup (61%), APOL1 risk variants were significantly correlated (defined as FDR <0.1) with decreased global mesangial hypercellularity, decreased condensation of cytoskeleton, and increased tubular microcysts. No significant correlations were detected in MCD cohort. Independent of risk alleles, glomerular APOL1 expression in FSGS patients was not correlated with morphologic features. CONCLUSIONS: While APOL1-associated FSGS is associated with two risk alleles, both one and two risk alleles are associated with cellular/tissue changes in this study of FSGS patients. Our lack of discovery of a large group of tissue differences in FSGS and no significant difference in MCD may be due to the lack of power but also supports investigating whether machine learning methods may more sensitively detect APOL1-associated changes.

Ultrastructural Characterization of Proteinuric Patients Predicts Clinical Outcomes.

BACKGROUND: The analysis and reporting of glomerular features ascertained by electron microscopy are limited to few parameters with minimal predictive value, despite some contributions to disease diagnoses. METHODS: We investigated the prognostic value of 12 electron microscopy histologic and ultrastructural changes (descriptors) from the Nephrotic Syndrome Study Network (NEPTUNE) Digital Pathology Scoring System. Study pathologists scored 12 descriptors in NEPTUNE renal biopsies from 242 patients with minimal change disease or FSGS, with duplicate readings to evaluate reproducibility. We performed consensus clustering of patients to identify unique electron microscopy profiles. For both individual descriptors and clusters, we used Cox regression models to assess associations with time from biopsy to proteinuria remission and time to a composite progression outcome (≥40% decline in eGFR, with eGFR<60 ml/min per 1.73 m2, or ESKD), and linear mixed models for longitudinal eGFR measures. RESULTS: Intrarater and interrater reproducibility was >0.60 for 12 out of 12 and seven out of 12 descriptors, respectively. Individual podocyte descriptors such as effacement and microvillous transformation were associated with complete remission, whereas endothelial cell and glomerular basement membrane abnormalities were associated with progression. We identified six descriptor-based clusters with distinct electron microscopy profiles and clinical outcomes. Patients in a cluster with more prominent foot process effacement and microvillous transformation had the highest rates of complete proteinuria remission, whereas patients in clusters with extensive loss of primary processes and endothelial cell damage had the highest rates of the composite progression outcome. CONCLUSIONS: Systematic analysis of electron microscopic findings reveals clusters of findings associated with either proteinuria remission or disease progression.

The motor protein Myo1c regulates transforming growth factor-ß-signaling and fibrosis in podocytes.

Transforming growth factor-ß (TGF-ß) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-ß signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-ß-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-ß-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-ß signaling through downregulation of canonical and non-canonical TGF-ß pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-ß signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-ß responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-ß-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.

Health-related quality of life in glomerular disease.

There is scant literature describing the effect of glomerular disease on health-related quality of life (HRQOL). The Cure Glomerulonephropathy study (CureGN) is an international longitudinal cohort study of children and adults with four primary glomerular diseases (minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and IgA nephropathy). HRQOL is systematically assessed using items from the Patient-Reported Outcomes Measurement Informative System (PROMIS). We assessed the relationship between HRQOL and demographic and clinical variables in 478 children and 1115 adults at the time of enrollment into CureGN. Domains measured by PROMIS items included global assessments of health, mobility, anxiety, fatigue, and sleep impairment, as well as a derived composite measure incorporating all measured domains. Multivariable models were created that explained 7 to 32% of variance in HRQOL. Patient-reported edema consistently had the strongest and most robust association with each measured domain of HRQOL in multivariable analysis (adjusted ß [95% CI] for composite PROMIS score in children, -5.2 [-7.1 to -3.4]; for composite PROMIS score in adults, -6.1 [-7.4 to -4.9]). Female sex, weight (particularly obesity), and estimated glomerular filtration rate were also associated with some, but not all, domains of HRQOL. Primary diagnosis, disease duration, and exposure to immunosuppression were not associated with HRQOL after adjustment. Sensitivity analyses and interaction testing demonstrated no significant association between disease duration or immunosuppression and any measured domain of HRQOL. Thus, patient-reported edema has a consistent negative association with HRQOL in patients with primary glomerular diseases, with substantially greater impact than other demographic and clinical variables.

CureGN Study Rationale, Design, and Methods: Establishing a Large Prospective Observational Study of Glomerular Disease.

RATIONALE & OBJECTIVES: Glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and immunoglobulin A (IgA) nephropathy, share clinical presentations, yet result from multiple biological mechanisms. Challenges to identifying underlying mechanisms, biomarkers, and new therapies include the rarity of each diagnosis and slow progression, often requiring decades to measure the effectiveness of interventions to prevent end-stage kidney disease (ESKD) or death. STUDY DESIGN: Multicenter prospective cohort study. SETTING & PARTICIPANTS: Cure Glomerulonephropathy (CureGN) will enroll 2,400 children and adults with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy (including IgA vasculitis) and a first diagnostic kidney biopsy within 5 years. Patients with ESKD and those with secondary causes of glomerular disease are excluded. EXPOSURES: Clinical data, including medical history, medications, family history, and patient-reported outcomes, are obtained, along with a digital archive of kidney biopsy images and blood and urine specimens at study visits aligned with clinical care 1 to 4 times per year. OUTCOMES: Patients are followed up for changes in estimated glomerular filtration rate, disease activity, ESKD, and death and for nonrenal complications of disease and treatment, including infection, malignancy, cardiovascular, and thromboembolic events. ANALYTICAL APPROACH: The study design supports multiple longitudinal analyses leveraging the diverse data domains of CureGN and its ancillary program. At 2,400 patients and an average of 2 years' initial follow-up, CureGN has 80% power to detect an HR of 1.4 to 1.9 for proteinuria remission and a mean difference of 2.1 to 3.0mL/min/1.73m2 in estimated glomerular filtration rate per year. LIMITATIONS: Current follow-up can only detect large differences in ESKD and death outcomes. CONCLUSIONS: Study infrastructure will support a broad range of scientific approaches to identify mechanistically distinct subgroups, identify accurate biomarkers of disease activity and progression, delineate disease-specific treatment targets, and inform future therapeutic trials. CureGN is expected to be among the largest prospective studies of children and adults with glomerular disease, with a broad goal to lessen disease burden and improve outcomes.

Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys.

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference-mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm-mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling to preserve glomerular function and podocyte viability in adult mice.

Slit diaphragm protein Neph1 and its signaling: a novel therapeutic target for protection of podocytes against glomerular injury.

Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.

Ret is critical for podocyte survival following glomerular injury in vivo.

Podocyte injury and loss directly cause proteinuria and the progression to glomerulosclerosis. Elucidation of the mechanisms of podocyte survival and recovery from injury is critical for designing strategies to prevent the progression of glomerular diseases. Glial cell line-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase, Ret, are upregulated in both nonimmune and immune-mediated in vitro and in vivo models of glomerular diseases. We investigated whether Ret, a known receptor tyrosine kinase critical for kidney morphogenesis and neuronal growth and development, is necessary for glomerular and podocyte development and survival in vivo. Since deletions of both GDNF and Ret result in embryonic lethality due to kidney agenesis, we examined the role of Ret in vivo by generating mice with a conditional deletion of Ret in podocytes (Ret(flox/flox); Nphs2-Cre). In contrast to the lack of any developmental and maintenance deficits, Ret(flox/flox); Nphs2-Cre mice showed a significantly enhanced susceptibility to adriamycin nephropathy, a rodent model of focal segmental glomerulosclerosis. Thus, these findings demonstrated that the Ret signaling pathway is important for podocyte survival and recovery from glomerular injury in vivo.

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease.

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 h. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multicenter observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria, and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared with other diagnoses. Thus these results do not support a pathological role for suPAR in FSGS.

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible.

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis.

Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.

Solution structure analysis of cytoplasmic domain of podocyte protein Neph1 using small/wide angle x-ray scattering (SWAXS).

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.