Autoantibodies Targeting Nephrin in Podocytopathies.

BACKGROUND: Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. METHODS: We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. RESULTS: The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. CONCLUSIONS: In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).

Protective role for kidney TREM2high macrophages in obesity- and diabetes-induced kidney injury.

Diabetic kidney disease (DKD), the most common cause of kidney failure, is a frequent complication of diabetes and obesity, and yet to date, treatments to halt its progression are lacking. We analyze kidney single-cell transcriptomic profiles from DKD patients and two DKD mouse models at multiple time points along disease progression-high-fat diet (HFD)-fed mice aged to 90-100 weeks and BTBR ob/ob mice (a genetic model)-and report an expanding population of macrophages with high expression of triggering receptor expressed on myeloid cells 2 (TREM2) in HFD-fed mice. TREM2high macrophages are enriched in obese and diabetic patients, in contrast to hypertensive patients or healthy controls in an independent validation cohort. Trem2 knockout mice on an HFD have worsening kidney filter damage and increased tubular epithelial cell injury, all signs of worsening DKD. Together, our studies suggest that strategies to enhance kidney TREM2high macrophages may provide therapeutic benefits for DKD.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Pathologic-genomic correlation identified a novel variant in FN1 and established the diagnosis of recurrent fibronectin glomerulopathy in the kidney allograft.

Fibronectin glomerulopathy is a rare inherited kidney disease, characterized by abnormal accumulation of fibronectin in the glomeruli. We report an exceptional case of recurrent fibronectin glomerulopathy first diagnosed in the kidney allograft. The presence of IgA staining in the native kidney biopsy and the reported family history of IgA nephropathy had led to initial pretransplant diagnosis of IgA nephropathy. At 4.5 years posttransplant, the patient presented with kidney insufficiency and minimal proteinuria. The allograft biopsy revealed glomerular deposits with very weak staining for immunoglobulins and vague filamentous material. Immunostaining for fibronectin was positive, and genetic studies showed a variant of unknown significance in the fibronectin 1 gene. Proteomic analyses of the glomeruli in the native kidney biopsy demonstrated large amount of fibronectin with abundant accumulation of the peptide synthesized by the detected variant. These findings established the diagnosis of recurrent fibronectin glomerulopathy secondary to a novel variant in the fibronectin 1 gene. This report sheds light on recurrent fibronectin glomerulopathy in the allograft, highlights the diagnostic pitfalls of the disease, and underscores the importance of pathologic-genomic correlation to establish the correct diagnosis.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Uncovering a novel role of focal adhesion and interferon-gamma in cellular rejection of kidney allografts at single cell resolution.

Background: Kidney transplant recipients are currently treated with nonspecific immunosuppressants that cause severe systemic side effects. Current immunosuppressants were developed based on their effect on T-cell activation rather than the underlying mechanisms driving alloimmune responses. Thus, understanding the role of the intragraft microenvironment will help us identify more directed therapies with lower side effects. Methods: To understand the role of the alloimmune response and the intragraft microenvironment in cellular rejection progression, we conducted a Single nucleus RNA sequencing (snRNA-seq) on one human non-rejecting kidney allograft sample, one borderline sample, and T-cell mediated rejection (TCMR) sample (Banff IIa). We studied the differential gene expression and enriched pathways in different conditions, in addition to ligand-receptor (L-R) interactions. Results: Pathway analysis of T-cells in borderline sample showed enrichment for allograft rejection pathway, suggesting that the borderline sample reflects an early rejection. Hence, this allows for studying the early stages of cellular rejection. Moreover, we showed that focal adhesion (FA), IFNg pathways, and endomucin (EMCN) were significantly upregulated in endothelial cell clusters (ECs) of borderline compared to ECs TCMR. Furthermore, we found that pericytes in TCMR seem to favor endothelial permeability compared to borderline. Similarly, T-cells interaction with ECs in borderline differs from TCMR by involving DAMPS-TLRs interactions. Conclusion: Our data revealed novel roles of T-cells, ECs, and pericytes in cellular rejection progression, providing new clues on the pathophysiology of allograft rejection.

Lupus Cardiomyopathy and Nephritis Associated With Adalimumab and Cytomegalovirus Infection in a Patient With Seronegative Rheumatoid Arthritis: A Case of Rhupus Syndrome.

Tumor necrosis factor-alpha (TNF-α) inhibitors are associated with lupus-like disease, known as anti-TNF-α-induced lupus (ATIL). Cytomegalovirus (CMV) was reported to exacerbate lupus in the literature. To date, systemic lupus erythematosus (SLE) triggered by adalimumab in the setting of CMV infection has never been described. We present an unusual case of a 38-year-old female with a past medical history of seronegative rheumatoid arthritis (SnRA) who developed SLE associated with the use of adalimumab and CMV infection. She had severe SLE features including lupus nephritis and cardiomyopathy. The medication was discontinued. She was initiated on pulse steroid therapy and discharged with an aggressive regimen for SLE, including prednisone, mycophenolate mofetil, and hydroxychloroquine. She remained on the medications until a year later upon follow-up. ATIL from adalimumab usually manifests only mild symptoms of SLE such as arthralgia, myalgia, and pleurisy. Nephritis is very rare, and cardiomyopathy is unprecedented. Concomitant CMV infection might contribute to disease severity. Patients with SnRA may have an increased risk of developing SLE later when exposed to such medications and infection.

The role of HLA antigens in recurrent primary focal segmental glomerulosclerosis.

Primary focal segmental glomerulosclerosis (FSGS), typically characterized by diffuse podocyte foot process effacement and nephrotic syndrome (diffuse podocytopathy), is generally attributed to a circulating permeability factor. Primary FSGS can recur after transplantation where it manifests as diffuse foot process effacement in the early stages, with subsequent evolution of segmental sclerotic lesions. Previous published literature has been limited by the lack of stringent selection criteria to define primary FSGS. Although immunogenetic factors play an important role in many glomerular diseases, their role in recurrent primary FSGS post-transplantation has not been systematically investigated. To address this, we retrospectively studied a multicenter cohort of 74 kidney allograft recipients with end stage kidney disease due to primary FSGS, confirmed by clinical and histologic parameters. After adjusting for race/ethnicity, there was a numeric higher frequency of HLA-A30 antigen in primary FSGS (19%) compared to each of 22,490 healthy controls (7%, adjusted OR=2.0, P=0.04) and 296 deceased kidney donors (10%, OR=2.1, P=0.03). Within the group of transplant patients with end stage kidney disease due to primary FSGS, donor HLA-A30 was associated with recurrent disease (OR=9.1, P=0.02). Multivariable time-to-event analyses revealed that recipients who self-identified as Black people had lower risk of recurrent disease, probably reflecting enrichment of these recipients with APOL1 high-risk genotypes. These findings suggest a role for recipient and donor immunogenetic makeup in recurrent primary FSGS post-transplantation. Further larger studies in well-defined cohorts of primary FSGS that include high-resolution HLA typing and genome-wide association are necessary to refine these hereditary signals.

Clonal dynamics of alloreactive T cells in kidney allograft rejection after anti-PD-1 therapy.

Kidney transplant recipients are at particular risk for developing tumors, many of which are now routinely treated with immune checkpoint inhibitors (ICIs); however, ICI therapy can precipitate transplant rejection. Here, we use TCR sequencing to identify and track alloreactive T cells in a patient with melanoma who experienced kidney transplant rejection following PD-1 inhibition. The treatment was associated with a sharp increase in circulating alloreactive CD8+ T cell clones, which display a unique transcriptomic signature and were also detected in the rejected kidney but not at tumor sites. Longitudinal and cross-tissue TCR analyses indicate unintended expansion of alloreactive CD8+ T cells induced by ICI therapy for cancer, coinciding with ICI-associated organ rejection.

Expression of Class II Human Leukocyte Antigens on Human Endothelial Cells Shows High Interindividual and Intersubclass Heterogeneity.

SIGNIFICANCE STATEMENT: Donor-specific antibodies against class II HLA are a major cause of chronic kidney graft rejection. Nonetheless, some patients presenting with these antibodies remain in stable histological and clinical condition. This study describes the use of endothelial colony-forming cell lines to test the hypothesis of the heterogeneous expression of HLA molecules on endothelial cells in humans. Flow cytometry and immunofluorescence staining revealed substantial interindividual and interlocus variability, with HLA-DQ the most variable. Our data suggest that the expression of HLA class II is predicted by locus. The measurement of endothelial expression of HLA class II in the graft could present a novel paradigm in the evaluation of the alloimmune risk in transplantation and certain diseases. BACKGROUND: HLA antigens are important targets of alloantibodies and allospecific T cells involved in graft rejection. Compared with research into understanding alloantibody development, little is known about the variability in expression of their ligands on endothelial cells. We hypothesized individual variability in the expression of HLA molecules. METHODS: We generated endothelial colony forming cell lines from human peripheral blood mononuclear cells ( n =39). Flow cytometry and immunofluorescence staining were used to analyze the cells, and we assessed the relationship between HLA-DQ expression and genotype. Two cohorts of kidney transplant recipients were analyzed to correlate HLA-DQ mismatches with the extent of intragraft microvascular injury. RESULTS: Large variability was observed in the expression of HLA class II antigens, not only between individuals but also between subclasses. In particular, HLA-DQ antigens had a low and heterogeneous expression, ranging from 0% to 85% positive cells. On a within-patient basis, this expression was consistent between endothelial cell colonies and antigen-presenting cells. HLA-DQ5 and -DQ6 were associated with higher levels of expression, whereas HLA-DQ7, -DQ8, and -DQ9 with lower. HLA-DQ5 mismatches among kidney transplant recipients were associated with significant increase in graft microvascular. CONCLUSION: These data challenge the current paradigm that HLA antigens, in particular HLA class II, are a single genetic and post-translational entity. Understanding and assessing the variability in the expression of HLA antigens could have clinical monitoring and treatment applications in transplantation, autoimmune diseases, and oncology.

The pathologic spectrum of adenovirus nephritis in the kidney allograft.

Adenovirus nephritis (ADVN) is a rare and understudied complication of kidney transplantation. Unlike BK virus nephropathy (BKVN), our knowledge of clinicopathologic manifestations of ADVN remains rudimentary and essentially limited to case reports. To expand on this, we retrospectively studied 11 kidney transplant recipients with ADVN and compared their allograft biopsies to 33 kidney transplant recipients with BKVN using conventional microscopy and the 770 gene Nanostring Banff Human Organ Transplant Profiling Panel. Patients with ADVN had a median age of 44 years, were predominantly male, and developed ADVN at a median of 31 months post-transplantation. Eight patients presented with fever and ten had hematuria. The most common histologic manifestations included granulomas (82%), tubulocentric inflammation (73%), and tubular degenerative changes consistent with acute tubular necrosis (73%). During a median follow-up of 55 months after biopsy, three patients developed allograft failure from subsequent acute rejection. All seven patients with available follow-up PCR showed resolution of viremia at a median of 30 days after diagnosis. Compared to BKVN, ADVN demonstrated more granulomas and less tubulointerstitial scarring. On follow-up, patients with ADVN had more rapid clearance of viral DNA from plasma. Transcriptomic analyses showed that ADVN had increased expression of several pro-inflammatory transcriptomes, mainly related to innate immunity, was associated with increased expression of transcripts with inhibitory effects on inflammatory response and showed higher enrichment with neutrophils, which can cause aggressive but short-lasting damage. Thus, we demonstrate that, despite its association with aggressive neutrophil-rich inflammation, ADVN does not often lead to allograft failure. Hence, preventing subsequent acute rejection following resolution of ADVN may improve allograft survival.

JAK inhibitor blocks COVID-19 cytokine-induced JAK/STAT/APOL1 signaling in glomerular cells and podocytopathy in human kidney organoids.

COVID-19 infection causes collapse of glomerular capillaries and loss of podocytes, culminating in a severe kidney disease called COVID-19-associated nephropathy (COVAN). The underlying mechanism of COVAN is unknown. We hypothesized that cytokines induced by COVID-19 trigger expression of pathogenic APOL1 via JAK/STAT signaling, resulting in podocyte loss and COVAN phenotype. Here, based on 9 biopsy-proven COVAN cases, we demonstrated for the first time, to the best of our knowledge, that APOL1 protein was abundantly expressed in podocytes and glomerular endothelial cells (GECs) of COVAN kidneys but not in controls. Moreover, a majority of patients with COVAN carried 2 APOL1 risk alleles. We show that recombinant cytokines induced by SARS-CoV-2 acted synergistically to drive APOL1 expression through the JAK/STAT pathway in primary human podocytes, GECs, and kidney micro-organoids derived from a carrier of 2 APOL1 risk alleles, but expression was blocked by a JAK1/2 inhibitor, baricitinib. We demonstrate that cytokine-induced JAK/STAT/APOL1 signaling reduced the viability of kidney organoid podocytes but was rescued by baricitinib. Together, our results support the conclusion that COVID-19-induced cytokines are sufficient to drive COVAN-associated podocytopathy via JAK/STAT/APOL1 signaling and that JAK inhibitors could block this pathogenic process. These findings suggest JAK inhibitors may have therapeutic benefits for managing cytokine-induced, APOL1-mediated podocytopathy.

High-resolution Slide-seqV2 spatial transcriptomics enables discovery of disease-specific cell neighborhoods and pathways.

High-resolution spatial transcriptomics enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remains unexplored. We applied Slide-seqV2 to mouse and human kidneys, in healthy and distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in tissue from nine distinct human kidneys, which revealed a cell neighborhood centered around a population of LYVE1+ macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially restricted kidney filter and blood-flow-regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially restricted subpopulation of epithelial cells, we discovered perturbations in 77 genes associated with the unfolded protein response. Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.

BACKGROUND: Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell-targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. METHODS: We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. RESULTS: In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. CONCLUSIONS: Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

A glomerular transcriptomic landscape of apolipoprotein L1 in Black patients with focal segmental glomerulosclerosis.

Apolipoprotein L1 (APOL1)-associated focal segmental glomerulosclerosis (FSGS) is the dominant form of FSGS in Black individuals. There are no targeted therapies for this condition, in part because the molecular mechanisms underlying APOL1's pathogenic contribution to FSGS are incompletely understood. Studying the transcriptomic landscape of APOL1 FSGS in patient kidneys is an important way to discover genes and molecular behaviors that are unique or most relevant to the human disease. With the hypothesis that the pathology driven by the high-risk APOL1 genotype is reflected in alteration of gene expression across the glomerular transcriptome, we compared expression and co-expression profiles of 15,703 genes in 16 Black patients with FSGS at high-risk vs 14 Black patients with a low-risk APOL1 genotype. Expression data from APOL1-inducible HEK293 cells and normal human glomeruli were used to pursue genes and molecular pathways uncovered in these studies. We discovered increased expression of APOL1 and nine other significant differentially expressed genes in high-risk patients. This included stanniocalcin, which has a role in mitochondrial and calcium-related processes along with differential correlations between high- and low-risk APOL1 and metabolism pathway genes. There were similar correlations with extracellular matrix- and immune-related genes, but significant loss of co-expression of mitochondrial genes in high-risk FSGS, and an NF-κB-down regulating gene, NKIRAS1, as the most significant hub gene with strong differential correlations with NDUF family (mitochondrial respiratory genes) and immune-related (JAK-STAT) genes. Thus, differences in mitochondrial gene regulation appear to underlie many differences observed between high- and low-risk Black patients with FSGS.

TRPC5 Channel Inhibition Protects Podocytes in Puromycin-Aminonucleoside Induced Nephrosis Models.

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.