Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Goose nephritic astrovirus infection increases autophagy, destroys intercellular junctions in renal tubular epithelial cells, and damages podocytes in the kidneys of infected goslings.

Goose nephritic astrovirus (GNAstV) has recently been identified, which causes kidney swelling and visceral gout in goslings. However, the pathological changes in kidney tissue due to GNAstV infection have not yet been described. In the study, fifty goslings were orally infected with GNAstV, and fifty goslings received PBS as a control. Kidney tissue was collected at different days following infection (dpi) to assess the injury. GNAstV infection reduced body weight, increased the relative weight of the kidney, and increased serum uric acid and creatinine levels. GNAstV was found within renal epithelial cells, and the viral load in the kidney peaked at 7 dpi. Pale and swollen kidney tissue was observed in infected goslings, especially at 5 and 7 dpi. GNAstV infection caused degeneration and necrosis of renal epithelial cells, structural destruction of the brush border, glycogen deposition in the glomerular mesangium, increased fibrosis, and infiltration of inflammatory cells into the renal interstitium. Moreover, swollen mitochondria, broken mitochondrial ridges, autophagosomes, and autophagolysosomes were observed under ultrahistopathological examination. GNAstV infection increased levels of LC3B, ATG5, and Beclin 1, and decreased p62, and downregulated WT1 mRNA and upregulated desmin mRNA. At early stages, GNAstV infection decreased expression of intercellular junction-related genes, including ZO-1, occludin, claudin-10, and catenin-α2. In conclusion, GNAstV infection causes renal epithelial cell autophagy, destruction of brush border and intercellular junctions, podocyte damage, and increased fibrosis, ultimately resulting in damage to the kidney.

Collapsing Focal Segmental Glomerulosclerosis in Viral Infections.

Collapsing glomerulopathy represents a special variant of the proteinuric kidney disease focal segmental glomerulosclerosis (FSGS). Histologically, the collapsing form of FSGS (cFSGS) is characterized by segmental or global condensation and obliteration of glomerular capillaries, the appearance of hyperplastic and hypertrophic podocytes and severe tubulointerstitial damage. Clinically, cFSGS patients present with acute kidney injury, nephrotic-range proteinuria and are at a high risk of rapid progression to irreversible kidney failure. cFSGS can be attributed to numerous etiologies, namely, viral infections like HIV, cytomegalovirus, Epstein-Barr-Virus, and parvovirus B19 and also drugs and severe ischemia. Risk variants of the APOL1 gene, predominantly found in people of African descent, increase the risk of developing cFSGS. Patients infected with the new Corona-Virus SARS-CoV-2 display an increased rate of acute kidney injury (AKI) in severe cases of COVID-19. Besides hemodynamic instability, cytokine mediated injury and direct viral entry and infection of renal epithelial cells contributing to AKI, there are emerging reports of cFSGS associated with SARS-CoV-2 infection in patients of mainly African ethnicity. The pathogenesis of cFSGS is proposed to be linked with direct viral infection of podocytes, as described for HIV-associated glomerulopathy. Nevertheless, there is growing evidence that the systemic inflammatory cascade, activated in acute viral infections like COVID-19, is a major contributor to the impairment of basic cellular functions in podocytes. This mini review will summarize the current knowledge on cFSGS associated with viral infections with a special focus on the influence of systemic immune responses and potential mechanisms propagating the development of cFSGS.

COVID-19-Associated Glomerular Disease.

BACKGROUND: Studies have documented AKI with high-grade proteinuria in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In some patients, biopsies have revealed collapsing glomerulopathy, a distinct form of glomerular injury that has been associated with other viruses, including HIV. Previous patient reports have described patients of African ancestry who developed nephrotic-range proteinuria and AKI early in the course of disease. METHODS: In this patient series, we identified six patients with coronavirus disease 2019 (COVID-19), AKI, and nephrotic-range proteinuria. COVID-19 was diagnosed by a positive nasopharyngeal swab RT-PCR for SARS-CoV-2 infection. We examined biopsy specimens from one transplanted kidney and five native kidneys. Three of the six patients underwent genetic analysis of APOL1, the gene encoding the APOL1 protein, from DNA extracted from peripheral blood. In addition, we purified genomic DNA from paraffin-embedded tissue and performed APOL1 genotype analysis of one of the native biopsies and the donor kidney graft. RESULTS: All six patients were of recent African ancestry. They developed COVID-19-associated AKI with podocytopathy, collapsing glomerulopathy, or both. Patients exhibited generally mild respiratory symptoms, and no patient required ventilator support. Genetic testing performed in three patients confirmed high-risk APOL1 genotypes. One APOL1 high-risk patient developed collapsing glomerulopathy in the engrafted kidney, which was transplanted from a donor who carried a low-risk APOL1 genotype; this contradicts current models of APOL1-mediated kidney injury, and suggests that intrinsic renal expression of APOL1 may not be the driver of nephrotoxicity and specifically, of podocyte injury. CONCLUSIONS: Glomerular disease presenting as proteinuria with or without AKI is an important presentation of COVID-19 infection and may be associated with a high-risk APOL1 genotype.

Motility of human renal cells is disturbed by infection with pathogenic hantaviruses.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) caused by pathogenic hantaviruses in Europe and Asia is often characterized by acute kidney injury (AKI) with massive proteinuria. Renal filtration depends on the integrity of epithelial and endothelial monolayers in the tubular and glomerular apparatus. Tubular and glomerular cells represent target cells of hantavirus infection. However, the detailed mechanisms of renal impairment induced by hantaviruses are not well understood. METHODS: We analyzed the cellular consequences of hantavirus infection by measuring adhesion and migration capacity of human renal cells infected with Puumala (PUUV) or Hantaan (HTNV) virus. The impact of hantaviral nucleocapsid proteins (N proteins) on motility was examined by transfection of podocytes. RESULTS: Infection of kidney cells with hantavirus species PUUV and HTNV causes a significant reduction of migration capacity. The impaired motility depends on viral replication and transfection of podocytes with N protein of PUUV or HTNV reveals that the expression of N protein alone is sufficient to deteriorate podocyte function. The cellular effects are more pronounced for the more pathogenic HTNV than for PUUV that causes a milder form of HFRS. CONCLUSIONS: The direct impairment of migration capacity of renal cells by hantaviral N proteins may contribute substantially to proteinuria observed in the clinical picture of hantavirus infection.

Epigenetic regulation of RCAN1 expression in kidney disease and its role in podocyte injury.

Mounting evidence suggests that epigenetic modification is important in kidney disease pathogenesis. To determine whether epigenetic regulation is involved in HIV-induced kidney injury, we performed genome-wide methylation profiling and transcriptomic profiling of human primary podocytes infected with HIV-1. Comparison of DNA methylation and RNA sequencing profiles identified several genes that were hypomethylated with corresponding upregulated RNA expression in HIV-infected podocytes. Notably, we found only one hypermethylated gene with corresponding downregulated RNA expression, namely regulator of calcineurin 1 (RCAN1). Further, we found that RCAN1 RNA expression was suppressed in glomeruli in human diabetic nephropathy, IgA nephropathy, and lupus nephritis, and in mouse models of HIV-associated nephropathy and diabetic nephropathy. We confirmed that HIV infection or high glucose conditions suppressed RCAN1 expression in cultured podocytes. This suppression was alleviated upon pretreatment with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine, suggesting that RCAN1 expression is epigenetically suppressed in the context of HIV infection and diabetic conditions. Mechanistically, increased expression of RCAN1 decreased HIV- or high glucose-induced nuclear factor of activated T cells (NFAT) transcriptional activity. Increased RCAN1 expression also stabilized actin cytoskeleton organization, consistent with the inhibition of the calcineurin pathway. In vivo, knockout of RCAN1 aggravated albuminuria and podocyte injury in mice with Adriamycin-induced nephropathy. Our findings suggest that epigenetic suppression of RCAN1 aggravates podocyte injury in the setting of HIV infection and diabetic nephropathy.

Phosphorodiamidate morpholino targeting the 5' untranslated region of the ZIKV RNA inhibits virus replication.

BACKGROUND: Zika virus (ZIKV) infection has been associated with microcephaly in infants. Currently there is no treatment or vaccine. Here we explore the use of a morpholino oligonucleotide targeted to the 5' untranslated region (5'-UTR) of the ZIKV RNA to prevent ZIKV replication. METHODS: Morpholino DWK-1 inhibition of ZIKV replication in human glomerular podocytes was examined by qRT-PCR, reduction in ZIKV genome copy number, western blot analysis, immunofluorescence and proinflammatory cytokine gene expression. RESULTS: Podocytes pretreated with DWK-1 showed reduced levels of both viral mRNA and ZIKV E protein expression compared to controls. We observed suppression in proinflammatory gene expression for IFN-ß (interferon ß) RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1α (macrophage inflammatory protein-1α), TNF-α (tumor necrosis factor-α) and IL1-α (interleukin 1-α) in ZIKV-infected podocytes pretreated with DWK-1. CONCLUSIONS: Morpholino DWK-1 targeting the ZIKV 5'-UTR effectively inhibits ZIKV replication and suppresses ZIKV-induced proinflammatory gene expression.

Zika Virus Infection of the Human Glomerular Cells: Implications for Viral Reservoirs and Renal Pathogenesis.

Background: Zika virus (ZIKV) infection in the human renal compartment has not been reported. Several clinical reports have describe high-level persistent viral shedding in the urine of infected patients, but the associated mechanisms have not been explored until now. The current study examined cellular components of the glomerulus of the human kidney for ZIKV infectivity. Methods: I infected primary human podocytes, renal glomerular endothelial cells (GECs), and mesangial cells with ZIKV. Viral infectivity was analyzed by means of microscopy, immunofluorescence, real-time reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qRT-PCR), and the proinflammatory cytokines interleukin 1ß, interferon ß, and RANTES (regulated on activation of normal T cells expressed and secreted) were assessed using qRT-PCR. Results: I show that glomerular podocytes, renal GECs, and mesangial cells are permissive for ZIKV infection. ZIKV infectivity was confirmed in all 3 cell types by means of immunofluorescence staining, RT-PCR, and qRT-PCR, and qRT-PCR analysis revealed increased transcriptional induction of interleukin 1ß, interferon ß, and RANTES in ZIKV-infected podocytes at 72 hours, compared with renal GECs and mesangial cells. Conclusions: The findings of this study support the notion that the glomerulus may serve as an amplification reservoir for ZIKV in the renal compartment. The impact of ZIKV infection in the human renal compartment is unknown and will require further study.

Hedgehog pathway plays a vital role in HIV-induced epithelial-mesenchymal transition of podocyte.

HIV-associated nephropathy (HIVAN) is characterized by heavy proteinuria, rapidly progressive renal failure, and distinct morphological features in the kidney. HIV-induced epithelial-mesenchymal transition (EMT) is critically important for the progression of kidney injury. In this study, we tested the role of hedgehog pathway in the HIV-induced EMT and fibrosis of kidney. We used the Tg26 mice, the abundantly used HIVAN mouse model, to investigate the activation of hedgehog pathway by HIV. Western blotting and real time PCR results showed that renal tissue expression of hedgehog pathway related molecules, including hedgehog homologous (Shh, Ihh, Dhh), PTCH, and Gli1, were increased in HIVAN (Tg26) mice; while immunofluorescent staining displayed localization PTCH expression in podocytes. For in vitro studies, we used recombinant sonic hedgehog (Shh) and HIV for their expression by podocytes. Both the methods activated the hedgehog pathway, enhanced the expression of EMT markers, and decreased impermeability. Overexpression of Gli1 by human podocytes also augmented their expression of EMT markers. On the other hand, the blockade of hedgehog pathway with Gant 58, a specific blocker for Gli1-induced transcription, dramatically decreased HIV-induced podocyte EMT and permeability. These results indicate that hedgehog pathway plays an important role in HIV-induced podocyte injury. The present study provides mechanistical insight into a new target for therapeutic strategy.

Transmembrane TNF-α Facilitates HIV-1 Infection of Podocytes Cultured from Children with HIV-Associated Nephropathy.

Studies have shown that podocytes and renal tubular epithelial cells from patients with HIV-associated nephropathy (HIVAN) express HIV-1 transcripts, suggesting that productive infection of renal epithelial cells precipitates development of HIVAN. However, podocytes and renal tubular epithelial cells do not express CD4 receptors, and it is unclear how these cells become productively infected in vivo We investigated the mechanisms underlying the infection by HIV-1 of podocytes cultured from the urine of children with HIVAN. We observed low-level productive infection on exposure of these cells to primary cell-free HIV-1 supernatants. However, envelope-defective recombinant HIV-1 did not infect the renal epithelial cell lines. Moreover, treatment of podocytes to inhibit endocytic transport or dynamin activity or remove cell surface heparan sulfate proteoglycans reduced infection efficiency. Transfection of CD4- 293T cells with a cDNA expression library developed from a podocyte cell line derived from a child with HIVAN led to the identification of TNF-α as a possible mediator of HIV-1 infection. Overexpression of transmembrane TNF-α in cultured CD4- renal tubular epithelial cells, 293T cells, and HeLa cells enabled the infection of these cells; exposure to soluble TNF-α did not. Immunohistochemistry showed TNF-α expression in podocytes of renal sections from children with HIVAN. Furthermore, we found that TNF-α enhanced NF-κB activation and integration of HIV-1 into the podocyte DNA. Finally, inhibition of dynamin activity blocked TNF-α-mediated infection. These data establish a role for transmembrane TNF-α in facilitating the viral entry and integration of HIV-1 into the DNA of renal epithelial cells.

Impact of APOL1 polymorphism and IL-1ß priming in the entry and persistence of HIV-1 in human podocytes.

BACKGROUND: Patients of African ancestry with untreated HIV-1 infection and carrying the G1 or G2 kidney disease risk variants (Vs) at the APOL1 gene have a tenfold higher risk of developing HIV-associated nephropathy (HIVAN) compared to those with the non-risk wild type (WT) G0 variant. However, the mechanistic contribution of the APOL1 allelic state to kidney injury in HIV-1 infection remains to be elucidated. RESULTS: Non-risk WT APOL1 is associated with lower intracellular levels of HIV-1 in conditionally immortalized human podocytes, while the over expression of G1 or G2 risk Vs significantly increases viral accumulation. The priming of podocytes with exogenous IL-1ß facilitates HIV-1 entry, via the up-regulation of DC-SIGN. The over expression of APOL1 G1 and G2 risk Vs in combination with an increase in IL-1ß levels causes a greater increase in viral concentration than either condition alone. In turn, HIV-1 and exogenous IL-1ß together induce a de novo secretion of endogenous IL-1ß and an increase of APOL1 gene expression. CONCLUSIONS: Our findings indicate that the presence of risk Vs of APOL1 is permissive of HIV-1 persistence in human podocytes in synergy with IL-1ß, a cytokine that characterizes the inflammatory milieu of acute and chronic phases of HIV-1 infection. The elucidation of these molecular mechanisms explains, at least in part, the higher frequency of HIVAN in populations carrying the risk polymorphic genetic variant of APOL1 gene.

Infection-Related Focal Segmental Glomerulosclerosis in Children.

Focal segmental glomerulosclerosis (FSGS) is the most common cause of steroid resistant nephrotic syndrome in children. It describes a unique histological picture of glomerular damage resulting from several causes. In the majority of patients the causing agent is still unknown, but in some cases viral association is evident. In adults, the most established FSGS causing virus is the human immune-deficiency virus, which is related to a collapsing variant of FSGS. Nevertheless, other viruses are also suspected for causing a collapsing or noncollapsing variant, for example, hepatitis B virus, parvovirus B19, and Cytomegalovirus. Although the systemic infection mechanism is different for these viruses, there are similarities in the pathomechanism for the induction of FSGS. As the podocyte is the key structure in the pathogenesis of FSGS, a direct infection of these cells or immediate damage through the virus or viral components has to be considered. Although viral infections are a very rare cause for FSGS in children, the treating pediatric nephrologist has to be aware of a possible underlying infection, as this has a relevant impact on therapy and prognosis.

HIV Promotes NLRP3 Inflammasome Complex Activation in Murine HIV-Associated Nephropathy.

Dysregulated growth and loss of podocytes are important features of HIV-associated nephropathy. Recently, HIV was reported to induce a new type of programed cell death, pyroptosis, in T lymphocytes through induction of Nod-like receptor protein 3 (NLRP3) inflammasome complexes. We evaluated the role of HIV in podocyte NLRP3 inflammasome formation both in vivo and in vitro. Renal cortical sections of HIV-transgenic mice (Tg26) displayed increased expression of NLRP3, ASC (a CARD protein), caspase-1, and IL-1ß proteins, confirming NLRP3 inflammasome complex formation in podocytes of Tg26 mice. Renal tissues of Tg26 mice also displayed enhanced mRNA levels and protein expressions of inflammasome markers (NLRP3, ASC, and caspase-1, and IL-1ß). Serum of Tg26 mice also showed elevated concentrations of IL-1ß cytokine compared with FVBN mice. HIV induced pyroptosis in a dose- and time-dependent manner within podocytes, a phenotype of inflammasome activation. Caspase-1 inhibitor not only attenuated podocyte expression of caspase-1 and IL-1ß but also provided protection against pyroptosis, suggesting that HIV-induced podocyte injury was mediated by caspase-1 activation. Interestingly, HIV-induced podocyte pyroptosis could be partially inhibited by Tempol (a superoxide dismutase-mimetic agent) and by glyburide (an inhibitor of potassium efflux). These findings suggest that generation of reactive oxygen species and potassium efflux contribute to HIV-induced pyroptosis and NLRP3 inflammasome activation in podocytes.

Epigenetic Modulation of Human Podocyte Vitamin D Receptor in HIV Milieu.

HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression.

Advances in the pathogenesis of HIV-associated kidney diseases.

Despite improved outcomes among persons living with HIV who are treated with antiretroviral therapy, they remain at increased risk for acute and chronic kidney diseases. Moreover, since HIV can infect renal epithelial cells, the kidney might serve as a viral reservoir that would need to be eradicated when attempting to achieve full virologic cure. In recent years, much progress has been made in elucidating the mechanism by which HIV infects renal epithelial cells and the viral and host factors that promote development of kidney disease. Polymorphisms in APOL1 confer markedly increased risk of HIV-associated nephropathy; however, the mechanism by which ApoL1 variants may promote kidney disease remains unclear. HIV-positive persons are at increased risk of acute kidney injury, which may be a result of a high burden of subclinical kidney disease and/or viral factors and frequent exposure to nephrotoxins. Despite the beneficial effect of antiretroviral therapy in preventing and treating HIVAN, and possibly other forms of kidney disease in persons living with HIV, some of these medications, including tenofovir, indinavir, and atazanavir can induce acute and/or chronic kidney injury via mitochondrial toxicity or intratubular crystallization. Further research is needed to better understand factors that contribute to acute and chronic kidney injury in HIV-positive patients and to develop more effective strategies to prevent and treat kidney disease in this vulnerable population.

Visualization of cytoskeletal dynamics in podocytes using adenoviral vectors.

Glomerular visceral epithelial cells (podocytes) play a key role in maintaining selective protein filtration in the kidney. Podocytes have a complex cell shape characterized by the presence of numerous actin-rich processes, which cover the surface of glomerular capillaries and are connected by specialized cell-cell adhesion complexes (slit diaphragms). Human genetic studies and experiments in knockout mouse models show that actin filaments and actin-associated proteins are indispensable for the maintenance of podocyte shape, slit diaphragm integrity, and normal glomerular filtration. The ability to examine cytoskeletal protein organization and dynamics in podocytes and to test the effects of disease-associated mutations on protein localization provides valuable information for researchers aiming to dissect the molecular mechanisms of podocyte dysfunction. We describe how adenovirus-mediated transduction of cultured podocytes with DNA constructs can be used to reliably introduce fluorescently tagged cytoskeletal markers for live cell imaging with high efficiency and low toxicity. This technique can be used to study the dynamic reorganization of the podocyte cytoskeleton and to test the effects of novel mutations on podocyte cytoskeletal dynamics.

The kidney as a reservoir for HIV-1 after renal transplantation.

Since the recent publication of data showing favorable outcomes for patients with HIV-1 and ESRD, kidney transplantation has become a therapeutic option in this population. However, reports have documented unexplained reduced allograft survival in these patients. We hypothesized that the unrecognized infection of the transplanted kidney by HIV-1 can compromise long-term allograft function. Using electron microscopy and molecular biology, we examined protocol renal transplant biopsies from 19 recipients with HIV-1 who did not have detectable levels of plasma HIV-1 RNA at transplantation. We found that HIV-1 infected the kidney allograft in 68% of these patients. Notably, HIV-1 infection was detected in either podocytes predominately (38% of recipients) or tubular cells only (62% of recipients). Podocyte infection associated with podocyte apoptosis and loss of differentiation markers as well as a faster decline in allograft function compared with tubular cell infection. In allografts with tubular cell infection, epithelial cells of the proximal convoluted tubules frequently contained abnormal mitochondria, and both patients who developed features of subclinical acute cellular rejection had allografts with tubular cell infection. Finally, we provide a novel noninvasive test for determining HIV-1 infection of the kidney allograft by measuring HIV-1 DNA and RNA levels in patients' urine. In conclusion, HIV-1 can infect kidney allografts after transplantation despite undetectable viremia, and this infection might influence graft outcome.

HIV compromises integrity of the podocyte actin cytoskeleton through downregulation of the vitamin D receptor.

Alterations in the podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on the podocyte actin cytoskeleton and the mechanism involved. We hypothesized that HIV may be compromising the actin cytoskeleton via downregulation of the vitamin D receptor (VDR) of conditionally immortalized differentiated human podocytes (CIDHPs). HIV-transduced podocytes (HIV/CIDHPs) not only displayed downregulation of VDR but also showed activation of the renin-angiotensin system (RAS) in the form of enhanced expression of renin and increased production of ANG II. Moreover, CIDHPs lacking VDR displayed enhanced ANG II production, and treatment of HIV/CIDHPs with EB1089 (vitamin D3; VD) attenuated ANG II production. HIV/CIDHPs as well as ANG II-treated CIDHPs exhibited enhanced expression of cathepsin (CTS) L. Additionally, losartan (an ANG II type I receptor blocker) inhibited both HIV- and ANG II-induced podocyte cathepsin L expression. Furthermore, VD downregulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV- and ANG II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization; on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphological evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression, and HIV/CIDHPs expressing CTSL exhibited downregulation of dynamin. These findings indicate that HIV-induced downregulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised the actin cytoskeleton.