Exploiting the neonatal crystallizable fragment receptor to treat kidney disease.

The neonatal Fc receptor (FcRn) was initially discovered as the receptor that allowed passive immunity in newborns by transporting maternal IgG through the placenta and enterocytes. Since its initial discovery, FcRn has been found to exist throughout all stages of life and in many different cell types. Beyond passive immunity, FcRn is necessary for intrinsic albumin and IgG recycling and is important for antigen processing and presentation. Given its multiple important roles, FcRn has been utilized in many disease treatments including a new class of agents that were developed to inhibit FcRn for treatment of a variety of autoimmune diseases. Certain cell populations within the kidney also express high levels of this receptor. Specifically, podocytes, proximal tubule epithelial cells, and vascular endothelial cells have been found to utilize FcRn. In this review, we summarize what is known about FcRn and its function within the kidney. We also discuss how FcRn has been used for therapeutic benefit, including how newer FcRn inhibiting agents are being used to treat autoimmune diseases. Lastly, we will discuss what renal diseases may respond to FcRn inhibitors and how further work studying FcRn within the kidney may lead to therapies for kidney diseases.

Identification of Four Mouse FcRn Splice Variants and FcRn-Specific Vesicles.

Research into the neonatal Fc receptor (FcRn) has increased dramatically ever since Simister and Mostov first purified a rat version of the receptor. Over the years, FcRn has been shown to function not only as a receptor that transfers immunity from mother to fetus but also performs an array of different functions that include transport and recycling of immunoglobulins and albumin in the adult. Due to its important cellular roles, several clinical trials have been designed to either inhibit/enhance FcRn function or develop of non-invasive therapeutic delivery system such as fusion of drugs to IgG Fc or albumin to enhance delivery inside the cells. Here, we report the accidental identification of several FcRn alternatively spliced variants in both mouse and human cells. The four new mouse splice variants are capable of binding immunoglobulins' Fc and Fab portions. In addition, we have identified FcRn-specific vesicles in which immunoglobulins and albumin can be stored and that are involved in the endosomal-lysosomal system. The complexity of FcRn functions offers significant potential to design and develop novel and targeted therapeutics.

Knockout of the neonatal Fc receptor alters immune complex trafficking and lysosomal function in cultured podocytes.

Podocytes are key to preventing the filtration of serum proteins into the urine. Recent evidence also suggests that in immune mediated kidney diseases, podocytes are the targets of immune complexes (ICs). The mechanisms whereby podocytes handle and respond to ICs remain unknown. The neonatal Fc receptor (FcRn) is involved in IgG handling in podocytes and is also required in dendritic cells to traffic ICs to the lysosome for proteolytic degradation of antigen and presentation on MHC II. Here we examine the role of FcRn in handling ICs in podocytes. We show that knockout of FcRn in podocytes results in decreased trafficking of ICs to the lysosome and increases IC trafficking to recycling endosomes. FcRn KO also alters lysosomal distribution, decreases lysosomal surface area and decreases cathepsin B expression and activity. We demonstrate that signaling pathways in cultured podocytes differ after treatment with IgG alone versus ICs and that podocyte proliferation in both WT and KO podocytes is suppressed by IC treatment. Our findings suggest that podocytes respond differentially to IgG versus ICs and that FcRn modifies the lysosomal response to ICs. Elucidating the mechanisms underlying podocyte handling of ICs may provide novel pathways to modulate immune mediated kidney disease progression.

Factor H related proteins modulate complement activation on kidney cells.

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney.

Minimal Change Disease Is Associated With Endothelial Glycocalyx Degradation and Endothelial Activation.

Introduction: Minimal change disease (MCD) is considered a podocyte disorder triggered by unknown circulating factors. Here, we hypothesized that the endothelial cell (EC) is also involved in MCD. Methods: We studied 45 children with idiopathic nephrotic syndrome (44 had steroid sensitive nephrotic syndrome [SSNS], and 12 had biopsy-proven MCD), 21 adults with MCD, and 38 healthy controls (30 children, 8 adults). In circulation, we measured products of endothelial glycocalyx (EG) degradation (syndecan-1, heparan sulfate [HS] fragments), HS proteoglycan cleaving enzymes (matrix metalloprotease-2 [MMP-2], heparanase activity), and markers of endothelial activation (von Willebrand factor [vWF], thrombomodulin) by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. In human kidney tissue, we assessed glomerular EC (GEnC) activation by immunofluorescence of caveolin-1 (n = 11 MCD, n = 5 controls). In vitro, we cultured immortalized human GEnC with sera from control subjects and patients with MCD/SSNS sera in relapse (n = 5 per group) and performed Western blotting of thrombomodulin of cell lysates as surrogate marker of endothelial activation. Results: In circulation, median concentrations of all endothelial markers were higher in patients with active disease compared with controls and remained high in some patients during remission. In the MCD glomerulus, caveolin-1 expression was higher, in an endothelial-specific pattern, compared with controls. In cultured human GEnC, sera from children with MCD/SSNS in relapse increased thrombomodulin expression compared with control sera. Conclusion: Our data show that alterations involving the systemic and glomerular endothelium are nearly universal in patients with MCD and SSNS, and that GEnC can be directly activated by circulating factors present in the MCD/SSNS sera during relapse.

Targeted Disruption of a Proximal Tubule-Specific TMEM174 Gene in Mice Causes Hyperphosphatemia and Vascular Calcification.

BACKGROUND: The proximal tubules play a critical role in phosphate (Pi) homeostasis by reabsorbing Pi via sodium-dependent Pi cotransporters. NPT2A is a major proximal-specific Pi cotransporter, whose expression is regulated by circulating hormones, such as parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). In this study, we aimed to find a novel regulator in Pi homeostasis. METHODS: Using RNA-seq and RT-qPCR analysis, we identified proximal tubule cell-enriched genes. We next used RNAi screening of the identified proximal tubular cell-enriched genes to identify a novel proximal tubule-specific gene that contributes to FGF23- and PTH-mediated inhibition of Pi uptake and NPT2 reduction. We created mice lacking this novel regulator of Pi homeostasis to examine whether the novel regulator contributes to Pi homeostasis in vivo. RESULTS: We identified 54 kidney-enriched genes, 19 of which are expressed in renal primary proximal tubule cells. One of the proximal tubule-specific genes, TMEM174, interacted with NPT2A, and its knockdown blocked the reduction of NPT2A protein by FGF23 and PTH treatments in human and opossum proximal tubule cells. TMEM174 KO mice had significantly increased levels of serum Pi, FGF23, and PTH, resulting in vascular calcification. CONCLUSIONS: TMEM174 is a novel regulator of Pi homeostasis that interacts with NPT2A.

Cefazolin Plus Ceftazidime versus Cefazolin Monotherapy in the Treatment of Culture-Negative Peritonitis: A Retrospective Cohort Study.

BACKGROUND: Based on current ISPD guidelines, it is unclear as to whether ceftazidime should be discontinued in subsequent management of culture-negative peritonitis if it is used as empirical gram-negative coverage. Herein, we aim to compare the clinical outcomes of cefazolin plus ceftazidime versus cefazolin alone. METHODS: This was a retrospective cohort study. Adult peritoneal dialysis (PD) patients who were diagnosed with culture-negative peritonitis between 2014 and 2020 were included. Patients were categorized into two groups according to treatment regimen. Primary response rate, peritonitis relapse rate, and time to primary response were compared. Factors that predicted primary response were determined using Cox regression analysis. RESULTS: A total of 58 patients were included in the study. Of these, 42 received cefazolin plus ceftazidime and 16 received cefazolin monotherapy. Overall, the mean age was 65.7±10.4 years. Most of the patients (81.3%) were prescribed continuous ambulatory peritoneal dialysis. Initial effluent WBC was 4211±10357 in the combination group and 3833±6931 cell/mm3 in the monotherapy group (p=0.89). There was no significant difference in primary response at day 5 between the two groups (95.2% in the combination group vs93.7% in the monotherapy group, p=0.82). However, cumulative probability of primary response by the Kaplan-Meier analysis in the combination group was higher than in the monotherapy group (p=0.02). Adjusted HR of serum potassium level to predict a primary response was 1.83 according to multivariate analysis (p=0.03). There was no difference between the two groups in terms of peritonitis relapse or catheter removal. CONCLUSION: This is the first study to compare clinical outcomes between cefazolin plus ceftazidime versus cefazolin monotherapy in culture-negative peritonitis. Our results suggest that if peritonitis is resolving at day 3, discontinuation of ceftazidime could yield favorable treatment outcomes and might be appropriate for subsequent management. However, the risk of not having gram-negative coverage should be considered.

Surgical procedures suppress autophagic flux in the kidney.

Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.

Generating a Podocyte-Specific Neonatal F Receptor (FcRn) Knockout Mouse.

Proteinuria is a widely used marker of renal disease and is strongly associated with renal and cardiovascular outcomes. The molecular mechanisms underlying filtration of serum proteins through the glomerular filtration barrier (GFB) remain to be determined. Since the GFB is a complex structure, studies of albumin or IgG trafficking in cultured cells in vitro may not fully recapitulate these processes in vivo. In other epithelial cells including renal proximal tubular cells, the neonatal Fc receptor (FcRn) is required to divert albumin and IgG from the degradative pathway which allows these proteins to be recycled or transcytosed. To examine the role of podocyte FcRn in albumin and IgG trafficking in vivo, we detail the creation of a podocyte-specific FcRn knockout mouse and describe methods for examining intraglomerular detection of albumin and IgG in these mice.

Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis.

Podocytes have been proposed to be antigen presenting cells (APCs). In traditional APCs, the neonatal Fc receptor (FcRn) is required for antigen presentation and global knockout of FcRn protects against glomerulonephritis. Since podocytes express FcRn, we sought to determine whether the absence of podocyte FcRn ameliorates immune-mediated disease. We examined MHCII and costimulatory markers expression in cultured wild type (WT) and FcRn knockout (KO) podocytes. Interferon gamma (IFNγ) induced MHCII expression in both WT and KO podocytes but did not change CD80 expression. Neither WT nor KO expressed CD86 or inducible costimulatory ligand (ICOSL) at baseline or with IFNγ. Using an antigen presentation assay, WT podocytes but not KO treated with immune complexes induced a modest increase in IL-2. Induction of the anti-glomerular basement membrane (anti-GBM) model resulted in a significant decrease in glomerular crescents in podocyte-specific FcRn knockout mouse (podFcRn KO) versus controls but the overall percentage of crescents was low. To examine the effects of the podocyte-specific FcRn knockout in a model with a longer autologous phase, we used the nephrotoxic serum nephritis (NTS) model. We found that the podFcRn KO mice had significantly reduced crescent formation and glomerulosclerosis compared to control mice. This study demonstrates that lack of podocyte FcRn is protective in immune mediated kidney disease that is dependent on an autologous phase. This study also highlights the difference between the anti-GBM model and NTS model of disease.

Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype.

Glomerular endothelial cells (GEnC) are a specialized microvascular subset of endothelial cells that, when injured, result in many types of diseases within the kidney. Thus, techniques to study GEnC in a cell culture system are important to investigate mechanisms of GEnC injury. Studies of endothelial cell function in culture have predominately relied on using macrovascular endothelial cells from vascular areas other than the glomerulus. Over the last 15 years, glomerular endothelial cells lines have been created but were isolated by targeting cells expressing CD31. Some studies identified endothelial cells isolated from the microvasculature do not express CD31 and some suggest that CD31+ cells are phenotypically different than endothelial cells found in capillaries. Here we detail our method of isolation, purification, and conditional immortalization of mouse glomerular endothelial cells targeting endothelial cells that do not express CD31.•This method allows for isolation, purification, and conditional immortalization of glomerular endothelial cells for continued passage of GEnCs beyond that of primary cell culture.•This method can be used in genetically modified mice to investigate how a modification of a specific gene or protein affects the glomerular endothelium at the cellular level.

Regulation of the Actin Cytoskeleton in Podocytes.

Podocytes are an integral part of the glomerular filtration barrier, a structure that prevents filtration of large proteins and macromolecules into the urine. Podocyte function is dependent on actin cytoskeleton regulation within the foot processes, structures that link podocytes to the glomerular basement membrane. Actin cytoskeleton dynamics in podocyte foot processes are complex and regulated by multiple proteins and other factors. There are two key signal integration and structural hubs within foot processes that regulate the actin cytoskeleton: the slit diaphragm and focal adhesions. Both modulate actin filament extension as well as foot process mobility. No matter what the initial cause, the final common pathway of podocyte damage is dysregulation of the actin cytoskeleton leading to foot process retraction and proteinuria. Disruption of the actin cytoskeleton can be due to acquired causes or to genetic mutations in key actin regulatory and signaling proteins. Here, we describe the major structural and signaling components that regulate the actin cytoskeleton in podocytes as well as acquired and genetic causes of actin dysregulation.

IL-6-mediated hepatocyte production is the primary source of plasma and urine neutrophil gelatinase-associated lipocalin during acute kidney injury.

Neutrophil gelatinase associated lipocalin (NGAL, Lcn2) is the most widely studied biomarker of acute kidney injury (AKI). Previous studies have demonstrated that NGAL is produced by the kidney and released into the urine and plasma. Consequently, NGAL is currently considered a tubule specific injury marker of AKI. However, the utility of NGAL to predict AKI has been variable suggesting that other mechanisms of production are present. IL-6 is a proinflammatory cytokine increased in plasma by two hours of AKI and mediates distant organ effects. Herein, we investigated the role of IL-6 in renal and extra-renal NGAL production. Wild type mice with ischemic AKI had increased plasma IL-6, increased hepatic NGAL mRNA, increased plasma NGAL, and increased urine NGAL; all reduced in IL-6 knockout mice. Intravenous IL-6 in normal mice increased hepatic NGAL mRNA, plasma NGAL and urine NGAL. In mice with hepatocyte specific NGAL deletion (Lcn2hep-/-) and ischemic AKI, hepatic NGAL mRNA was absent, and plasma and urine NGAL were reduced. Since urine NGAL levels appear to be dependent on plasma levels, the renal handling of circulating NGAL was examined using recombinant human NGAL. After intravenous recombinant human NGAL administration to mice, human NGAL in mouse urine was detected by ELISA during proximal tubular dysfunction, but not in pre-renal azotemia. Thus, during AKI, IL-6 mediates hepatic NGAL production, hepatocytes are the primary source of plasma and urine NGAL, and plasma NGAL appears in the urine during proximal tubule dysfunction. Hence, our data change the paradigm by which NGAL should be interpreted as a biomarker of AKI.

An in vitro model of antibody-mediated injury to glomerular endothelial cells: Upregulation of MHC class II and adhesion molecules.

Chronic active antibody-mediated rejection is a major cause of allograft failure in kidney transplantation. Microvascular inflammation and transplant glomerulopathy are defining pathologic features of chronic active antibody-mediated rejection and are associated with allograft failure. However, the mechanisms of leukocyte infiltration and glomerular endothelial cell injury remain unclear. We hypothesized MHC class II ligation on glomerular endothelial cells (GEnC) would result in upregulation of adhesion molecules and production of chemoattractants. A model of endothelial cell activation in the presence of antibodies to MHC classes I and II was used to determine the expression of adhesion molecules and chemokines. Murine GEnC were activated with IFNγ, which upregulated gene expression of ß2-microglobulin (MHC class I), ICAM1, VCAM1, CCL2, CCL5, and IL-6. IFNγ stimulation of GEnC increased surface expression of MHC class I, MHC class II, ICAM1, and VCAM1. Incubation with antibodies directed at MHC class I or class II did not further enhance adhesion molecule expression. Multispectral imaging flow cytometry and confocal microscopy demonstrated MHC molecules co-localized with the adhesion molecules ICAM1 and VCAM1 on the GEnC surface. GEnC secretion of chemoattractants, CCL2 and CCL5, was increased by IFNγ stimulation. CCL2 production was further enhanced by incubation with sensitized plasma. Endothelial activation induces de novo expression of MHC class II molecules and increases surface expression of MHC class I, ICAM1 and VCAM1, which are all co-localized together. Maintaining the integrity and functionality of the glomerular endothelium is necessary to ensure survival of the allograft. IFNγ stimulation of GEnC propagates an inflammatory response with production of chemokines and co-localization of MHC and adhesion molecules on the GEnC surface, contributing to endothelial cell function as antigen presenting cells and an active player in allograft injury.

Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton.

The neonatal Fc receptor (FcRn) has been shown to be required for antigen presentation in dendritic cells, and global knockout of FcRn attenuates immune-mediated kidney disease. Podocytes express interleukin-6 (IL-6) receptor and produce IL-6 under proinflammatory conditions. Here we examined the role of FcRn in the IL-6-mediated inflammatory response in podocytes. We examined IL-6 production by ELISA and expression by qPCR in wild type (WT) and FcRn knockout (KO) podocytes after treatment with proinflammatory stimuli as well as IL-6-mediated signaling via the JAK/STAT pathway. We also examined podocyte motility in cultured WT and KO podocytes after a proinflammatory challenge. We found that FcRn KO podocytes produced minimal amount of IL-6 after treatment with albumin, IgG, or immune complexes whereas WT podocytes had a robust response. FcRn KO podocytes also had minimal expression of IL-6 compared with WT. By Western blotting, there was significantly less phosphorylated STAT3 in KO podocytes after treatment with IFNγ or immune complexes. In a scratch assay, FcRn KO podocytes showed increased motility comparted KO, suggesting a defect in actin dynamics. Cultured FcRn KO podocytes also demonstrated abnormal stress fibers compared with WT and the defect could be rescued by IL-6 treatment. This study shows that in podocytes, FcRn modulates the IL-6 mediated response to proinflammatory stimuli and regulates podocytes actin structure, motility and synaptopodin expression.

Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo.

Proteinuria is strongly associated with kidney disease progression but the mechanisms underlying podocyte handling of serum proteins such as albumin and IgG remain to be elucidated. We have previously shown that albumin and IgG are transcytosed by podocytes in vitro. In other epithelial cells, the neonatal Fc receptor (FcRn) is required to salvage albumin and IgG from the degradative pathway thereby allowing these proteins to be transcytosed or recycled. Here we directly examine the role of FcRn in albumin and IgG trafficking in podocytes by studying handling of these proteins in FcRn knockout (KO) podocytes in vitro and in a podocyte-specific FcRn knockout mice in vivo. In vitro, we find that knockout of FcRn leads to IgG accumulation in podocytes but does not alter albumin trafficking. Similarly, in vivo, podocyte-specific knockout of FcRn does not result in albumin accumulation in podocytes in vivo as measured by mean albumin fluorescence intensity whereas these mice demonstrate significant intraglomerular accumulation of IgG over time. In addition we find that podocyte-specific FcRn KO mice demonstrate mesangial expansion as they age and activation of mesangial cells as demonstrated by increased expression of α-smooth muscle actin. Taken together, these results suggest that trafficking pathways for albumin and IgG differ in podocytes and that sustained disruption of trafficking of plasma proteins alters glomerular structure.

Shank2 Regulates Renal Albumin Endocytosis.

Albuminuria is a strong and independent predictor of kidney disease progression but the mechanisms of albumin handling by the kidney remain to be fully defined. Previous studies have shown that podocytes endocytose albumin. Here we demonstrate that Shank2, a large scaffolding protein originally identified at the neuronal postsynaptic density, is expressed in podocytes in vivo and in vitro and plays an important role in albumin endocytosis in podocytes. Knockdown of Shank2 in cultured human podocytes decreased albumin uptake, but the decrease was not statistically significant likely due to residual Shank2 still present in the knockdown podocytes. Complete knockout of Shank2 in podocytes significantly diminished albumin uptake in vitro. Shank2 knockout mice develop proteinuria by 8 weeks of age. To examine albumin handling in vivo in wild-type and Shank2 knockout mice we used multiphoton intravital imaging. While FITC-labeled albumin was rapidly seen in the renal tubules of wild-type mice after injection, little albumin was seen in the tubules of Shank2 knockout mice indicating dysregulated renal albumin trafficking in the Shank2 knockouts. We have previously found that caveolin-1 is required for albumin endocytosis in cultured podocytes. Shank2 knockout mice had significantly decreased expression and altered localization of caveolin-1 in podocytes suggesting that disruption of albumin endocytosis in Shank2 knockouts is mediated via caveolin-1. In summary, we have identified Shank2 as another component of the albumin endocytic pathway in podocytes.

Renal control of calcium, phosphate, and magnesium homeostasis.

Calcium, phosphate, and magnesium are multivalent cations that are important for many biologic and cellular functions. The kidneys play a central role in the homeostasis of these ions. Gastrointestinal absorption is balanced by renal excretion. When body stores of these ions decline significantly, gastrointestinal absorption, bone resorption, and renal tubular reabsorption increase to normalize their levels. Renal regulation of these ions occurs through glomerular filtration and tubular reabsorption and/or secretion and is therefore an important determinant of plasma ion concentration. Under physiologic conditions, the whole body balance of calcium, phosphate, and magnesium is maintained by fine adjustments of urinary excretion to equal the net intake. This review discusses how calcium, phosphate, and magnesium are handled by the kidneys.

Podocytes degrade endocytosed albumin primarily in lysosomes.

Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.