Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.
ABSTRACT: Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-? activation in podocytes; however, it is not clear how TGF-? signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-? signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS.
Project description:The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.
Project description:Focal segmental glomerulosclerosis is a major cause of end stage renal disease. Many patients prove unresponsive to available therapies. An improved understanding of the molecular basis of the disease process could provide insights leading to novel therapeutic approaches. In this study we carried out an RNA-seq analysis of the altered gene expression patterns of podocytes, mesangial cells and glomerular endothelial cells of the bigenic Cd2ap+/-, Fyn-/- mutant mouse model of FSGS. In the podocytes we observed upregulation of many genes related to the Tgf? family/pathway, including Gdnf, Tgf?1, Tgf?2, Snai2, Vegfb, Bmp4, and Tnc. The mutant podocytes also showed upregulation of Acta2, a marker of smooth muscle and associated with myofibroblasts, which are implicated in driving fibrosis. GO analysis of the podocyte upregulated genes identified elevated protein kinase activity, increased expression of growth factors, and negative regulation of cell adhesion, perhaps related to the observed podocyte loss. Both podocytes and mesangial cells showed strong upregulation of aldehyde dehydrogenase genes involved in the synthesis of retinoic acid. Similarly, the Cd2ap+/-, Fyn-/- mesangial cells, as well as podocytes in other genetic models, and the glomeruli of human FSGS patients, all show upregulation of the serine protease Prss23, with the common thread suggesting important functionality. Another gene with strong upregulation in the Cd2ap+/-, Fyn-/- mutant mesangial cells as well as multiple other mutant mouse models of FSGS was thrombospondin, which activates the secreted inactive form of Tgf?. The Cd2ap+/-, Fyn-/- mutant endothelial cells showed elevated expression of genes involved in cell proliferation, angioblast migration, angiogenesis, and neovasculature, all consistent with the formation of new blood vessels in the diseased glomerulus. The resulting global definition of the perturbed molecular pathways in the three major cell types of the mutant glomerulus provide deeper understanding of the molecular pathogenic pathways.
Project description:The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1?, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).
Project description:<h4>Introduction</h4>The podocyte is thought to be the mainly affected cell type in focal segmental glomerulosclerosis (FSGS). However, recent studies have also indicated a role for glomerular endothelial cells and podocyte-endothelial crosstalk in FSGS development. An experimental model for podocyte injury showed that increased endothelin-1 (ET-1) signaling between podocytes and endothelial cells induces endothelial oxidative stress and subsequent podocyte loss. In the current study, we investigated endothelial endothelin receptor A (ET<sub>A</sub>R) expression in patients with FSGS and its association with podocyte injury and glomerular oxidative stress.<h4>Methods</h4>We selected 39 biopsy samples of patients with FSGS and 8 healthy control subjects, and stained them for ET<sub>A</sub>R, nephrin and 8-oxo-guanine, a DNA lesion caused by oxidative damage. Glomeruli with ET<sub>A</sub>R-positive endothelium and with nephrin loss were scored, and the 8-oxo-guanine-positive glomerular area was measured.<h4>Results</h4>The mean percentage of glomeruli with ET<sub>A</sub>R-positive endothelial cells in patients with FSGS was higher compared to that in healthy control subjects (52% vs. 7%; <i>P</i> < 0.001). The presence of glomerular ET<sub>A</sub>R-positive endothelium was strongly associated with nephrin loss both on the biopsy level (rho = 0.47; <i>P</i> < 0.01), as on the level of individual glomeruli (odds ratio = 2.0; <i>P</i> < 0.001). Moreover, glomeruli with ET<sub>A</sub>R-positive endothelium showed more 8-oxo-guanine-positive staining (1.9% vs. 2.4%; <i>P</i> = 0.037). Finally, 8-oxo-guanine positivity in glomeruli was associated with increased levels of proteinuria.<h4>Conclusion</h4>Taking together our findings, we show that ET<sub>A</sub>R is increased in glomerular endothelial cells of patients with FSGS and associated with podocyte damage and glomerular oxidative stress. These findings support the hypothesis that ET-1 signaling in glomerular endothelial cells contributes to disease development in patients with FSGS.
Project description:Podocyte injury is a common hallmark in various glomerular diseases. The level of LRRC55 was increased in podocytes of patients with focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN), and membranous nephropathy (MN). Upregulated LRRC55 and increased intracellular Ca2+ led to BK channel activation and the loss of intracellular potassium, resulting in apoptosome formation and caspase-3 activation in angiotensin II (Ang II)-treated podocytes. Knockout of Lrrc55 or the BK channel prevented the BK current and ameliorated podocyte injury in Ang II-treated mice. Upstream, NFATc3 regulated the expression of LRRC55. Increased LRRC55 expression in podocytes was also evident in animal models of FSGS, DN, and MN. Treatment with losartan or LRRC55 siRNA suppressed LRRC55 expression, prevented BK channel activation, and attenuated podocyte injury in animal models of FSGS, DN, and MN. In conclusion, upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes in FSGS, DN, and MN. LRRC55 inhibition may represent a new therapeutic approach for podocyte injury.
Project description:BACKGROUND:Focal segmental glomerulosclerosis (FSGS) is a histologic pattern of injury demonstrated by renal biopsy that can arise from a diverse range of causes and mechanisms. It has an estimated incidence of 7 per 1 million and is the most common primary glomerular disorder leading to end-stage renal disease in the United States. This review focuses on damage to the podocyte and the consequences of this injury in patients with FSGS, the genetics of FSGS, and approaches to treatment with a focus on the effects on podocytes. SUMMARY:The podocyte is central to the glomerular filtration barrier and is particularly vulnerable because of its highly differentiated post-mitotic phenotype. The progressive structural changes involved in the pathology of FSGS include podocyte foot process effacement, death of podocytes and exposure of the glomerular basement membrane, filtration of nonspecific plasma proteins, expansion of capillaries, misdirected filtration at points of synechiae, and mesangial matrix proliferation. Although damage to and death of podocytes can result from single-gene disorders, evidence also suggests a role for soluble factors, such as soluble urokinase-type plasminogen activator receptor, cardiotrophin-like cytokine-1, and anti-CD40 antibodies, that promote FSGS recurrence post transplant. Several classes of medications, including corticosteroids, calcineurin inhibitors, endothelin receptor antagonists, adrenocorticotropic hormone, and rituximab, have been shown to be effective for the treatment of FSGS and have been demonstrated to have significant protective effects on podocytes. Key Messages: Greater understanding of podocyte biology is essential to the identification of new treatment targets and medications for the management of patients with FSGS.
Project description:TGF-β-activated kinase 1 (TAK1) is a key intermediate in signal transduction induced by TGF-β or inflammatory cytokines, such as TNF-α and IL-1, which are potent inducers of podocyte injury responses that lead to proteinuria and glomerulosclerosis. Nevertheless, little is known about the physiologic and pathologic roles of TAK1 in podocytes. To examine the in vivo role of TAK1, we generated podocyte-specific Tak1 knockout mice (Nphs2-Cre(+):Tak1(fx/fx); Tak1(∆/∆)). Targeted deletion of Tak1 in podocytes resulted in perinatal lethality, with approximately 50% of animals dying soon after birth and 90% of animals dying within 1 week of birth. Tak1(∆/∆) mice developed proteinuria from P1 and exhibited delayed glomerulogenesis and reduced expression of Wilms' tumor suppressor 1 and nephrin in podocytes. Compared with Tak1(fx/fx) mice, Tak1(∆/∆) mice exhibited impaired formation of podocyte foot processes that caused disruption of the podocyte architecture with prominent foot process effacement. Intriguingly, Tak1(∆/∆) mice displayed increased expression of vascular endothelial growth factor within the glomerulus and abnormally enlarged glomerular capillaries. Furthermore, 4- and 7-week-old Tak1(∆/∆) mice with proteinuria had increased collagen deposition in the mesangium and the adjacent tubulointerstitial area. Thus, loss of Tak1 in podocytes is associated with the development of proteinuria and glomerulosclerosis. Taken together, our data show that TAK1 regulates the expression of Wilms' tumor suppressor 1, nephrin, and vascular endothelial growth factor and that TAK1 signaling has a crucial role in podocyte differentiation and attainment of normal glomerular microvasculature during kidney development and glomerular filtration barrier homeostasis.
Project description:Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. We expressed enhanced green fluorescent protein-tagged to ribosomal protein L10a in podocytes under the control of the collagen-1?1 promoter, enabling one-step podocyte-specific mRNA isolation over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed Col1?1-eGFP-L10a mice with the Actn4(-/-) and Actn4(+/K256E) models of FSGS and analyzed podocyte transcriptional profiles at 2, 6, and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression data sets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and defines novel biomarkers and therapeutic targets for human glomerular disease.
Project description:Abstract Glomerular podocytes play a key role in proteinuric diseases. Accumulating evidence suggests that cGMP signaling has podocyte protective effects. The major source of cGMP generation in podocytes is natriuretic peptides. The natriuretic peptide clearance receptor (NPRC) binds and degrades natriuretic peptides. As a result, NPRC inhibits natriuretic peptide‐induced cGMP generation. To enhance cGMP generation in podocytes, we blocked natriuretic peptide clearance using the specific NPRC ligand ANP(4‐23). We then studied the effects of NPRC blockade in both cultured podocytes and in a mouse transgenic (TG) model of focal segmental glomerulosclerosis (FSGS) created in our laboratory. In this model, a single dose of the podocyte toxin puromycin aminonucleoside (PAN) causes robust albuminuria in TG mice, but only mild disease in non‐TG animals. We found that natriuretic peptides protected cultured podocytes from PAN‐induced apoptosis, and that ANP(4‐23) enhanced natriuretic peptide‐induced cGMP generation in vivo. PAN‐induced heavy proteinuria in vehicle‐treated TG mice, and this increase in albuminuria was reduced by treatment with ANP(4‐23). Treatment with ANP(4‐23) also reduced the number of mice with glomerular injury and enhanced urinary cGMP excretion, but these differences were not statistically significant. Systolic BP was similar in vehicle and ANP(4‐23)‐treated mice. These data suggest that: 1. Pharmacologic blockade of NPRC may be useful for treating glomerular diseases such as FSGS, and 2. Treatment outcomes might be improved by optimizing NPRC blockade to inhibit natriuretic peptide clearance more effectively. The effects of blocking natriuretic peptide clearance from the circulation was examined in a mouse model of focal segmental glomerulosclerosis (FSGS). We found that blockade of the natriuretic clearance receptor protected glomerular podocytes from apoptosis and inhibited albuminuria in FSGS.
Project description:The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.