Glomerular Epithelial Cell Injury Research Articles

Swollen Feet: Considering the Paradoxical Roles of Interleukins in Nephrotic Syndrome.

Interleukins are a family of 40 bioactive peptides that act through cell surface receptors to induce a variety of intracellular responses. While interleukins are most commonly associated with destructive, pro-inflammatory signaling in cells, some also play a role in promoting cellular resilience and survival. This review will highlight recent evidence of the cytoprotective actions of the interleukin 1 receptor (IL-1R)- and common gamma chain receptor (IL-Rγc)-signaling cytokines in nephrotic syndrome (NS). NS results from the injury or loss of glomerular visceral epithelial cells (i.e., podocytes). Although the causes of podocyte dysfunction vary, it is clear that pro-inflammatory cytokines play a significant role in regulating the propagation, duration and severity of disease. Pro-inflammatory cytokines signaling through IL-1R and IL-Rγc have been shown to exert anti-apoptotic effects in podocytes through the phosphoinositol-3-kinase (PI-3K)/AKT pathway, highlighting the potential utility of IL-1R- and IL-Rγc-signaling interleukins for the treatment of podocytopathy in NS. The paradoxical role of interleukins as drivers and mitigators of podocyte injury is complex and ill-defined. Emerging evidence of the cytoprotective role of some interleukins in NS highlights the urgent need for a nuanced understanding of their pro-survival benefits and reveals their potential as podocyte-sparing therapeutics for NS.

Bioinformatic analysis of CHEK1 as a marker of glomerular epithelial cell injury in diabetic nephropathy.

Diabetic nephropathy (DN) is considered to be a kidney disease caused by diabetes. In recent years, the incidence of DN has been on the rise, which is also a major challenge in the treatment and prognosis of the disease. Therefore, the search for new biomarkers of DN is urgent and has important clinical significance for reducing the morbidity and mortality of DN. In this study, two datasets GSE1009 and GSE142153 were selected to extract expression profile-based data from DN glomerular samples, and 238 differentially expressed genes (DEGs) were screened. Then, through enrichment analysis, the biological function of DEGs involved in DN disease was preliminarily explored. Subsequently, the STRING website was used to construct a protein-protein interaction map (PPI) to find 10 key genes (CHEK1, ITGB3, COL4A2, COL4A5, COL4A3, COL4A4, CCNB2, CCNB1, TPX2, KIF11), Which play an important role in the progression of DN disease and are closely related to other genes. CHEK1 was the focus of this study, and the expression level of CHEK1 in glomerular epithelial cell models was verified by qRT-PCR. Our results suggest that CHEK1 is a potential biomarker of the degree of damage to DN glomerular epithelial cells.

Matrix metalloproteinase-9 regulates afferent arteriolar remodeling and function in hypertension-induced kidney disease

This study tested if matrix metalloproteinase (MMP)-9 promoted microvascular pathology that initiates hypertensive (HT) kidney disease in salt-sensitive (SS) Dahl rats. SS rats lacking Mmp9 (Mmp9-/-) and littermate control SS rats were studied after one week on a normotensive 0.3% sodium chloride (Pre-HT SS and Pre-HT Mmp9-/-) or a hypertension-inducing diet containing 4.0% sodium chloride (HT SS and HT Mmp9-/-). Telemetry-monitored blood pressure of both the HT SS and HT Mmp9-/- rats increased and did not differ. Kidney microvessel transforming growth factor-beta 1 (Tgfb1) mRNA did not differ between Pre-HT SS and Pre-HT Mmp9-/- rats, but with hypertension and expression of Mmp9 and Tgfb1 increased in HT SS rats, along with phospho-Smad2 labeling of nuclei of vascular smooth muscle cells, and with peri-arteriolar fibronectin deposition. Loss of MMP-9 prevented hypertension-induced phenotypic transformation of microvascular smooth muscle cells and the expected increased microvascular expression of pro-inflammatory molecules. Loss of MMP-9 in vascular smooth muscle cells invitro prevented cyclic strain-induced production of active TGF-β1 and phospho-Smad2/3 stimulation. Afferent arteriolar autoregulation was impaired in HT SS rats but not in HT Mmp9-/- rats or the HT SS rats treated with doxycycline, an MMP inhibitor. HT SS but not HT Mmp9-/- rats showed decreased glomerular Wilms Tumor 1 protein-positive cells (a marker of podocytes) along with increased urinary podocin and nephrin mRNA excretion, all indicative of glomerular damage. Thus, our findings support an active role for MMP-9 in a hypertension-induced kidney microvascular remodeling process that promotes glomerular epithelial cell injury in SS rats.

317-OR: Metabolomic Analyses Identify Novel Predictors of Diabetic Kidney Disease in Youth-Onset Type 2 Diabetes

Background: Diabetic kidney disease (DKD) develops by young adulthood in up to 50% of people with youth-onset type 2 diabetes (Y-T2D), increasing risk of dialysis and premature death. Understanding mechanisms responsible for early DKD is key to management and prevention; accordingly, we sought to identify metabolite signatures of DKD in Y-T2D. Methods: We measured 57 metabolites in 374 baseline plasma and urine samples from the Treatment Options for type 2 Diabetes in Adolescents and Youth (TODAY) study, using mass spectrometry with a targeted ZipChip based assay. Urine albumin-to-creatinine ratio (UACR) was assessed annually for up to 15 years. Incident moderate and severe albuminuria were defined as UACR ≥30 and ≥300 mg/g, respectively, on ≥2 of 3 measures. We evaluated prediction of moderate and severe albuminuria in separate Cox proportional hazards models adjusted for HbA1c, triglycerides, systolic blood pressure, and estimated insulin sensitivity. Urine metabolites were normalized by urine creatinine. Results: Participants were 14±2 years of age, 37% male; 43% developed either moderate or severe albuminuria. Four urine metabolites predicted time to moderate albuminuria, while 8 urine metabolites predicted time to severe albuminuria, with 3 metabolites in common: 2-hydroxybutyric acid (moderate: HR: 0.82 per 1 SD [95% CI 0.70, 0.96]; severe: 0.70 [0.55, 0.88]), glycine (moderate: 0.81 [0.69, 0.94]; severe: 0.62 [0.45, 0.85]), citric acid (moderate: 0.78 [0.67, 0.91]; severe: 0.76 [0.58, 0.99]). Four plasma metabolites predicted time to moderate albuminuria and severe albuminuria, such as glutamic acid for moderate albuminuria (1.23 [1.03, 1.47]) and citric acid for severe albuminuria (1.39 [1.09, 1.77]). Conclusion: Higher urine metabolites involved in mitigating oxidative stress (glycine), glomerular epithelial cell injury (2-hydroxybutyric acid) and preserving mitochondrial function (citrate) predicted lower risk of albuminuria in Y-T2D. Disclosure L.Pyle: None. L.M.Laffel: Advisory Panel; Medtronic, Lilly Diabetes, Novo Nordisk, Vertex Pharmaceuticals Incorporated, Roche Diagnostics, Provention Bio, Inc., Consultant; Dexcom, Inc., Janssen Pharmaceuticals, Inc., Medscape. A.S.Shah: None. K.Drews: None. J.R.Ryder: None. R.Gubitosi-klug: None. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. T.B.Vigers: None. L.El ghormli: None. I.De boer: Advisory Panel; AstraZeneca, Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Otsuka America Pharmaceutical, Inc., Bayer Inc., Consultant; George Clinical, Gilead Sciences, Inc., Medscape, Research Support; Dexcom, Inc. R.G.Nelson: None. S.Waikar: None. H.L.Heerspink: Consultant; AstraZeneca, Boehringer Ingelheim International GmbH, Bayer Inc., Eli Lilly and Company, Chinook Therapeutics Inc., CSL Behring, Gilead Sciences, Inc., George Clinical, Merck & Co., Inc., Janssen Research & Development, LLC, Traveere Pharmaceuticals, Novo Nordisk. N.White: None. K.L.Tommerdahl: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (U01DK61242)

Podocyte-specific Transcription Factors: Could MafB become a Therapeutic Target for Kidney Disease?

The increasing number of patients with chronic kidney disease (CKD) is being recognized as an emerging global health problem. Recently, it has become clear that injury and loss of glomerular visceral epithelial cells, known as podocytes, is a common early event in many forms of CKD. Podocytes are highly specialized epithelial cells that cover the outer layer of the glomerular basement membrane. They serve as the final barrier to urinary protein loss through the formation and maintenance of specialized foot-processes and an interposed slit-diaphragm. We previously reported that the transcription factor MafB regulates the podocyte slit diaphragm protein production and transcription factor Tcf21. We showed that the forced expression of MafB was able to prevent CKD. In this review, we discuss recent advances and offer an updated overview of the functions of podocyte-specific transcription factors in kidney biology, aiming to present new perspectives on the progression of CKD and respective therapeutic strategies.

Inhibition of the Wnt/β-catenin signaling pathway reduces autophagy levels in complement treated podocytes.

In idiopathic membranous nephropathy, the complement membrane attack complex, more commonly referred to as complement 5b-9 (C5b-9), induces glomerular epithelial cell injury and proteinuria. C5b-9 can also activate numerous mechanisms that restrict or facilitate injury. Recent studies suggest that autophagy and the canonical Wnt signaling pathway serve an important role in repairing podocyte injury. However, the effect of C5b-9 on these pathways and the relationship between them remains unclear. The aim of the present study was to show the effect of C5b-9 on the Wnt/β-catenin signaling pathway and autophagy in podocytes in vitro. Levels of relevant indicators were detected by immunofluorescence staining and capillary western immunoassay. C5b-9 serum significantly activated the Wnt/β-catenin signaling pathway and promoted autophagy. Treatment with Dickkopf-related protein 1 (DKK1), a Wnt/β-catenin pathway blocker, protected podocytes from injury and significantly inhibited autophagy. The results indicated that inhibition of the Wnt/β-catenin pathway physiologically activated autophagy. The results indicated that C5b-9 resulted in a decrease in Akt in podocytes. However, the podocytes preincubated with DKK1 and then attacked by C5b-9 showed an increase in Akt levels. This may explain the observation that blocking the Wnt/β-catenin signaling pathway attenuated C5b-9 podocyte damage, while inhibiting autophagy. The results of the present study also suggest that regulation of these two pathways may serve as a novel method for the treatment of idiopathic membranous nephropathy.

The Role of Glomerular Epithelial Injury in Kidney Function Decline in Patients With Diabetic Kidney Disease in the TRIDENT Cohort

IntroductionAlthough diabetic kidney disease (DKD) is responsible for more than half of all chronic and end-stage kidney disease (ESKD), the association of light (LM) and electron microscopic (EM) structural changes with clinical parameters and prognosis in DKD is incompletely understood.MethodsThis is an interim analysis of 62 patients diagnosed with biopsy-confirmed DKD from the multicenter TRIDENT (Transformative Research in Diabetic Nephropathy) study. Twelve LM and 8 EM descriptors, representing changes in glomeruli, tubulointerstitium, and vasculature were analyzed for their relationship with clinical measures of renal function. Patients were followed every 6 months.ResultsMultivariable linear regression analysis revealed that estimated glomerular filtration rate (eGFR) upon enrollment correlated the best with interstitial fibrosis. On the other hand, the rate of kidney function decline (eGFR slope) correlated the most with glomerular lesions including global glomerulosclerosis and mesangiolysis. Unbiased clustering analysis based on histopathologic data identified 3 subgroups. The first cluster, encompassing subjects with the mildest histologic lesions, had the most preserved kidney function. The second and third clusters had similar degrees of kidney dysfunction and structural damage, but differed in the degree of glomerular epithelial cell and podocyte injury (podocytopathy DKD subtype). Cox proportional hazard analysis showed that subjects in cluster 2 had the highest risk to reach ESKD (hazard ratio: 17.89; 95% confidence interval: 2.13–149.79). Glomerular epithelial hyperplasia and interstitial fibrosis were significant predictors of ESKD in the multivariate model.ConclusionThe study highlights the association between fibrosis and kidney function and identifies the role of glomerular epithelial changes and kidney function decline.

Glomerular Epithelial Cells-Targeted Heme Oxygenase-1 Over Expression in the Rat: Attenuation of Proteinuria in Secondary But Not Primary Injury

Background/Aims: Induction of heme oxygenase 1 (HO-1) in glomerular epithelial cells (GEC) in response to injury is poor and this may be a disadvantage. We, therefore, explored whether HO-1 overexpression in GEC can reduce proteinuria induced by puromycin aminonucleoside (PAN) or in anti-glomerular basement membrane (GBM) antibody (Ab)-mediated glomerulonephritis (GN). Methods: HO-1 overexpression in GEC (GEC^HO-1) of Sprague-Dawley rats was achieved by targeting a FLAG-human (h) HO-1 using transposon-mediated transgenesis. Direct GEC injury was induced by a single injection of PAN. GN was induced by administration of an anti-rat GBM Ab and macrophage infiltration in glomeruli was assessed by immunohistochemistry and western blot analysis, which was also used to assess glomerular nephrin expression. Results: In GEC^HO-1 rats, FLAG-hHO-1 transprotein was co-immunolocalized with nephrin. Baseline glomerular HO-1 protein levels were higher in GEC^HO-1 compared to wild type (WT) rats. Administration of either PAN or anti-GBM Ab to WT rats increased glomerular HO-1 levels. Nephrin expression markedly decreased in glomeruli of WT or GEC^HO-1 rats treated with PAN. In anti-GBM Ab-treated WT rats, nephrin expression also decreased. In contrast, it was preserved in anti-GBM Ab-treated GEC^HO-1 rats. In these, macrophage infiltration in glomeruli and the ratio of urine albumin to urine creatinine (Ualb/Ucreat) were markedly reduced. There was no difference in Ualb/Ucreat between WT and GEC^HO-1 rats treated with PAN. Conclusion: Depending on the type of injury, HO-1 overexpression in GEC may or may not reduce proteinuria. Reduced macrophage infiltration and preservation of nephrin expression are putative mechanisms underlying the protective effect of HO-1 overexpression following immune injury.

Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.

Calcium-independent Phospholipase A2γ Enhances Activation of the ATF6 Transcription Factor during Endoplasmic Reticulum Stress

Injury of visceral glomerular epithelial cells (GECs) causes proteinuria in many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated GEC injury. Because iPLA2γ is localized at the endoplasmic reticulum (ER), this study addressed whether the cytoprotective effect of iPLA2γ involves the ER stress unfolded protein response (UPR). In cultured rat GECs, overexpression of the full-length iPLA2γ, but not a mutant iPLA2γ that fails to associate with the ER, augmented tunicamycin-induced activation of activating transcription factor-6 (ATF6) and induction of the ER chaperones, glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78). Augmented responses were inhibited by the iPLA2γ inhibitor, (R)-bromoenol lactone, but not by the cyclooxygenase inhibitor, indomethacin. Tunicamycin-induced cytotoxicity was reduced in GECs expressing iPLA2γ, and the cytoprotection was reversed by dominant-negative ATF6. GECs from iPLA2γ knock-out mice showed blunted ATF6 activation and chaperone up-regulation in response to tunicamycin. Unlike ATF6, the two other UPR pathways, i.e. inositol-requiring enzyme 1α and protein kinase RNA-like ER kinase pathways, were not affected by iPLA2γ. Thus, in GECs, iPLA2γ amplified activation of the ATF6 pathway of the UPR, resulting in up-regulation of ER chaperones and cytoprotection. These effects were dependent on iPLA2γ catalytic activity and association with the ER but not on prostanoids. Modulating iPLA2γ activity may provide opportunities for pharmacological intervention in glomerular diseases associated with ER stress.

Expression of Ser729 phosphorylated PKCepsilon in experimental crescentic glomerulonephritis: an immunohistochemical study.

PKCε, a DAG-dependent, Ca2+- independent kinase attenuates extent of fibrosis following tissue injury, suppresses apoptosis and promotes cell quiescence. In crescentic glomerulonephritis (CGN), glomerular epithelial cells (GEC) contribute to fibro-cellular crescent formation while they also transdifferentiate to a mesenchymal phenotype. The aim of this study was to assess PKCε expression in CGN. Using an antibody against PKC-ε phosphorylated at Ser729, we assessed its localization in rat model of immune-mediated rapidly progressive CGN. In glomeruli of control animals, pPKCε was undetectable. In animals with CGN, pPKCε was expressed exclusively in glomerular epithelial cells (GEC) and in GEC comprising fibrocellular crescents that had acquired a myofibroblasttype phenotype. In non-immune GEC injury induced by puromycin aminonucleoside and resulting in proteinuria of similar magnitude as in CGN, pPKCε expression was absent. There was constitutive pPKCε expression in distal convoluted tubules, collecting ducts and thick segments of Henley’s loops in both control and experimental animals. We propose that pPKCε expression occurring in GEC and in fibrocellular crescentic lesions in CGN may facilitate PKCε dependent pathologic processes.

Complement-mediated Activation of Calcium-independent Phospholipase A2γ: ROLE OF PROTEIN KINASES AND PHOSPHORYLATION

In experimental membranous nephropathy, complement C5b-9-induces glomerular epithelial cell (GEC) injury and proteinuria. The effects of C5b-9 are mediated via signaling pathways, including calcium-independent phospholipase A(2)γ (iPLA(2)γ), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The iPLA(2)γ pathway is cytoprotective. This study addresses the mechanisms of iPLA(2)γ activation. iPLA(2)γ activity was monitored by quantifying prostaglandin E(2) (PGE(2)) production. In GECs, iPLA(2)γ localized at the endoplasmic reticulum and mitochondria. Complement-mediated production of PGE(2) was amplified in GECs that overexpress iPLA(2)γ, compared with control cells, and was blocked by the iPLA(2)γ inhibitor bromoenol lactone in both iPLA(2)γ-overexpressing and control GECs. In GECs that overexpress iPLA(2)γ, complement-mediated PGE(2) production was reduced by inhibitors of MAP/ERK kinase 1 (MEK1) and p38 but not JNK. In COS-1 cells that overexpress iPLA(2)γ and cyclooxygenase-1, PGE(2) production was induced by co-expression of constitutively active MEK1 or MAPK-interacting kinase 1 (MNK1) as well as by stimulation with epidermal growth factor (EGF) + ionomycin. Complement- and EGF + ionomycin-stimulated iPLA(2)γ activity was attenuated by the S511A/S515A double mutation. Moreover, complement and EGF + ionomycin enhanced phosphorylation of Ser-511. Thus, complement-mediated activation of iPLA(2)γ is mediated via ERK and p38 pathways, and phosphorylation of Ser-511 and/or Ser-515 plays a key role in the catalytic activity and signaling of iPLA(2)γ. Defining the mechanisms by which complement activates iPLA(2)γ provides opportunities for development of novel therapeutic approaches to GEC injury and proteinuria.

Hyperglycemia Decreases the Expression of ATP Synthase <b><i>β</i></b> Subunit and Enolase 2 in Glomerular Epithelial Cells

Glomerular epithelial cells (GECs) are known to play a key role in maintaining the structure and function of the glomerulus. GEC injury induced by hyperglycemia is present in early-stage diabetic nephropathy (DN), which is the most common cause of renal failure. In an attempt to identify target proteins involved in the pathogenesis of GEC injury at early DN, we performed the proteomic analysis using primary cultures of GECs, prepared from the dissected rat glomeruli. The protein expression profiles in the two-dimensional electrophoresis gels were compared between GECs treated for three days with normal glucose (5 mM) and those with high glucose (30 mM) concentrations. These concentrations correspond to blood glucose concentrations under normoglycemia and hyperglycemia, respectively. Proteins with differential expression levels were identified using ESI-Q-TOF tandem mass spectrometry. The primary GECs cultured in hyperglycemic conditions showed cellular hypertrophy and increased production of reactive oxygen species, both of which reflect the GEC injury. Our proteomic analysis identified eight proteins with differential expression profiles, depending on glucose concentrations. Among them, we selected ATP synthase β subunit and enolase 2 that are related to energy metabolism and are down-regulated under hyperglycemia, and confirmed that hyperglycemia decreased the expression levels of ATP synthase β subunit and enolase 2 proteins by western blotting analysis. Hyperglycemia may impair mitochondrial function and alter glycolysis in GECs by down-regulating the expression of ATP synthase β subunit and enolase 2. The present study may provide a better understanding of the pathogenic mechanisms of GEC injury in early DN.

Protective effects of <I>Tongluo</I> recipe against injury of glomerular visceral epithelial cells in diabetic rats

Protective effects of <I>Tongluo</I> recipe against injury of glomerular visceral epithelial cells in diabetic rats

Renoprotective Action of a Matrix Metalloproteinase Inhibitor in Progressive Mesangioproliferative Nephritis

Background/Aim: Matrix metalloproteinases (MMPs) play pivotal roles in extracellular matrix turnover and are involved in chronic kidney disease. The renoprotective action of a synthetic MMP inhibitor, compound A, was investigated in chronic nephritis. Methods: Nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. The effects of compound A on proteinuria, blood urea nitrogen, and matrix-related gene expressions were evaluated. Collagen accumulation, as assessed by periodic acid-Schiff staining and hydroxyproline content, was determined. The integrity of glomerular epithelial cells and glomerular basement membrane was evaluated with desmin immunohistochemistry and electron microscopic detection of anionic charge sites, respectively. Results: Treatment with compound A notably attenuated proteinuria, ameliorated blood urea nitrogen, and prevented glomerulosclerosis. Gene upregulation of collagen and transforming growth factor β1 in the cortex was prevented in the treated animals. Glomerular epithelial cell injury was milder, and glomerular basement membrane anionic sites were protected with the treatment. Conclusion: A novel MMP inhibitor, compound A, exerts protective effects in progressive glomerulonephritis. Compound A ameliorates various aspects of renal injuries and may have therapeutic potential toward kidney diseases.

Complement modulates the function of the ubiquitin–proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells

In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin–proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFPu (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFPu levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFPu was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFPu degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.

Effects of <I>Tongluo</I> recipe on injury of glomerular visceral epithelial cells in streptozotocin-induced diabetic rats

Effects of <I>Tongluo</I> recipe on injury of glomerular visceral epithelial cells in streptozotocin-induced diabetic rats

Podocyte Injury Associated with Mutantα-Actinin-4

Focal segmental glomerulosclerosis (FSGS) is an important cause of proteinuria and nephrotic syndrome in humans. The pathogenesis of FSGS may be associated with glomerular visceral epithelial cell (GEC; podocyte) injury, leading to apoptosis, detachment, and “podocytopenia”, followed by glomerulosclerosis. Mutations in α-actinin-4 are associated with FSGS in humans. In cultured GECs, α-actinin-4 mediates adhesion and cytoskeletal dynamics. FSGS-associated α-actinin-4 mutants show increased binding to actin filaments, compared with the wild-type protein. Expression of an α-actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric FSGS. GECs that express mutant α-actinin-4 show defective spreading and motility, and such abnormalities could alter the mechanical properties of the podocyte, contribute to cytoskeletal disruption, and lead to injury. The potential for mutant α-actinin-4 to injure podocytes is also suggested by the characteristics of this mutant protein to form microaggregates, undergo ubiquitination, impair the ubiquitin-proteasome system, enhance endoplasmic reticulum stress, and exacerbate apoptosis.

Role of smooth muscle protein SM22α in glomerular epithelial cell injury

Podocytes are considered terminally differentiated cells in the mature kidney under normal conditions. In the face of injury, podocytes may proceed along several possible pathways, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. There is mounting evidence that transdifferentiation into a dysregulated phenotype may also be a potential cell fate. We have previously reported that the transcript of SM22α, an actin-binding protein considered one of the earliest markers of smooth muscle differentiation, is upregulated nearly 70-fold in glomeruli of rats with passive Heymann nephritis (PHN). In contrast, the SM22α transcript is absent in normal adult rat glomeruli. The purpose of this study was to define SM22α's expression during kidney development and its role in glomerular diseases characterized by podocyte injury and proteinuria. During glomerulogenesis and podocyte differentiation, SM22α was expressed in glomeruli. This expression disappeared with glomerular maturation. Along with SM22α induction in PHN, confirmed at both mRNA and protein levels, SM22α was also induced across a broad range of proteinuric diseases, including experimental animal models (puromycin aminonucleoside nephropathy, adriamycin nephropathy, passive nephrotoxic nephritis, and diet-induced obesity) and human diseases (collapsing glomerulopathy, diabetic nephropathy, classic focal segmental glomerulosclerosis, IgA nephropathy, minimal-change disease, membranous nephropathy, and membranoproliferative glomerulonephritis). Crescentic glomerulonephritis was induced in SM22α +/+ and SM22α -/- mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody 12.5 mg/20 g body wt × 2 doses (n = 12-15/group), with mice euthanized at 7 and 14 days. Compared with SM22α -/- mice, SM22α +/+ mice demonstrated worse disease by histopathological parameters. In addition, there was greater apoptosis (cleaved caspase-3 immunostaining), fewer podocytes (Wilms' tumor-1 immunostaining), and less proliferation (Ki-67 immunostaining) in diseased SM22α +/+ mice. Furthermore, there was decreased activation of Erk1/2 in diseased SM22α +/+ mice. We conclude that the de novo expression of SM22α in glomerular epithelial cells affects the course of crescentic glomerulonephritis.

Endoplasmic reticulum stress in glomerular epithelial cell injury

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in specific podocyte proteins. This study addresses mediation of GEC injury, focusing on endoplasmic reticulum (ER) stress. We studied signaling in cultured GECs in the presence or absence of the extracellular matrix (ECM). Adhesion to collagen supports cell survival, but adhesion to plastic (loss of contact with ECM) leads to apoptosis. Compared with collagen-adherent cells, GECs on plastic showed increased protein misfolding in the ER, and an adaptive-protective ER stress response, including increased expression of ER chaperones, increased phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), and a reduction in protein synthesis. Activation of these ER stress pathways counteracted apoptosis. However, tunicamycin (a potent stimulator of ER stress) changed the ER stress response from protective to cytotoxic, as tunicamycin induced the proapoptotic ER stress gene, C/EBP homologous protein-10, and exacerbated apoptosis in GECs adherent to plastic, but not collagen. In GECs adherent to plastic, adaptive ER stress was associated with an increase in polyubiquitinated proteins and "choking" of the proteasome. Furthermore, pharmacological inhibition of the proteasome induced ER stress in GECs. Finally, we show that ER stress (induction of ER chaperones and eIF2α phosphorylation) was evident in experimental FSGS in vivo. Thus interactions of GECs with ECM may regulate protein folding and induction of the ER stress response. FSGS is associated with induction of ER stress. Enhancing protective aspects of the ER stress response may reduce apoptosis and possibly glomerulosclerosis.