Kidney Injury Molecule-1 Is Upregulated in Renal Lipotoxicity and Mediates Palmitate-Induced Tubular Cell Injury and Inflammatory Response.

Diabetic nephropathy is increasingly recognized as a major contributor to kidney failure in patients with obesity and type 2 diabetes. This study was designed to identify the molecular mediators of kidney injury associated with metabolic syndrome with or without hyperglycemia. We compared renal gene expression profiles in Zucker lean (ZL), Zucker obese (ZO), and Zucker diabetic (ZD) rats using cDNA microarray with quantitative verification of selected transcripts by real-time PCR. Compared to the 20-week-old ZL control (glucose: 110 ± 8 mg/dL), both prediabetic ZO (glucose: 157 ± 11 mg/dL) and diabetic ZD (glucose: 481 ± 37 mg/dL) rats displayed hyperlipidemia and kidney injury with a high degree of proteinuria. cDNA microarray identified 25 inflammation and injury-related transcriptomes whose expression levels were similarly increased in ZO and ZD kidneys. Among them, kidney injury molecule-1 (KIM-1) was found to be the most highly upregulated in both ZO and ZD kidneys. Immunofluorescence staining of kidney sections revealed a strong correlation between lipid overload and KIM-1 upregulation in proximal tubules of ZO and ZD rats. In cultured primary renal tubular epithelial cells (TECs), administration of saturated fatty acid palmitate resulted in an upregulation of KIM-1, osteopontin, and CD44, which was greatly attenuated by U0126, an inhibitor of extracellular signal-regulated kinase (ERK)1/2. Moreover, knockdown of KIM-1 by siRNA interference inhibited palmitate-induced cleaved caspase-3, osteopontin, and CD44 proteins in primary TECs. Our results indicate that KIM-1 expression is upregulated in renal lipotoxicity and may play an important role in fatty acid-induced inflammation and tubular cell damage in obesity and diabetic kidney disease.

Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway.

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25-1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.

Kidney Injury Molecule-1 Enhances Endocytosis of Albumin in Renal Proximal Tubular Cells.

Receptor-mediated endocytosis plays an important role in albumin reabsorption by renal proximal tubule epithelial cells. Kidney injury molecule-1 (KIM-1) is a scavenger receptor that is upregulated on the apical membrane of proximal tubules in proteinuric kidney disease. In this study, we examined the cellular localization and functional role of KIM-1 in cultured renal tubule epithelial cells (TECs). Confocal immunofluorescence microscopy reveals intracellular and cell surface localization of KIM-1 in primary renal TECs. Albumin stimulation resulted in a redistribution of KIM-1 and tight junction protein zonula occludens-1 in primary TEC monolayer. An increase in albumin internalization was observed in both primary TECs expressing endogenous KIM-1 and rat kidney cell line (NRK-52E) overexpressing exogenous KIM-1. KIM-1-induced albumin accumulation was abolished by its specific antibody. Moreover, endocytosed KIM-1 and its cargo proteins were delivered from endosomes to lysosomes for degradation in a clathrin-dependent pathway. Supportive evidence includes (1) detection of KIM-1 in Rab5-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes, (2) colocalization of KIM-1 and clathrin in the intracellular vesicles, and (3) blockade of KIM-1-mediated albumin internalization by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. KIM-1 expression was upregulated by albumin but downregulated by transforming growth factor-ß1. Taken together, our data indicate that KIM-1 increases albumin endocytosis in renal tubule epithelial cells, at least partially via a clathrin-dependent mechanism. J. Cell. Physiol. 231: 896-907, 2016. © 2015 Wiley Periodicals, Inc.

Spatial control of proton pump H,K-ATPase docking at the apical membrane by phosphorylation-coupled ezrin-syntaxin 3 interaction.

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.

Altered expression profile of renal α(1D)-adrenergic receptor in diabetes and its modulation by PPAR agonists.

Alpha(1D)-adrenergic receptor (α(1D)-AR) plays important roles in regulating physiological and pathological responses mediated by catecholamines, particularly in the cardiovascular and urinary systems. The present study was designed to investigate the expression profile of α(1D)-AR in the diabetic kidneys and its modulation by activation of peroxisome proliferator-activated receptors (PPARs). 12-week-old Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with fenofibrate or rosiglitazone for 8-10 weeks. Gene microarray, real-time PCR, and confocal immunofluorescence microscopy were performed to assess mRNA and protein expression of α(1D)-AR in rat kidney tissue. Using microarray, we found that α(1D)-AR gene was dramatically upregulated in 22-week-old ZD rats compared to ZL controls. Quantitative PCR analysis verified a 16-fold increase in α(1D)-AR mRNA in renal cortex from ZD animals compared to normal controls. Chronic treatment with fenofibrate or rosiglitazone reduced renal cortical α(1D)-AR gene. Immunofluorescence staining confirmed that α(1D)-AR protein was induced in the glomeruli and tubules of diabetic rats. Moreover, dual immunostaining for α(1D)-AR and kidney injury molecule-1 indicated that α(1D)-AR was expressed in dedifferentiated proximal tubules of diabetic Zucker rats. Taken together, our results show that α(1D)-AR expression is upregulated in the diabetic kidneys. PPAR activation suppressed renal expression of α(1D)-AR in diabetic nephropathy.

Glomerular expression of kidney injury molecule-1 and podocytopenia in diabetic glomerulopathy.

BACKGROUND/AIMS: Studies have shown that kidney injury molecule-1 (KIM-1) is upregulated in damaged renal proximal tubules. In this study, we examined KIM-1 expression in glomerular epithelial cells in diabetic glomerulopathy. METHODS: Renal histology, immunostaining and Western blot for protein level, and real-time PCR for mRNA expression of KIM-1 and podocyte markers were evaluated in untreated or losartan-treated Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats. RESULTS: The diabetic rats showed an increased glomerular expression of KIM-1. KIM-1 staining was localized primarily in the hyperplastic parietal epithelium of Bowman's capsule in the early stages of diabetes with subsequent increase in KIM-1-positive cells in the glomerular tuft in the more advanced stages. The increase in glomerular KIM-1 was associated with a decrease in podocytes in Fa/Fa rats. Antiproteinuric treatment with losartan attenuated podocytopenia and decreased renal expression of KIM-1 in treated diabetic rats. In an in vitro study, albumin overload increased KIM-1 protein in the primary cultures of rat glomerular epithelial cells. CONCLUSION: These results show that glomerular KIM-1 expression was increased, in proportion to the extent of proteinuria and podocytopenia in the diabetic animals, supporting that KIM-1 could be used as a potential biomarker for glomerular injury in proteinuric kidney disease.

Fenofibrate attenuates tubulointerstitial fibrosis and inflammation through suppression of nuclear factor-κB and transforming growth factor-ß1/Smad3 in diabetic nephropathy.

Fibrates, the ligands of peroxisome proliferator-activated receptor-alpha, have been shown to have a renal protective action in diabetic models of renal disease, but the mechanisms underlying this effect are unknown. In the present study, we sought to investigate in greater detail the effect of fenofibrate and its mechanism of action on renal inflammation and tubulointerstitial fibrosis in an animal model of type 2 diabetes mellitus. Twelve-week-old non-diabetic Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with vehicle or fenofibrate for 10 weeks. mRNA and protein analyses were performed by real-time polymerase chain reaction, Western blot and immunostaining. The diabetic condition of ZD rats was associated with an increase in collagen and alpha-smooth muscle actin accumulation in the kidney, which was significantly reduced by fenofibrate. Chronic treatment of ZD rats with fenofibrate attenuated renal inflammation and tubular injury as evidenced by a decrease in mRNA and protein expression of secreted phosphoprotein-1, monocyte chemotactic protein-1 and kidney injury molecule-1 in the kidneys. Renal interstitial macrophage infiltration was also significantly reduced in the kidneys of fenofibrate-treated diabetic animals. Moreover, renal nuclear factor (NF)-kappaB DNA-binding activity, transforming growth factor (TGF)-beta1 and phospho-Smad3 proteins were significantly higher in ZD animals compared with ZL ones. This increase in NF-kappaB activity, TGF-beta1 expression and Smad3 phosphorylation was greatly attenuated by fenofibrate in the diabetic kidneys. Taken together, fenofibrate suppressed NF-kappaB and TGF-beta1/Smad3 signaling pathways and reduced renal inflammation and tubulointerstitial fibrosis in diabetic ZD animals.

Plasma 24,25-dihydroxyvitamin D concentration of Dahl salt-sensitive rats decreases during high salt intake.

Dahl salt-sensitive rats, but not salt-resistant rats, develop hypertension in response to high salt intake. We have previously shown an inverse relationship between plasma 25-hydroxyvitamin D (25-OHD) concentration and blood pressure of Dahl salt-sensitive rats during high salt intake. In this study, we report on the relationship between high salt intake and plasma 24,25-dihydroxyvitamin D (24,25-(OH)(2)D) concentration of Dahl salt-sensitive and salt-resistant rats. Rats were fed a high salt diet (8%) and sacrificed at day 2, 7, 14, 21, and 28. Plasma 24,25-(OH)(2)D concentrations of salt-sensitive rats were reduced to 50% of that at baseline at day 2-when blood pressure and plasma 25-OHD concentration were unchanged, but 25-OHD content in the kidney was 81% of that at baseline. Plasma 24,25-(OH)(2)D concentration was reduced further to 10% of that at baseline from day 7 to 14 of high salt intake, a reduction that was prevented in rats switched to a low salt (0.3%) diet at day 7. Exogenous 24,25-dihydroxycholecalciferol (24,25-(OH)(2)D(3)), administered at a level that increased plasma 24,25-(OH)(2)D concentration to five times normal, did not attenuate the salt-induced hypertension of salt-sensitive rats. Plasma 24,25-(OH)(2)D concentration of salt-resistant rats was gradually reduced to 50% of that at baseline at day 14 and returned to baseline value at day 28 of high salt intake. We conclude that the decrease in plasma 24,25-(OH)(2)D concentration in salt-sensitive rats during high salt intake is caused by decreased 25-OHD content in the kidney and also by another unidentified mechanism.

1A-779 attenuates angiotensin-(1-7) depressor response in salt-induced hypertensive rats.

Chronic infusion of angiotensin-(1-7) [Ang-(1-7)] lowers blood pressure in salt-induced and spontaneously hypertensive (SHR) rats. In the present study, we have examined the acute effect of Ang-(1-7) in salt-induced hypertension using Dahl salt-sensitive rats placed on low (0.3%) or high (8.0% NaCl) salt diets for 2 weeks. Rats fed a high salt diet showed a greater rise in BP than those fed a low salt diet. Ang-(1-7) (24 microg/kg) reduced mean arterial pressure (MAP), enhanced the release of prostacyclin and nitric oxide, and suppressed thromboxane A(2) levels. A-779 (48 microg/kg, i.v), a selective Ang-(1-7) antagonist, partially blocked these effects of Ang-(1-7). The Ang-(1-7)-induced depressor response observed in these animals was related to an increase in vasodilatory prostanoids, a decrease in the constrictor prostanoid thromboxane A(2), and an increase in nitric oxide levels in both plasma and isolated aortic rings.

Ischemia elicits a coordinated expression of pro-survival proteins in mouse myocardium.

Cardiomyocytes are post-mitotic, long-lived cells until disruptions to pro-survival factors occur after myocardial ischemia. To gain an understanding of the factors involved with ischemic injury, we examined expression changes in pro-survival and opposing pro-apoptotic signals at early and chronic periods of ischemia using an in vivo murine model. Alterations of pro-survival proteins such as the inhibitor of apoptosis protein on chromosome X (xIAP) and the apoptotic repressor protein (ARC) have not been evaluated in a murine model of cardiac ischemia. Early ischemia (1 day) resulted in a 50% reduction in ARC protein levels relative to sham-operated left ventricles, without significant changes in the expression of xIAP or other pro-survival factors. In contrast, a deficiency of xIAP expression was found in cardiac infarcts starting after 1 week, concomitant with significant evidence of apoptotic cell death and an up-regulation of pro-apoptotic signals including Bax, tumor necrosis factor-a, and caspase-8 activation. Chronic ischemia (after 2 weeks) was associated with elevated levels of other pro-survival factors such as Bcl-xL and the phosphorylated form of Akt, as part of the adaptive remodeling of the myocardium. Altogether, these findings suggest that strategies to increase IAP expression may promote myocyte survival after chronic ischemia.