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Cleveland, OH, United States

Schelling J.R.,Case Western Reserve University | Schelling J.R.,Rammelkamp Center for Education and Research
Pediatric Nephrology | Year: 2016

The longstanding focus in chronic kidney disease (CKD) research has been on the glomerulus, which is sensible because this is where glomerular filtration occurs, and a large proportion of progressive CKD is associated with significant glomerular pathology. However, it has been known for decades that tubular atrophy is also a hallmark of CKD and that it is superior to glomerular pathology as a predictor of glomerular filtration rate decline in CKD. Nevertheless, there are vastly fewer studies that investigate the causes of tubular atrophy, and fewer still that identify potential therapeutic targets. The purpose of this review is to discuss plausible mechanisms of tubular atrophy, including tubular epithelial cell apoptosis, cell senescence, peritubular capillary rarefaction and downstream tubule ischemia, oxidative stress, atubular glomeruli, epithelial-to-mesenchymal transition, interstitial inflammation, lipotoxicity and Na+/H+ exchanger-1 inactivation. Once a a better understanding of tubular atrophy (and interstitial fibrosis) pathophysiology has been obtained, it might then be possible to consider tandem glomerular and tubular therapeutic strategies, in a manner similar to cancer chemotherapy regimens, which employ multiple drugs to simultaneously target different mechanistic pathways. © 2015, IPNA. Source


Madhavan S.M.,Rammelkamp Center for Education and Research | O'Toole J.F.,Rammelkamp Center for Education and Research | Konieczkowski M.,Rammelkamp Center for Education and Research | Ganesan S.,MetroHealth Medical Center | And 3 more authors.
Journal of the American Society of Nephrology | Year: 2011

In patients of African ancestry, genetic variants in APOL1, which encodes apolipoprotein L1, associate with the nondiabetic kidney diseases, focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy (HIVAN), and hypertensive nephropathy. Understanding the renal localization of APOL1 may provide clues that will ultimately help elucidate the mechanisms by which APOL1 variants promote nephropathy. Here, we used immunohistology to examine APOL1 localization in normal human kidney sections and in biopsies demonstrating either FSGS (n = 8) or HIVAN (n = 2). Within normal glomeruli, APOL1 only localized to podocytes. Compared with normal glomeruli, fewer cells stained for APOL1 in FSGS and HIVAN glomeruli, even when expression of the podocyte markers GLEPP1 and synaptopodin appeared normal. APOL1 localized to proximal tubular epithelia in normal kidneys, FSGS, and HIVAN. We detected APOL1 in the arteriolar endothelium of normal and diseased kidney sections. Unexpectedly, in both FSGS and HIVAN but not normal kidneys, the media of medium artery and arterioles contained a subset of α-smooth muscle actin-positive cells that stained for APOL1. Comparing the renal distribution of APOL1 in nondiabetic kidney disease to normal kidney suggests that a previously unrecognized arteriopathy may contribute to disease pathogenesis in patients of African ancestry. Copyright © 2011 by the American Society of Nephrology. Source


Goel M.,Rammelkamp Center for Education and Research | Schilling W.P.,Rammelkamp Center for Education and Research
American Journal of Physiology - Renal Physiology | Year: 2010

The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial Ca2+ flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca2+ entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the Ca2+ permeability of the apical membrane of fura-2-loaded IMCD-3 cells using the Mn2+ quench technique. Mn 2+ influx across the apical membrane was increased 12- to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an IC50 value <100 nM. In contrast, Mn 2+ influx was only increased ∼2-fold by basolateral ATP. Mn 2+ influx was also activated by apical, but not basolateral, 1-stearoyl-2-acetyl-snglycerol (SAG), a known activator of TRPC3 channels. Apical ATPand SAG-induced Mn2+ influx was increased by overexpression of TRPC3 and completely blocked by expression of the dominant negative TRPC3 construct. Mn2+ influx was also stimulated ∼2-fold by thapsigargin applied to either the apical or basolateral side. Thapsigargin-induced flux was blocked by BTP2 but was unaffected by overexpression of TRPC3 or by dominant negative TRPC3. Apical ATP, but not basolateral ATP, increased transepithelial 45Ca2+ flux. These results demonstrate that the apical membrane of IMCD-3 cells has two distinct Ca2+ influx pathways: 1) a store-operated channel activated by thapsigargin and basolateral ATP and 2) TRPC3 channels activated by apical ATP. Only activation of TRPC3 leads to net transepithelial apical-to-basolateral Ca2+ flux. Furthermore, these results demonstrate that native TRPC3 is not a store-operated channel in IMCD-3 cells. Copyright © 2010 the American Physiological Society. Source


Lock J.T.,Rammelkamp Center for Education and Research | Sinkins W.G.,Case Western Reserve University | Schilling W.P.,Rammelkamp Center for Education and Research | Schilling W.P.,Case Western Reserve University
Journal of Physiology | Year: 2012

• In non-excitable cells, oxidative stress increases inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R) activity, which can cause Ca 2+ oscillations under basal conditions and enhance agonist-stimulated changes in cytosolic free Ca 2+ concentration. • Protein S-glutathionylation, the reversible modification of cysteine thiols by glutathione, is elevated in response to oxidative stress, but the consequence of glutathionylation for IP 3R function is not known. • In this study we provide evidence that Ca 2+-induced Ca 2+-release (CICR) via the IP 3R is enhanced by oxidant-induced glutathionylation in cultured aortic endothelial cells. • Our results suggest glutathionylation may represent a fundamental mechanism for regulating IP 3R activity during physiological redox signalling and during pathological oxidative stress. In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca 2+ concentration ([Ca 2+] i) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R) to IP 3. Although thiol modification of the IP 3R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein-glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H 2O 2), on oscillations in [Ca 2+] i in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca 2+, acute treatment with either diamide or H 2O 2 increased the number of BAECs exhibiting asynchronous Ca 2+ oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca 2+ oscillations and BAECs to bradykinin-stimulated Ca 2+ oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca 2+ transient suggesting that Ca 2+-induced Ca 2+ release (CICR) via the IP 3R is enhanced by glutathionylation. Similar to diamide, H 2O 2 increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP 3R 1 is increased in cells challenged with H 2O 2. Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP 3R to Ca 2+, i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP 3R activity during physiological redox signalling and during pathologicalical oxidative stress. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society. Source


Lock J.T.,Case Western Reserve University | Sinkins W.G.,Rammelkamp Center for Education and Research | Schilling W.P.,Rammelkamp Center for Education and Research | Schilling W.P.,Case Western Reserve University
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2011

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+] i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca 2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5- bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N′-bis(2- chloroethyl)-N-nitrosourea or depletion of glutathione by L-buthionine- sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca 2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively. Copyright © 2011 the American Physiological Society. Source

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