Intracellular targets in heme protein-induced renal injury

Intracellular targets in heme protein-induced renal injury. We examined two potential intracellular targets in the glycerol model of acute renal failure, namely, the mitochondrion and the nucleus. Within three hours, alterations in mitochondrial function are already apparent. With either glutamate/malate or succinate/rotenone, state 3 and uncoupled respirations were decreased at three hours, and at 24 hours, such decrements were quite pronounced; in the presence of glutamate/malate, state 2 respiration was also depressed at 24 hours, while with succinate/rotenone state 2 was increased. Marked ultrastructural changes were observed in mitochondria studied at three hours, including the novel finding of degenerate mitochondria in autophagic vacuoles. Since the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to concentrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persistent decline in oxygen consumption until oxygen consumption was virtually undetectable. With higher concentrations of heme, the early increase in oxygen consumption was blunted and the progressive decline in oxygen consumption was hastened. The antioxidant iron chelator, deferoxamine, prevented the early rise in oxygen consumption but did not prevent or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not observed at any time point. At 3 and 24 hours there was DNA injury by the TUNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in the glycerol kidneys at 24 hours but not at three hours. At neither time point was evidence of apoptosis observed by light or electron microscopy. In studies undertaken in cell culture models, heme, at concentrations of 10 μM, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line derived from the distal tubule. At concentrations of 50 μM, heme induced approximately 20% positivity in MDCK cells but none in LLC-PK1 cells by the TUNEL technique. We conclude that mitochondria and nuclei are prominent targets for injury in the glycerol model of acute renal failure. The presence of TUNEL-positive cells in the distal nephron but not at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults

Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype

Altres ajuts: E.S. and M.D. were supported by the generous contribution of Asdent Patients Association. This work was supported in part by grants from Ministerio de Ciencia e Innovación, the Fundación Senefro (SEN2019 to A.M.), and Red de Investigación Renal REDinREN (12/0021/0013). K.A.N. is supported by National Institutes of Health (NIH) DK 47060. A.M. group holds the Quality Mention from the Generalitat de Catalunya.Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial-mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGF β)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGF β -induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair

Renal response to repetitive exposure to heme proteins: Chronic injury induced by an acute insult

Renal response to repetitive exposure to heme proteins: Chronic injury induced by an acute insult.BackgroundRenal diseases are conventionally classified into acute and chronic disorders. We questioned whether acute, reversible, renal insults may be induced to incite a chronic scarring process, employing as an acute insult the glycerol model of heme protein-induced renal injury.MethodsRats were subjected to weekly injections of hypertonic glycerol for up to six months. Renal function was serially determined, and the effect of such insults on renal histology and renal expression of collagen and fibrogenic cytokines was assessed.ResultsAfter the first injection of glycerol, which, expectedly, induced a prompt fall in the glomerular filtration rate (GFR), subsequent injections encountered a remarkable renal resistance in that the fall in GFR was markedly blunted. This resistance to acute decline in renal function in rats subjected to repetitive injections of glycerol was accompanied by less necrosis and apoptosis of renal tubular epithelial cells after such injections. The attenuation in the fall in GFR in response to repetitive exposure to glycerol-induced heme protein injury was maintained for up to six months. A progressive decline in GFR appeared after three months and was accompanied by histologic tubulointerstitial injury, the latter assessed at six months. These kidneys demonstrated up-regulation of collagen I, III, and IV in conjunction with increased expression of the oxidant-inducible, chemotactic cytokine, monocyte chemoattractant protein-1 (MCP-1), and the oxidant-inducible, fibrogenic cytokine, transforming growth factor-β1 (TGF-β1). The exposure of the kidney to a single injection of hypertonic glycerol increased the expression of both cytokines some three to five days following this exposure, while the exposure of NRK 49F cells in culture to an iron-dependent model of oxidative stress also increased expression of TGF-β1 and collagen mRNAs.ConclusionsWe conclude that this nephrotoxic insult, repetitively administered, encounters a resistance in the kidney such that the expected fall in GFR does not occur. However, with time, such resistance is accompanied by a decrease in GFR, the latter associated with chronic tubulointerstitial disease. Thus, a long-term cost is exacted, either along with, or as a consequence of, such resistance. We suggest that chronic up-regulation of such oxidant-inducible genes such as TGF-β1 and MCP-1 contributes to tubulointerstitial disease, and iron-mediated oxidative stress may directly induce TGF-β1