Rosiglitazone Affects Nitric Oxide Synthases and Improves Renal Outcome in a Rat Model of Severe Ischemia/Reperfusion Injury

Background. Nitric oxide (NO)-signal transduction plays an important role in renal ischemia/reperfusion (I/R) injury. NO produced by endothelial NO-synthase (eNOS) has protective functions whereas NO from inducible NO-synthase (iNOS) induces impairment. Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg) was administered i.p. to SD-rats (f) subjected to bilateral renal ischemia (60 min). Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NOx-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3) was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion) and reduces histomorphological injury. Additionally, RGZ reduces NOx plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury

Acidic Environment Leads to ROS-Induced MAPK Signaling in Cancer Cells

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pHe, pHi) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pHi in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pHe. Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H2O2 enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment

Regulation of expression of renal organic anion transporters OAT1 and OAT3 in a model of ischemia/reperfusion injury

Background: Recently, we gained evidence that impairment of rOat1 and rOat3 expression induced by ischemic acute kidney injury (AKI) is mediated by COX metabolites and this suppression might be critically involved in renal damage. Methods: (i) Basolateral organic anion uptake into proximal tubular cells after model ischemia and reperfusion (I/R) was investigated by fluorescein uptake. The putative promoter sequences from hOAT1 (SLC22A6) and hOAT3 (SCL22A8) were cloned into a reporter plasmid, transfected into HEK cells and (ii) transcriptional activity was determined after model ischemia and reperfusion as a SEAP reporter gen assay. Inhibitors or antagonists were applied with the beginning of reperfusion. Results: By using inhibitors of PKA (H89) and PLC (U73122), antagonists of E prostanoid receptor type 2 (AH6809) and type 4 (L161,982), we gained evidence that I/R induced down regulation of organic anion transport is mediated by COX1 metabolites via E prostanoid receptor type 4. The latter signaling was confirmed by application of butaprost (EP2 agonist) or TCS2510 (EP4 agonist) to control cells. In brief, the latter signaling was verified for the transcriptional activity in the reporter gen assay established. Therein, selective inhibitors for COX1 (SC58125) and COX2 (SC560) were also applied. Conclusion: Our data show (a) that COX1 metabolites are involved in the regulation of renal organic anion transport(ers) after I/R via the EP4 receptor and (b) that this is due to transcriptional regulation of the respective transporters. As the promoter sequences cloned were of human origin and expressed in a human renal epithelial cell line we (c) hypothesize that the regulatory mechanisms described after I/R is meaningful for humans as well

The Nephrotoxic Ifosfamide-Metabolite Chloroacetaldehyde Interferes with Renal Extracellular Matrix Homeostasis

BACKGROUND/AIMS Chronic renal proximal tubule dysfunction after therapy with the antineoplastic agent ifosfamide (IFO) is often attributed to the metabolite chloroacetaldehyde (CAA). Chronic IFO-nephropathy is reported to result in tubulointerstitial fibrosis and inflammation. METHODS To elucidate possible effects of CAA on extracellular matrix homeostasis, we investigated the action of CAA on markers of extracellular matrix (ECM) homeostasis in human proximal tubule cells (RPTEC) by use of direct ELISA for extracellular collagens and gelatin zymography. RESULTS An increase in type III collagen and a decrease in type IV collagen abundance in the media of RPTEC could be observed after exposure to CAA in clinically relevant concentrations. CAA increased intracellular type III and decreased intracellular type IV collagen. MMP-2 activity was decreased but MMP-9 activity unchanged. The enhanced CAA-induced collagen III formation could be attenuated by the intracellular Ca(2+)-chelator BAPTA-AM, the PKA-antagonist H-89 and by extracellular acidification. CAA-induced collagen III abundance was enhanced by db-cAMP and IBMX and by protein overload. CONCLUSIONS CAA exerts profibrotic effects on RPTEC dependent on Ca(2+) and cAMP/PKA-signaling. These effects are enhanced by additional protein burden and attenuated by acidification. © 2014 S. Karger AG, Basel

Transforming growth factor-β1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells

Transforming growth factor (TGF)-β1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial-mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF-β1-induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin-cubilin-dependent receptor-mediated endocytosis. In this study we used a well established cell culture model (proximal-tubule-derived opossum kidney (OK) cells) in order to test the hypothesis that TGF-β1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-β1 led to a time- and dose-dependent downregulation of megalin-cubilin-mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF-β1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-β1-induced downregulation of megalin-cubilin-mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF-β1. In conclusion our data indicate that enhanced levels of TGF-β1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-β1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis