Yuki Shibayama

Prorenin induces vascular smooth muscle cell proliferation and hypertrophy via epidermal growth factor receptor-mediated extracellular signal-regulated kinase and Akt activation pathway

Background It is widely acknowledged that the (pro)renin receptor mediates angiotensin (Ang) II-dependent and Ang II-independent effects of prorenin. Method We examined the effect of prorenin on vascular smooth muscle cell (VSMC) signal transduction, proliferation, and hypertrophy. Results Recombinant rat prorenin dose-dependently increased extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylation in rat VSMCs. Prorenin also significantly increased cell number, and [3H]-thymidine and [3H]-leucine incorporation, which were attenuated by pretreatment with inhibitors for ERK kinase and phosphatidylinositol 3 kinase. Prorenin was also found to stimulate epidermal growth factor (EGF) receptor and Src phosphorylation. Pretreatment of VSMCs with an EGF receptor tyrosine kinase inhibitor and a Src inhibitor significantly attenuated the prorenin-induced increase in ERK 1/2 and Akt phosphorylation, as well as DNA and protein synthesis. Prorenin-induced phosphorylation of the EGF receptor, ERK 1/2, and Akt, as well as DNA and protein synthesis were all blocked by (pro)renin receptor siRNA, but not by an Ang II type 1 receptor blocker, candesartan, nor an Ang-converting enzyme inhibitor, captopril. Conclusion These results reveal that prorenin directly stimulates VSMC proliferative and hypertrophic changes, dependent on the (pro)renin receptor, independent of Ang II. Furthermore, EGF receptor-mediated ERK 1/2 and Akt activation contributes to prorenin-dependent proliferative and hypertrophic effects in VSMCs.

Oxidative Stress-Induced Glomerular Mineralocorticoid Receptor Activation Limits the Benefit of Salt Reduction in Dahl Salt-Sensitive Rats

Background Mineralocorticoid receptor (MR) antagonists attenuate renal injury in salt-sensitive hypertensive rats with low plasma aldosterone levels. We hypothesized that oxidative stress causes MR activation in high-salt-fed Dahl salt-sensitive rats. Furthermore, we determined if MR activation persisted and induced renal injury, even after switching from a high- to a normal-salt diet. Methods and Findings High-salt feeding for 4 weeks increased dihydroethidium fluorescence (DHE, an oxidant production marker), p22phox (a NADPH oxidase subunit) and serum and glucocorticoid-regulated kinase-1 (SGK1, an MR transcript) in glomeruli, compared with normal-salt feeding, and these changes persisted 4 weeks after salt withdrawal. Tempol treatment (0.5 mmol/L) during high-salt feeding abolished the changes in DHE fluorescence, p22phox and SGK1. Dietary salt reduction after a 4-week high-salt diet decreased both blood pressure and proteinuria, but was associated with significantly higher proteinuria than in normal control rats at 4 weeks after salt reduction. Administration of tempol during high-salt feeding, or eplerenone, an MR antagonist (100 mg/kg/day), started after salt reduction, recovered proteinuria to normal levels at 4 weeks after salt reduction. Paraquat, a reactive oxygen species generator, enhanced MR transcriptional activity in cultured rat mesangial cells and mouse podocytes. Conclusions These results suggest that oxidative stress plays an important role in glomerular MR activation in Dahl salt-sensitive rats. Persistent MR activation even after reducing salt intake could limit the beneficial effects of salt restriction.

(Pro)renin receptor is crucial for Wnt/β-catenin-dependent genesis of pancreatic ductal adenocarcinoma

Although Wnt/β-catenin signaling is known to be aberrantly activated in PDAC, mutations of CTNNB1, APC or other pathway components are rare in this tumor type, suggesting alternative mechanisms for Wnt/β-catenin activation. Recent studies have implicated the (pro)renin receptor ((P)RR) is related to the Wnt/β-catenin signaling pathway. We therefore investigated the possible role of (P)RR in pancreatic carcinogenesis. Plasma s(P)RR levels were significantly (P < 0.0001) higher in patients with PDAC than in healthy matched controls. We also identified aberrant expression of (P)RR in premalignant PanIN and PDAC lesions and all the PDAC cell lines examined. Inhibiting (P)RR with an siRNA attenuated activation of Wnt/β-catenin signaling pathway and reduced the proliferative ability of PDAC cells in vitro and the growth of engrafted tumors in vivo. Loss of (P)RR induced apoptosis of human PDAC cells. This is the first demonstration that (P)RR may be profoundly involved in ductal tumorigenesis in the pancreas.

High glucose augments angiotensinogen in human renal proximal tubular cells through hepatocyte nuclear factor-5

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5’-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy.

(Pro)renin receptor is crucial for glioma development via the Wnt/β-catenin signaling pathway

OBJECTIVE The (pro)renin receptor (PRR) plays an essential role in the early development of the central nervous system by activating the Wnt/β-catenin signaling pathway. The authors investigated the potential role of the PRR in the pathogenesis of glioma. METHODS The authors performed immunohistochemical analysis to detect both the PRR and isocitrate dehydrogenase 1 with mutations involving arginine 132 ( IDH1R132H) in paraffin sections of 31 gliomas. Expression of the PRR and Wnt pathway components in cultured human glioma cell lines (U251MG, U87MG, and T98G) was measured using Western blotting. The effects of PRR short interfering RNA (siRNA) on glioma cell proliferation (WST-1 assay and direct cell counting) and apoptosis (flow cytometry and the caspase-3 assay) were also examined. RESULTS PRR expression was significantly higher in glioblastoma than in normal tissue or in lower grade glioma, regardless of IDH1R132H mutation. PRR expression was also higher in human glioblastoma cell lines than in human astrocytes. PRR expression showed a significant positive correlation with the Ki-67 labeling index, while it had a significant negative correlation with the survival time of glioma patients. Treatment with PRR siRNA significantly reduced expression of Wnt2, activated β-catenin, and cyclin D1 by human glioblastoma cell lines, and it reduced the proliferative capacity of these cell lines and induced apoptosis. CONCLUSIONS This is the first evidence that the PRR has an important role in development of glioma by aberrant activation of the Wnt/β-catenin signaling pathway. This receptor may be both a prognostic marker and a therapeutic target for glioma.

IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression

ABSTRACT Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan‐phosphodiesterase (PDE) inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX) dose and time dependently downregulated hypoxia‐inducible factor 1&agr; (HIF‐1&agr;) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8‐Br‐cAMP agonized the repression of HIF‐1&agr; promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF‐1&agr; promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl2 induced increased HIF‐1&agr; protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl2 and hypoxia induced mRNA expressions of two pro‐fibrogenic factors, platelet‐derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, &bgr;‐catenin; as well as protected against hypoxia induced cell‐death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF‐1&agr;, and thus may attenuate hypoxia induced renal fibrosis. HIGHLIGHTSIBMX dowregulates the mRNA expression of HIF‐1&agr; in tubular epithelial cells.Inhibiting PKA abrogates while activating cAMP enhances the effect of IBMX.IBMX reduces CoCl2 induced enhanced HIF‐1&agr; protein in the nucleus.IBMX downregulates hypoxia or CoCl2 induced enhanced PDGFB and LOX mRNAs.Effects of cAMP‐PKA mediated pathway on HIF‐1&agr; expression may contribute against renal fibrosis.

OS 15-05 HEPATOCYTE NUCLEAR FACTOR -5 ACTS AS A GLUCOSE-RESPONSIVE ELEMENT OF HUMAN ANGIOTENSINOGEN PROMOTER.

Objective: Previous studies have shown that angiotensinogen (AGT) synthesis is enhanced by high glucose (HG) in rat renal proximal tubular cells. We aimed to investigate the glucose-responsive elements within human AGT (hAGT) promoter in human immortalized proximal tubular HK-2 cells. Design and Method: HK-2 cells were treated with normal glucose (NG: 5.5 mmol/L) and high glucose (HG: 15 mmol/L). Human AGT promoter region was cloned from -4358 to +122. Consecutive 5’-end deletion mutant constructs and different site-direct mutagenesis products were transfected into HK-2, followed by promoter activity measurement by dual luciferase assay. Chromatin immunoprecipitation (CHIP) assay was used to confirm protein-DNA binding. Results: Compared to NG, hAGT mRNA and protein levels were significantly increased by HG treatment for 48 hours. Region from -22 to -1,896, whose deletion attenuated the effects of HG on hAGT promoter activity, was selected as glucose-responsive region. Among corresponding transcriptional factors of glucose-responsive regions, hepatocyte nuclear factor (HNF)-5 is involved in glucose metabolism and AGT regulation. Compared to NG, HG effect on promoter activity was negligible in HK-2 transfected with HNF-5 point mutated hAGT promoter, while significantly displayed with intact hAGT promoter transfection. Reduced protein level after HNF-5 point mutation was confirmed by CHIP assay. Conclusions: These data suggest that HG enhances human proximal tubular AGT through the activation of HNF-5.

OS 15-07 ROLE OF (PRO) RENIN RECEPTOR IN THE ACTIVATION OF WNT/ β-CATENIN PATHWAY IN COLON CANCER.

Objective: Constitutive activation of Wnt/&bgr;-catenin pathway plays an important role in the pathogenesis of colon cancer; however, recent studies have also indicated that elevated Wnt levels are essential. In the present study, we investigated the potential role of (pro) renin receptor in the pathophysiology of colon cancer. Design and Method: (P) RR expression in human colon tissues was measured by immunohistochemistry and Western blot analysis. Human colon cancer cell lines DLD-1 with adenomatous polyposis coli (APC) truncated mutation, and HCT116 with&bgr;-catenin activating mutation, were transfected with scramble siRNA and siRNA against (pro) renin receptor. Results: (Pro) renin receptor expression was significantly increased in colon cancer tissue. (Pro) renin receptor knockdown significantly decreased protein levels of the main components of Wnt/&bgr;-catenin pathway (Wnt, phosphorylated-LRP6, active &bgr;-catenin and cyclin D1) in both DLD-1 and HCT116 cells. Wnt signaling activity, measured by Tcf/Wnt signaling reporter assay, was also significantly decreased by (pro) renin receptor knockdown. WST-1 assay showed that (pro) renin receptor knockdown markedly reduced cell proliferation activity. Conclusions: These data suggest that (pro) renin receptor promotes colon cancer through Wnt/&bgr;-catenin pathway despite of intrinsic its constitutive activation.