Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury

Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury. Recent evidence indicates that transforming growth factor-β1 (TGF-β1) plays an important role in renal fibrosis via stimulation of extracellular matrix synthesis. The present study was undertaken to investigate the role of angiotensin II type I receptor (AT1 receptor) in hypertension-induced renal injury. Twenty-two-week-old stroke-prone spontaneously hypertensive rats (SHRSP), which had established hypertension and moderate renal damage, were orally given TCV-116, a selective non-peptide AT1 receptor antagonist (0.1, 1 or 10 mg/kg/day), enalapril (10 mg/kg/day) or vehicle once a day for 10 weeks. At the end point of the treatment, we examined renal function, the gene expressions of TGF-β1 and extracellular matrix components in the interstitium [collagen types I (COI) and III (COIII), fibronectin (FN)] and the basement membrane (COIV and laminin), and renal microscopic morphology in rats aged 32 weeks. In vehicle-treated 32 week-old SHRSP with renal dysfunction and nephrosclerosis, renal mRNA levels for TGF-β1, COI, COIII, FN, COIV were all several-fold higher than in WKY. Thus, renal TGF-β1 gene expression was enhanced in SHRSP, which may contribute to the increased renal expressions of COI, COIII, FN, COIV in SHRSP. Treatment with TCV-116 (0.1 mg/kg/day) in SHRSP, in spite of no reduction of blood pressure, decreased renal mRNA levels for TGF-β1, COI, COIII, FN, COIV, being accompanied by the significant decrease in urinary protein and albumin excretion, blood urea nitrogen and plasma creatinine. Treatment with TCV-116 (10 mg/kg/day) in SHRSP decreased mRNAs for TGF-β1, COI, COIII, FN and COIV to almost the same levels as WKY, being associated with normalization of urinary protein and albumin excretion and the prevention of nephrosclerosis, as judged by microscopic histological observations. On the other hand, the effects of enalapril (10 mg/kg/day) on the above mentioned mRNA levels, renal function and renal morphology were weaker than those of TCV-116 (10 mg/kg/day) and were as much as TCV-116 (1 mg/kg/day). These results suggest that independently of hypotensive action, AT1 receptor antagonist has a potent renal protective effect by inhibiting the gene expression of renal TGF-β1 and extracellular matrix components

Post-treatment with JP-1302 protects against renal ischemia/reperfusion-induced acute kidney injury in rats

Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors. Keywords: JP-1302, Ischemia/reperfusion, Acute kidney injury, α2C-adrenoceptor, Norepinephrin

Hsc70 Contributes to Cancer Cell Survival by Preventing Rab1A Degradation under Stress Conditions

<div><p>Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.</p></div

Functional annotation of upregulated or downregulated proteins in Rab1A knockdown cells.

<p>HT29 cells were transfected with siRNA for <i>Rab1A</i> or scramble control. Protein identification in Rab1A knockdown cells was performed through quantitative proteomics by stable isotope labeling, using iTRAQ. (A) Upregulated proteins with iTRAQ ratio ≥1.2. (B) Downregulated proteins with iTRAQ ratio <0.8.</p

Rab1A knockdown induced cell death not including apoptosis.

<p>HT29 cells transfected with <i>Hsc70</i>, <i>Rab1A</i>, <i>Ran</i>, or <i>control</i> siRNA were subjected to serum depletion, 5-FU, or vehicle treatment for 24 h. (A) Rab1A knockdown had little effect on the induction of apoptosis. The induction of apoptosis was analyzed by staining with Hoechst 33258. White arrowheads indicate apoptotic nuclei with condensed chromatin. Scale bar, 50 µm. (B, C) The decrease in cell number by Rab1A knockdown was not due to apoptosis. Numbers of total cells and apoptotic cells were quantified by counting Hoechst-stained cells and cells with nuclear condensation in (A), respectively. *, <i>p</i><0.05, **, <i>p</i><0.01 vs. control/veh; ^†, <i>p</i><0.05, ^††, <i>p</i><0.01 vs. control/SD; ^##, <i>p</i><0.01 vs. Rab1A/SD by two-way ANOVA followed by Bonferroni/Dunn post hoc test; values are the means ± S.D. (<i>n</i> = 3). (D) Rab1A suppression did not induce apoptosis. Apoptosis was determined by the cleavages of PARP-1 and caspase-3, detected by immunoblotting. β-actin was used as a loading control. Veh, vehicle. SD, serum depletion. FU, 5-fluorouracil. Immunoblotting data are representative of at least three separate experiments yielding similar results.</p

Hsc70 prevented Rab1A degradation.

<p>(A) Hsc70-Rab1A interaction through chaperone activity. HT29 cells were subjected to serum depletion for 24 h, and then lysed. To verify the interaction between Hsc70 and Rab1A in a chaperone-dependent manner, anti-Hsc70 or anti-Rab1A immunoprecipitant was eluted with ATP, followed by elution with SDS sample buffer and immunoblotting in order to confirm the bait proteins. (B) Hsc70 knockdown decreased the level of Rab1A protein under stress conditions. HT29 cells transfected with <i>Hsc70</i> or <i>control</i> siRNA were subjected to serum depletion, 5-FU, or vehicle treatment for 24 h. Immunoblotting for endogenous Rab1A and Hsc70 proteins. β-actin was used as a loading control. (C) Hsc70 knockdown did not decrease <i>Rab1A</i> mRNA level. After knockdown of Hsc70 and in the control, <i>Rab1A</i> mRNA levels were determined by qPCR at 48 h post-transfection. (D) Hsc70 knockdown promoted the ubiquitination of Rab1A. After Hsc70 knockdown or control cells were lysed, immunoprecipitation (IP) with anti-Rab1A or anti-ubiquitin antibodies was performed, followed by immunoblotting with anti-ubiquitin or anti-Rab1A antibodies. (E) MG132 treatment inhibited Rab1A degradation. Hsc70 knockdown cells were subjected to serum depletion or 5-FU treatment, and then MG132 (10 µM) or vehicle was added for the last 8 h before sampling. SD, serum depletion. FU, 5-fluorouracil. Ub, ubiquitin, IgG LC, immunoglobulin light chain. Data (except in C) are representative of at least two separate experiments yielding similar results.</p