Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

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

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

Rab1A-knockdown-induced cell death was caused by inhibition of autophagy but not ER-Golgi traffic.

<p>(A) BFA induced cell death differently from Rab1A knockdown. HT29 cells were transfected with <i>Hsc70</i> or <i>control</i> siRNA and untransfected cells were treated with 5 µg/ml BFA or DMSO at the onset of IncuCyte cell growth assay. Images were captured to monitor proliferation by IncuCyte inside a cell culture incubator. (B) Phase-contrast images at 48 h after the onset of measurement. Scale bar, 200 µm. (C) Inhibition of autophagy was not caused by the interruption of ER-Golgi traffic. Rab1A knockdown or control cells were subjected to serum depletion, 5-FU, or vehicle treatment for 24 h. Untransfected cells were treated with 5 µg/mL BFA for 6 h, and immunoblotted with anti-p62, LC3B, or Rab1A. β-actin was used as a loading control. (D) Hsc70 knockdown allowed autophagosome formation. HT29 cells transfected with <i>Hsc70</i>, <i>Rab1A</i>, <i>Ran</i>, or <i>control</i> siRNA were subjected to serum depletion or vehicle treatment for 24 h, and immunoblotted. Immunoblotting data are representative of at least two separate experiments yielding similar results.</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 interactors identified in serum-depleted and 5-FU-treated HT29 cells.

<p>(A) A schematic view of the identification of Hsc70 interactors. Cells were exposed to serum depletion (SD), 5-FU, or DMSO for 6 h. Hsc70 interactors were isolated with anti-Hsc70 antibodies from the cell extract, and were identified through subsequent analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Identification and functional classification of Hsc70 interactors that were associated with changes in stress response. A Venn diagram depicting Hsc70 interactors that were identified at >95% confidence score (1.3 ProtScore) using ProteinPilot 2.0 software. Column graphs indicate the number and functionality of identified proteins.</p