Background/Aims: Renalase, a novel flavoprotein expressed in the kidney and heart, reduces renal tubular necrosis and apoptosis, which suggests that it might protect against necrosis and/or apoptosis in myocardial ischemia reperfusion injury (MIRI). The present study thus explored the effects of renalase on Sprague-Dawley (SD) rats subjected to MIRI. Methods: We used Lentivirus-mediated RNA interference (RNAi) to inhibit the renalase gene expression in the heart tissue via pericardial cavity injection. The MIRI animal modal was established by blocking the left anterior descending artery for 45mins followed by 4h of reperfusion. Real-time PCR and western blotting were used to detect renalase expression in the heart tissue. Double staining and TUNEL were used to detect the necrosis and apoptosis in the myocardial cells, respectively. Results: All rats subjected to MIRI exhibited lower levels of renalase in the heart tissue than did the sham-operated group (P<0.05, n=6). The (RNAi) group rats exhibited lower renalase levels than did the controls and also exhibited more serious necrosis (7.12±0.56% vs. 3.32±0.93%, P<0.05, n=6) and apoptosis (151.8±8.2% vs. 66.8±6.5%, P<0.05, n=6); however, pretreatment with the recombinant renalase significantly reduced myocardial cell necrosis (1.51±0.12% vs. 4.13±0.02%, P<0.05, n=6) and apoptosis (21.3±5.0% vs. 52.6±10.4%, P<0.05, n=6) relative to the control rats. Conclusions: Exogenous recombinant renalase protein reduced myocardial cell necrosis and apoptosis. Recombinant renalase protein might be a new cardiovascular drug for ischemia/IR injury.
We have previously shown that the Na/K‐ATPase (NKA) acts as a receptor for cardiotonic steroids such as ouabain and salt to regulate Src kinase activities (Tian et al., Mol Biol Cell, 2006, 17:317; Ye et al., JBC, 2011). Here, we test the physiological role of this newly discovered signaling mechanism. Activation of NKA signaling by ouabain leads to endocytosis of both NKA and NHE3, which causes a reduction of transcellular Na+ transport in LLC‐PK1 cell monolayer. Expression of the N‐terminus of NKA α1 subunit (NT), which functions as a dominant negative mutant, specifically blocks the NKA signaling and prevents ouabain‐induced inhibition of transcellular Na+ transport. These in vitro experiments suggest that the signaling NKA be involved in the regulation of renal sodium handling and therefore, may play a role in the control of blood pressure (BP). To test this hypothesis in vivo, we have generated NT‐YFP transgenic mice (NT mice) and measured BP after mice were fed with either normal (0.5% NaCl) or high salt diet (4.0%). The high salt diet increased BP from 110 mmHg to 160 mmHg in NT mice, but not in age‐matched littermates. Moreover, renal functional curves reveal a salt‐sensitive hypertension phenotype in NT mice.
Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. CTS are classically known as specific inhibitors of Na/K‐ATPase (NKA) α1 ion‐pumping function, which is critical for Na+ and water reabsorption in the renal proximal tubule (PT). At much lower concentrations, closer to the range of those reported for endogenous CTS in the blood, they also initiate NKA/Src‐mediated signaling in renal cell cultures. To investigate the respective roles of PT NKA α1/Src signaling and ion‐pumping function in physiological salt handling, we developed a PT‐specific NKA α1 knockout (RPTα1−/−) mouse with PT‐specific rescue by wild‐type NKA α1 (RPT α1WT) or a mutant NKA α1 with intact ion‐pumping function and blunted NKA/Src signaling (RPTα1Y260A).The PT‐specific NKA α1 knockout mouse was generated by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed ATP1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce α1WT or α1Y260A expression at the Rosa26 locus. Mice were born with the expected Mendelian ratio and appeared phenotypically normal, with no detectable change in water and food intake, or body weight. Renal phenotyping was further assessed using metabolic cages in 2–4 month‐old male and female mice.PT‐specific KO and rescue was confirmed by immunohistochemistry in PT cells isolated from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice. Kidney size, morphology and structure appeared overall normal following periodic acid shift (PAS) and trichrome staining. Functionally, in contrast with the predicted outcome based solely on the classical enzymatic function of NKA, a significant decrease in daily urine output (0.55±0.2 vs 1.76±0.3 mL/24h in RPTα1+/+, n=8–10, p<0.01) and absolute Na+ excretion (0.13±0.07 vs 0.35±0.0.5 mmol/24h in RPTα1+/+, n=6, p<0.01) were observed in the absence of PT NKA. The urinary clearance of lithium was significantly decreased in RPTα1−/− (1.42±0.3 vs 4.13±0.5 mL/min in RPTα1+/+, n=3, p<0.05), suggesting that the observed increase in Na+ reabsorption originated in the PT. The urinary output was rescued in the RPTα1WT (2.12±0.3 vs 2.16±0.4 mL/24h in RPTα1WTcontrol, n=7–10, p>0.05) but not in the RPTα1Y260A (0.42±0.1 vs 1.56±0.2 mL/24h in RPTα1Y260Acontrol, n=6–8, p<0.01), which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption.These results support a previously unascertained physiological role of NKA/Src signaling in PT Na+ and water reabsorption, and warrant further investigation to assess the mechanism and possible impact on blood pressure regulation.Support or Funding InformationMarshall Institute of Interdisciplinary Research FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BACKGROUND. Two sequelae of non-alcoholic steatohepatitis (NASH), ESLD and HCC, have become the leading causes for liver transplantation in the Western. The present study aims to approach the cellular metabolic disturbances involved in NASH progression that are associated with microbiota community changes. METHODS. Metabolic effects and microbiota community changes were explored in the murine with NASH progression by blocking the Na/K-ATPase/Src/reactive oxygen amplification loop using the synthetic targeting peptide pNaKtide. DNA from the terminal ileum microbiota habitat was obtained and amplified by PCR to develop DNA bacterial phylogenic sequence analysis of wild type and treated animals at 12, 24 and 48 weeks. Induced changes by pSrc normalization at 24 weeks were correlated with liver morphological changes, intestinal CD4+/CD8+ ratio, and liver macrophage CD14+ expression. Differences among groups were evaluated by ANOVA/t-test and Principal Component Analysis (PCA). RESULTS. Microbiota communities varied significantly at all time points (12, 24 and 48 weeks), with an increase of Verrucomicrobia and a decrease of Bacteroidetes and Firmicutes in the HFD group. Microbiota community changes regressed to their wild-type state at 24 weeks on treated animals, and those changes were associated with a decrease in liver inflammation and senescence, lower ileum CD4+/CD8+ T cells and higher liver CD14+ cells (p<0.05). Concomitantly, the metabolic disturbances in our diet-induced NASH model were normalized by NKA/Src signaling blockage and exercise with a paucity of apoptotic activity, mitigation of cell senescence, and regression of liver fibrosis (p<0.01). CONCLUSIONS. pSrc inhibition at caveolar α1-Na/K-ATPase rescinded NASH-related metabolic disturbances establishing resident physiological microbiota communities with concomitant paucity on apoptotic activity and regression of liver fibrosis; effects that were associated with both gut and liver T-lymphocyte responses.
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