► Renal calpain 10 decreased 4weeks after streptozotocin-induced diabetes in rats. ► Renal mitochondrial dysfunction was detected in diabetic rats. ► siRNA knockdown of renal calpain 10 in rats ...revealed similar mitochondrial defects. ► Renal calpain 10 loss may be responsible for mitochondrial dysfunction in diabetes.
We showed that renal calpain 10, a mitochondrial and cytosolic Ca2+-regulated cysteine protease, is specifically decreased in kidneys of diabetic rats and mice, and is associated with diabetic nephropathy. The goals of this study were to examine renal calpain 10 and mitochondrial dysfunction in streptozotocin-induced hyperglycemic rats and determine the effects of siRNA-mediated knock down of renal calpain 10 on mitochondrial function. Four weeks after streptozotocin injection, calpain 10 protein and mRNA were decreased and calpain 10 substrates accumulated. We detected increased state 2 respiration in isolated renal mitochondria and increased markers of mitochondrial fission and mitophagy. All changes were prevented by daily insulin injection. Compared to scrambled siRNA, calpain 10 siRNA resulted in a marked decrease in renal calpain 10 at 2, 5 and 7days. In concert with the loss of renal calpain 10, calpain 10 substrates accumulated, mitochondrial fusion decreased, mitochondrial fission and mitophagy increased. In summary, insulin-sensitive hyperglycemia induced loss of renal calpain 10 is correlated with renal mitochondrial dysfunction, fission and mitophagy, and specific depletion of renal calpain 10 produces similar mitochondrial defects. These results provide evidence that diabetes-induced renal mitochondrial dysfunction and renal injury may directly result from the loss of renal calpain 10.
Ischemia is a leading cause of acute renal failure (ARF), a disease associated with high morbidity and mortality. Disruption of intercellular adhesion in the proximal tubules is linked to ARF, ...although the molecular mechanism(s) remains unclear. Our previous studies showed that ischemia is associated with cadherin cleavage and loss in NRK cells, putatively due to a matrix metalloproteinase (MMP) (7). In the current studies, a MMP required for E-cadherin cleavage and N-cadherin loss was identified. Chemical inhibitors against a number of soluble MMPs (1, 2, 3, 8, 9) failed to completely attenuate ischemia-induced cadherin loss. Under ischemic conditions, there was an increase in active membrane-type (MT)1-MMP but a decrease in MMP-2 protein expression. Plating cells on fibronectin protected against ischemia-induced loss of cadherins and, interestingly, no increase in active MT1-MMP levels was seen in ischemic cells on fibronectin-coated dishes. In addition, L cells stably expressing E- (LE) or N-cadherin (LN), but lacking MT1-MMP expression, were resistant to ischemia-induced cadherin loss. The role of MT1-MMP in ischemia-induced cadherin loss was confirmed by either blocking MT1-MMP activity with a neutralizing antibody or expression with shRNA constructs which protected full-length E- and N-cadherin during ischemia. Using shRNA constructs to suppress MT1-MMP expression, ischemia-induced disruption of cadherin function was ablated, and cell-cell contacts were preserved. These results demonstrate that ischemia induces increased expression of active MT1-MMP and subsequent disruption of cadherin/catenin complexes, implying that MT1-MMP plays a role in ischemia-induced ARF.
Calpains, Ca2+-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in ...Ca2+-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75kDa (calpain 10a), 56kDa (calpain 10c or 10d), and 50kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.
Our previous studies showed that renal proximal tubular cells (RPTC) express Ca2+-independent phospholipase A2γ (iPLA2γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA2γ prevents and/or ...repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA2γ in mitochondrial and cell function using an iPLA2γ-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA2γ expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA2γ. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA2γ-shRNA-treated cells. At 48 h after exposure to the iPLA2γ shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA2γ expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA2γ expression in RPTC. These results suggest that iPLA2γ is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA2γ from oxidative stress.
Although ischemia has been shown to disrupt cell adhesion, the underlying molecular mechanism is unknown. In these studies, we adapted a model of ischemia-reperfusion to normal rat kidney (NRK) ...cells, examined disruption of the cadherin/catenin complex, and identified a role for matrix metalloproteinases (MMPs) in ischemia-induced cleavage of cadherins. In NRK cells, ischemia was induced by applying a thin layer of PBS solution supplemented with calcium and magnesium and a layer of mineral oil, which restricts exposure to oxygen. NRK cells exhibited extracellular 80-kDa and intracellular 40-kDa E-cadherin fragments after 4 h of ischemia, and at 6 h the expression of full-length E-cadherin decreased. While no fragments of N-cadherin, alpha-catenin, and gamma-catenin were observed at any time point, the detectable levels of these proteins decreased during ischemia. Ischemia was detected by an increase in pimonidazole adducts, as well as an increase in glucose transporter-1 protein expression. Ischemia did not decrease cell number, but there was a decrease in ATP levels. In addition, there was no evidence of cleaved caspase 3 or 9 during 6 h of ischemia. The MMP inhibitors GM-6001 and TAPI-O inhibited cleavage and/or loss of E- and N-cadherin protein expression. Tissue inhibitors of metalloproteinases (TIMP)-3 and to a lesser extent TIMP-2, but not TIMP-1, inhibit ischemic cleavage and/or loss of E- and N-cadherin. These results demonstrate that ischemia induces a selective metalloproteinase-dependent cleavage of E-cadherin and decrease in N-cadherin that are associated with a disruption of junctional contacts.
Ethically cleared to launch? Rahimzadeh, Vasiliki; Fogarty, Jennifer; Caulfield, Timothy ...
Science (American Association for the Advancement of Science),
09/2023, Letnik:
381, Številka:
6665
Journal Article
Recenzirano
Rules are needed for human research in commercial spaceflight
Massive public and private investment in scientific research has enabled the commercial spaceflight industry to expand opportunities in ...space beyond primarily government-sponsored missions (
1
). Commercial companies endeavor to fly thousands of commercial spaceflight participants (cSFPs) and workers to space in the decades ahead (
2
). Although the future of safe commercial spaceflight depends on rigorous and inclusive research, the ethical conduct of such research is complicated by scientific uncertainty, high attendant risks (
3
), and poorly defined rules for research ethics oversight within the commercial spaceflight industry. Now is the opportune time to develop clear rules for ethical cSFP research while space activities are ramping up and the regulatory environment for commercial spaceflight is actively being shaped. We propose an ethical framework based on terrestrial human research that is anchored in four guiding principles—social responsibility, scientific excellence, proportionality, and global stewardship—and is applicable to the responsible conduct of research in commercial spaceflight.
Abstract only
Aging is associated with abnormalities in kidney function, but the exact mechanisms or causes are unknown. We examined calpain 10 protein expression in aging kidneys from rats, mice, ...and humans. Calpain 10 protein expression decreased in the kidney of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. However, there was no change in calpain 10 protein expression in the liver nor in calpain 1 or 2 protein expression in the kidney or liver in normal and caloric restricted Fischer 344 rats. A decrease in calpain 10 protein expression was also seen in aging mice and human kidneys in the absence of a change in calpain 1 or 2. We next examined the functional consequences of calpain 10 protein loss. A single intravenous dose of a siRNA directed against calpain 10 decreases calpain 10 protein in rat kidney cortex. Calpain 10 siRNA had no effect on kidney cortex calpain 1, demonstrating calpain isoform specificity, and calpain 10 siRNA had no effect on liver calpain 10 levels, demonstrating renal specificity. Importantly, serum creatinine levels increased at days 7 and 10 in rats treated with calpain 10 siRNA, suggesting loss of calpain 10 results in decreased renal function. In summary, we have shown calpain 10 protein levels are decreased in aging rat, mice, and human kidney tissues and loss of calpain 10 in vivo results in decreased kidney function.
Although ischemia is associated with disruption of cadherin-mediated adhesion in renal cell lines, the impact of decreased cadherin function on the transcriptional activity of β-catenin remains ...poorly defined. In these studies, we used a simulated ischemia model in normal rat kidney (NRK) cells to disrupt cadherin function. Cell viability; cadherin/catenin expression, function, and localization; and β-catenin-mediated transcriptional activity were assessed during ischemia/reperfusion. Following 6 hr of ischemia, a decrease in the expression of E- and N-cadherin was seen that correlated with altered cell morphology indicative of decreased intercellular adhesion. While ischemia was associated with activation of glycogen synthase kinase 3 beta (GSK-3β), this did not correlate with increased phosphorylation of β-catenin as assessed by Western blots using phosphoryl-specific antibodies. β-Catenin was not localized to the nucleus by immunofluorescence in ischemic NRK cells, but rather a strong perinuclear signal was seen in reperfused cells. This was consistent with the finding that neither ischemia nor reperfusion activated the transcriptional activity of β-catenin as assessed by the TCF-optimal promoter (TOPFlash) construct. However, NRK cells possess a competent Wnt pathway, as challenge with lithium chloride elicited a ten-fold increase in luciferase activity. These results suggest that ischemia-induced disruption of cadherin/catenin complexes is not sufficient to stimulate β-catenin transcriptional activity in NRK cells.
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