Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury.
Background The redox‐sensitive chaperone DJ ‐1 and uncoupling protein 2 are protective against mitochondrial oxidative stress. We previously reported that renal‐selective depletion and germline deletion of DJ ‐1 increases blood pressure in mice. This study aimed to determine the mechanisms involved in the oxidative stress–mediated hypertension in DJ ‐1 −/− mice. Methods and Results There were no differences in sodium excretion, renal renin expression, renal NADPH oxidase activity, and serum creatinine levels between DJ ‐1 −/− and wild‐type mice. Renal expression of nitro‐tyrosine, malondialdehyde, and urinary kidney injury marker‐1 were increased in DJ ‐1 −/− mice relative to wild‐type littermates. mRNA expression of mitochondrial heat shock protein 60 was also elevated in kidneys from DJ ‐1 −/− mice, indicating the presence of oxidative stress. Tempol‐treated DJ ‐1 −/− mice presented higher serum nitrite/nitrate levels than vehicle‐treated DJ ‐1 −/− mice, suggesting a role of the NO system in the high blood pressure of this model. Tempol treatment normalized renal kidney injury marker‐1 and malondialdehyde expression as well as blood pressure in DJ ‐1 −/− mice, but had no effect in wild‐type mice. The renal Ucp2 mRNA expression was increased in DJ ‐1 −/− mice versus wild‐type and was also normalized by tempol. The renal‐selective silencing of Ucp2 led to normalization of blood pressure and serum nitrite/nitrate ratio in DJ ‐1 −/− mice. Conclusions The deletion of DJ ‐1 leads to oxidative stress–induced hypertension associated with downregulation of NO function, and overexpression of Ucp2 in the kidney increases blood pressure in DJ ‐1 −/− mice. To our knowledge, this is the first report providing evidence of the role of uncoupling protein 2 in blood pressure regulation.
Protein nucleic acid deglycase (DJ‐1) exerts a protective role against oxidative stress. DJ‐1−/− mice have increased systolic BP (30±3%) relative to wild‐type (WT) littermates. This study aimed to determine the mechanisms involved in the oxidative stress‐mediated hypertension due to DJ‐1 germline deletion. At baseline, no differences were found in sodium excretion, renal expression of renin, NADPH activity or serum creatinine levels between DJ‐1−/− and WT mice (n=5–12). Histological assessment by H&E and Picro Sirius demonstrated no kidney morphological abnormalities associated to DJ‐1 deletion. However, renal expression of nitrotyrosine was elevated in DJ‐1−/− mice (+176.8±31% vs. WT mice, n=5), and urinary excretion of the renal damage marker KIM‐1 was increased in DJ‐1−/− mice (148±22% vs WT, n=4), indicating the presence of oxidative stress and kidney damage in DJ‐1−/− mice. In addition, mRNA expression of mitochondrial heat shock protein mtHSP60, but not mtHSP40, was increased in kidneys from DJ‐1−/− mice (2.9±0.1 fold changes of WT, n=4), evidencing mitochondrial oxidative stress in these mice. Conversely, levels of renal endoplasmic reticulum (ER) stress and renal inflammation were similar between the genotypes, indicating that inflammation was not altered in this animal model, despite the high mitochondrial ROS production. Two week treatment with the membrane‐permeable radical scavenger Tempol normalized BP (vehicle vs. tempol: 118±2 vs. 100±1%, n=4, p<0.05), renal malondialdehyde production (160±23% vs 109±15% vs WT, n=4, p<0.05) and KIM‐1 excretion (‐58±3% vs WT, n=4, p<0.05) in DJ‐1−/− mice, but had no effect in WT mice. Serum nitrite/nitrate levels in DJ‐1−/− mice treated with tempol were significantly elevated compared to vehicle‐treated DJ‐1−/− mice (172±30% DJ‐1−/− tempol vs. vehicle, n=4, p<0.05), suggesting a role of nitric oxide on the high blood pressure observed in this model. To examine if lack of DJ‐1 leads to exaggerated renal inflammation and renal damage after an insult, unilateral ureter obstruction (UUO) was performed in DJ‐1−/− and WT mice. After 2 weeks of UUO, increased cortical T‐cell infiltration was observed in WT mice (225±101%; sham vs. UUO, n=3–4; p<0.05) and it was dramatically exaggerated in the DJ‐1−/− mice (67±14%; WT UUO vs. DJ‐1−/− UUO, n=3; p<0.05). In conclusion, deletion of DJ‐1 leads to mitochondrial oxidative stress‐induced hypertension possibly, due to decreased nitric oxide bioavailability, without altering sodium excretion, renal NAPDH activity, inflammation, morphology or ER stress levels. Our results suggest a protective role of DJ‐1 against renal infiltration of T‐cells in the UUO model of kidney disease and may have important implications in the prevention of renal diseases.Support or Funding InformationFunded by NIH T32 DK007545 to CDM and NIH/NHLBI 5P01 HL074940‐10) and NIH/NHLBI 5P01 HL068686‐11 to PAJThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DJ-1 -/- mice, relative to wild-type (WT) littermates, have increased blood pressure (BP) ( DJ-1 -/- :130±4 vs WT:100±3 mmHg, n=6/8). and renal expressions of nitro-tyrosine (+76±31% of WT mice, n=5) and malondialdenyde (+63±23% of WT mice, n=4). Tempol, a superoxide dismutase mimetic, decreased the BP of DJ-1 -/- mice ( DJ-1 -/- : before tempol:119±3; after tempol:100±1 mmHg vs WT, n=4) and renal malondialdehyde production ( DJ-1 -/- : before tempol:+40±5%; after tempol:-24±5% vs WT, n=4) but increased serum nitrate/nitrite levels (+72±30%, n=4), indicating the presence of both oxidative and nitrosative stress. Lack of DJ-1 makes some cells vulnerable to endoplasmic reticulum (ER) stress. However, renal mRNA expression of ER stress markers, GRP94, ATF-4, ATF-6, sXBP-1, CHOP, caspase-12, and caspase-3 was not different between DJ-1 -/- and WT (n=7) mice. Markers of inflammation, IL-6, TNF α, MCP-1, NFκB, and T-cell and macrophage infiltration, were also not increased in the kidney of DJ-1 -/- mice. By contrast, renal mitochondrial (mt) HSP60, but not mtHSP40, was increased in DJ-1 -/- mice (2.9±0.1 fold, n=4) but there were no changes in the renal mRNA expressions of Nix/BNIP3L, BNIP3, PINK, FIS1, MFN1, MFN2, PPRC1, NRF-1, and PGC1, indicating that mt oxidative stress did not affect mt function. The renal expression of UCP2, which is involved in the control of mt-reactive oxygen species production, was elevated in DJ-1 -/- mice (4.1±1.1 fold of WT, n=4). Silencing UCP2 in mouse renal proximal tubule cells (-0.46.5±0.01 fold) increased the expression of ER stress and apoptosis markers CHOP (2±0.4 fold), ATF4 (2.6±0.6 fold), caspase-3 (2.3±0.4 fold), and caspase-12 (1.7±0.2 fold)(n=3). There were no differences in renal renin expression, sodium excretion, and serum creatinine between DJ-1 -/- and WT mice (n=5). There were no abnormalities in renal morphology, including fibrosis, in the kidneys of DJ-1 -/- mice. However, urinary KIM-1 was increased in DJ-1 -/- mice (148±22% of WT mice, n=4) and decreased by tempol (-58±3%, n=4); renal UCP2 expression was also partially normalized by tempol (1.8±0.2 fold of WT, n=4). UCP2 may protect from the development of renal ER stress and damage in the mt oxidative/nitrosative stress associated with DJ-1 depletion.
DJ-1 and uncoupling protein 2 (UCP2) exert protective roles against mitochondrial (MT) oxidative stress. DJ-1 -/- mice have increased systolic blood pressure (BP) (+30±3% vs WT, n=6). This study determined the mechanisms involved in the oxidative stress-mediated hypertension due to DJ-1 germline deletion. There were no differences in sodium excretion, renal renin expression, NADPH oxidase activity and serum creatinine between DJ-1 -/- and WT mice (n=5). However, renal expression of nitro-tyrosine was increased in DJ-1 -/- mice (+176.8±31% vs WT mice, n=5). Tempol, a radical scavenger, normalized the BP (tempol: 118±2% vs 100±1% vs WT, n=4) and renal malondialdehyde (tempol: 160±23% vs 109±15% vs WT, n=4) in DJ-1 -/- mice. Tempol-treated DJ-1 -/- mice had higher serum nitrite/nitrate levels than placebo-treated (172±30% vs WT, n=4). Heat shock protein mtHSP60 was increased in DJ-1 -/- mice (2.9±0.1-fold increase vs WT, n=4), indicating MT stress. However, there were no changes in the renal mRNA expression of mitophagy, MT fusion and MT biosynthesis markers indicating that MT function was not altered. Renal expression of UCP2 was increased in DJ-1 -/- mice (4.1±1.1-fold change vs WT, n=4), and was partially normalized by tempol (1.8±0.2-fold change vs WT, n=4), UCP2 may have a protective role on MT function in this model. UCP2 was selectively silenced via sub-capsular infusion of UCP2 siRNA in the kidney (WT: 63%±7 vs control: DJ-1 -/- :60%±6 vs control; n=4). mRNA expression of mitophagy markers BNIP3 (-0.65±6-fold) and PINK1 (1.55±0.3-fold), MT fusion markers FIS1 (-0.29±0.03-fold) and NFN2 (1.42±0.06-fold), and MT biosynthesis marker PPRC1 (1.71±0.07-fold) were altered by UCP2 silencing in DJ-1 -/- mice (n=4). Renal-selective silencing of UCP2 normalized BP in DJ-1 -/- mice ( DJ-1 -/- mice: 122±5 vs 98±7 mmHg, n=4), and the serum nitrite and nitrate concentrations (-40±9% vs WT, n=4). In conclusion, deletion of DJ-1 leads to oxidative stress-induced hypertension associated with down-regulation of NO synthesis. UCP2 has protective properties against the development of MT dysfunction in MT oxidative stress conditions . However, excessive and chronic over expression of UCP2 could have deleterious consequences on BP regulation.
A lack of diurnal variation in blood pressure is a risk factor for end‐organ disease. Time‐of‐day‐dependent oscillations in biological processes are mediated by a number of oscillating transcription factors, collectively termed the “molecular clock,” that control transcription of various regulators of physiological function. In particular, the genetic disruption of BMAL1 is known to impair blood pressure rhythms in mice. The goal of this feasibility study was to determine if human buccal cell BMAL1 mRNA is expressed in a time‐of‐day‐dependent manner. Following IRB approval, we recruited 11 healthy, adult volunteers (7 females, 4 males; 23‐50 years old) who were not on any medications, and had normal sleep patterns. Volunteers were instructed to swab for buccal cells at 6 AM, 12 noon, 6 PM, and 10 PM for three consecutive days and to return the swabs after the third day for RNA extraction and subsequent analysis by qRT‐PCR. We consistently obtained greater than 1 μg of total RNA in all buccal samples from all participants. BMAL1 mRNA displayed a reproducible rhythmic pattern in 9 of the 11 volunteers over the 3 day period (e.g. Day 1, 6 AM 2248±413, 12 PM 1149±336, 6 PM 715±211,10 PM 904±385 copy number/μg total RNA). These data suggest that human buccal cells exhibit rhythms in the circadian “molecular clock,” and may prove useful as a biomarker for determining mechanisms of circadian rhythm disorders such as nocturnal hypertension.
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