Renal Endoplasmic Reticulum Stress Research Articles

Inhibition of endoplasmic reticulum stress restores the balance of renal RAS components and lowers blood pressure in the spontaneously hypertensive rats

ABSTRACT Background Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of hypertension. However, the underlying mechanisms for lowering blood pressure (BP) by suppressing ER stress remain unclear. Here, we hypothesized that inhibition of ER stress could restore the balance between RAS components and lower BP in spontaneously hypertensive rats (SHRs). Methods Wistar-Kyoto (WKY) rats and SHRs received vehicle or 4-PBA, an ER stress inhibitor, in the drinking water for 4 weeks. BP was measured by tail-cuff plethysmography, and the expression of RAS components was examined by Western blot. Results Compared with vehicle-treated WKY rats, vehicle-treated SHRs exhibited higher blood pressure and increased renal ER stress and oxidative stress, accompanied by impaired diuresis and natriuresis. Moreover, SHRs had higher ACE and AT1R and lower AT2R, ACE2, and MasR expressions in the kidney. Interestingly, 4-PBA treatment improved impaired diuresis and natriuresis and lowered blood pressure in SHRs, accompanied by reducing ACE and AT1R protein expression and increasing AT2R, ACE2, and MasR expression in the kidneys of SHRs. In addition, these changes were associated with the reduction of ER stress and oxidative stress. Conclusions These results suggest that the imbalance of renal RAS components was associated with increased ER stress in SHRs. Inhibition of ER stress with 4-PBA reversed the imbalance of renal RAS components and restored the impaired diuresis and natriuresis, which, at least in part, explains the blood pressure-lowering effects of 4-PBA in hypertension.

Bilirubin improves renal function by reversing the endoplasmic reticulum stress and inflammation in the kidneys of type 2 diabetic rats fed high-fat diet

Diabetic kidney disease (DKD), as a chronic diabetes-induced complication, is considered the most frequent leading cause of end-stage renal disease (ESRD). Regarding the observed protective effects of bilirubin, as a potential endogenous antioxidant/anti-inflammatory compound, against DKD progression, we planned to evaluate the effects of bilirubin administration on endoplasmic reticulum (ER) stress and inflammation in type 2 diabetic (T2D) rats fed high-fat diet (HFD). In this regard, thirty 8-week adult male Sprague Dawley rats were divided into five groups (n = 6). T2D and obesity were induced by streptozotocin (STZ) (35 mg/kg) and HFD (700 kcal/day), respectively. Bilirubin treatment was carried out for 6- and 14-week intervals (10 mg/kg/day), intraperitoneally. Then, the expression levels of ER stress-related genes (i.e. binding immunoglobulin protein (Bip), C/EBP homologous protein (Chop), and spliced x-box-binding protein 1 (sXbp1), as well as nuclear factor-κB (NF-κB) were analyzed using quantitative Real-time PCR experiments. Moreover, histopathological and stereological changes of kidney and its related structures were investigated for the studied rats. Bip, Chop, and NF-κB expression levels were significantly decreased under bilirubin treatment, while sXbp1 was up-regulated following the bilirubin administration. More interestingly, glomerular constructive damages seen in HFD-T2D rats, were considerably improved in the animals received bilirubin. Stereological assessments also revealed that bilirubin could desirably reverse the mitigation of kidney's total volume and its related structures, such as cortex, glomeruli, and convoluted tubules. Taken together, bilirubin has potential protective/ameliorative effects on DKD progression, especially through alleviating the renal ER stress and inflammatory responses in T2D rats with injured kidneys. In this era, clinical benefits of mild hyperbilirubinemia can be considered in human DKD.

PRE-084 ameliorated kidney injury by reducing endoplasmic reticulum stress in the rat model of adenine-induced chronic kidney disease.

Endoplasmic reticulum (ER) stress plays an important role in the development of chronic kidney disease (CKD). Sigma-1 receptors (σ1Rs) are novel chaperone proteins that regulate ER stress. However, effect of σ1R activation on renal ER stress is yet unexplored. So, in the present study we investigated the effects of PRE-084, aσ1R agonist on renal injury and ER stress in the rat model of CKD. CKD group rats were fed adenine for 28 days and CKD treatment group rats were additionally administered PRE-084 intraperitoneally at 1, 3 and 10mg/kg body weight dose from Day 22-28. ER stress markers were evaluated using molecular biology techniques such as immunohistochemistry and Western blot. Marked kidney injury was observed in CKD rats as revealed by biochemical and histological findings. Expression of ER stress proteins such as phosphorylated protein kinase R-like ER kinase (p-PERK), cleaved activating transcription factor-6 (ATF-6f), phosphorylated inositol requiring enzyme1α (p-IRE1α) and caspase-12 were higher in CKD rats. Nevertheless, CKD rats treated with PRE-084 particularly at 10mg/kg dose showed considerably lesser kidney injury along with higher expression of σ1R and marked reduction of all the ER stress proteins studied. Results reveal that PRE-084 likely ameliorated the adenine-induced kidney injury by lowering ER stress through increased σ1R expression.

Epigallocatechin-3-gallate ameliorates renal endoplasmic reticulum stress-mediated inflammation in type 2 diabetic rats.

Epigallocatechin-3-gallate (EGCG), an essential polyphenolic constituent found in tea leaves, possesses various potent biological activities. This research was undertaken to investigate the impact of EGCG against endoplasmic reticulum (ER) stress-mediated inflammation and to clarify the underlying molecular mechanism in type 2 diabetic kidneys. The male rats were randomized into four groups: normal, diabetic, low-dose EGCG, and high-dose EGCG. In type 2 diabetic rats, hyperglycemia and hyperlipidemia noticeably caused renal structural damage and dysfunction and aggravated ER stress. Meanwhile, sustained ER stress activated the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and then upregulated the contents of inflammatory cytokines in the diabetic kidney. Following supplementation with 40 mg/kg and 80 mg/kg EGCG, hyperglycemia, hyperlipidemia, and renal histopathological alterations and dysfunction were noticeably ameliorated; renal ER stress, NLRP3 inflammasome, and inflammatory response were markedly repressed in the EGCG treatment groups. In summary, the current study highlighted the renoprotective effects of EGCG in type 2 diabetes and its mechanisms are mainly associated with the repression of ER stress-mediated NLRP3 inflammasome overactivation.

TLR4 Promotes ER stress and Unfolded Protein Response in Hypertension‐Induced Kidney Injury

Hypertension is the second leading cause of chronic kidney injury in the world. Endoplasmic reticulum (ER) is an important cell organelle that is involved in the synthesis, folding, and modification of various proteins to maintain homeostasis. ER stress is reported in hypertension‐induced damage of kidney, heart and brain. Further, TLR4 activation and signaling is linked to the development and progression of hypertension induced damage in various organs; however, the interplay between TLR4 and ER stress in renal pathology remains unknown. In the present study, we investigated whether TLR4 activation contributes to renal ER stress and inflammation in Angiotensin‐II (Ang‐II) induced hypertension and whether TLR4 deficiency protects the kidney by suppressing ER stress. C3H/HeouJ (Normal TLR4, TLR4N) and C3H/HeJ (Dysfunctional TLR4, TLR4M) aged 12‐14 weeks were treated with Ang‐II (1000 ng/Kg/min. x 28 days) using osmotic pumps. We found renal function was impaired in TLR4N mice compared to TLR4M. The expression of ER stress markers (ATF6, p‐IRE1α and p‐PERK), renal inflammation (TNFα, IL‐1β, CCL2) and transcription factor p‐NFkB was upregulated in TLR4N kidneys compared to TLR4M. Further, ER stress in TLR4N mice was associated with mRNA changes in GRP78, sXBP1, ATF4, CHOP genes that are involved in unfolded protein response. In TLR4N mice, renal inflammation was predominant in the tubular area and ER stress was observed in both glomerular and tubular area. TLR4M mice showed reduced ER stress and inflammation in the kidney. Taken together, our results suggest significant interaction between TLR4 and ER stress that may act as an obligatory step in mediating renal inflammation and damage, and TLR4 deficiency attenuates injury by regulating ER stress and unfolded protein response in Ang‐II‐induced hypertension.

Curcumin protects rats against gentamicin-induced nephrotoxicity by amelioration of oxidative stress, endoplasmic reticulum stress and apoptosis

Context Gentamicin (GM) is an aminoglycoside antibiotic which is commonly used against Gram-negative bacterial infection; however, serious complications including nephrotoxicity could limit its clinical use. Objective The present study examined the protective effects of curcumin (CUR) on endoplasmic reticulum (ER) stress-mediated apoptosis through its antioxidative property in GM-induced nephrotoxicity in rats. Materials and methods Male Sprague-Dawley rats (n = 3) were divided into six groups to receive normal saline (control), GM (100 mg/kg/day), co-treatment with GM and CUR (100, 200 and 300 mg/kg/day) and CUR (200 mg/kg/day) alone for 15 days by gavage feeding. Then, the renal function, kidney injury as well as oxidative stress, antioxidative markers and ER stress-mediated apoptosis were evaluated. Results Pre-treatment of CUR rescued the nephrotoxicity in GM-treated rats. Several nephrotoxicity hallmarks were reversed in the CUR-pre-treatment group. At the dose of 200 mg/kg/day, it could significantly lower serum creatinine (from 0.95 to 0.50 mg/dL), blood urea nitrogen (from 35.00 to 23.50 mg/dL) and augmented creatinine clearance (from 0.83 to 1.71 mL/min). The normalized expression of oxidative stress marker, malondialdehyde was decreased (from 13.00 to 5.98) in line with the increase of antioxidant molecules including superoxide dismutase (from 5.59 to 14.24) and glutathione (from 5.22 to 12.53). Furthermore, the renal ER stress and apoptotic protein biomarkers were lowered in CUR treatment. Discussion and conclusions Our findings pave the way for the application of CUR as a supplement in the prevention of nephrotoxicity and other kidney diseases in the future.

Investigation of the Crosstalk between GRP78/PERK/ATF-4 Signaling Pathway and Renal Apoptosis Induced by Nephropathogenic Infectious Bronchitis Virus Infection.

This study aims to explore the crosstalk between GRP78/PERK/ATF-4 signaling pathway and renal apoptosis induced by nephropathogenic infectious bronchitis virus (NIBV). Hy-Line brown chickens were divided into two groups (Con, n = 100 and Dis, n = 200). At 28 days of age, each chicken in the Dis group was intranasally injected with SX9 strain (10-5/0.2 ml). Venous blood and kidney tissues were collected at 1, 5, 11, 18 and 28 days postinfection. Our results showed that NIBV infection upregulated the levels of creatinine, uric acid, and calcium (Ca2+) levels. Histopathological examination revealed severe hemorrhage and inflammatory cell infiltration near the renal tubules. Meanwhile, NIBV virus particles and apoptotic bodies were observed by ultramicro electron microscope. In addition, RT-qPCR and Western blot showed that NIBV upregulated the expression of GRP78, PERK, eIF2α, ATF-4, CHOP, Caspase-3, Caspase-9, P53, Bax, and on the contrary, downregulated the expression of Bcl-2. Furthermore, immunofluorescence localization analysis showed that the positive expression of Bcl-2 protein was significantly decreased. Correlation analysis indicated that endoplasmic reticulum (ER) stress gene expression, apoptosis gene expression, and renal injury were potentially related. Taken together, NIBV infection can induce renal ER stress and apoptosis by activating of GRP78/PERK/ATF-4 signaling pathway, leading to kidney damage. IMPORTANCE Nephropathogenic infectious bronchitis virus (NIBV) induced renal endoplasmic reticulum stress in chickens. NIBV infection induced kidney apoptosis in chickens. GRP78/PERK/ATF-4 signaling pathway is potentially related to renal apoptosis induced by NIBV.

A deleterious interplay between endoplasmic reticulum stress and its functional linkage to mitochondria in nephrolithiasis

Hyperoxaluria is one of the leading causes of calcium oxalate stone formation in the kidney. Since hyperoxaluria produces Endoplasmic Reticulum (ER) stress in the kidney, it is thus likely that the adaptive unfolded protein response might affect the mitochondrial population as ER and mitochondria share close physical and functional interactions mandatory for several biological processes. Thus this work was designed to study the putative effects of endoplasmic reticulum stress on the renal mitochondria during hyperoxaluria-induced nephrolithiasis. The results showed that hyperoxaluria induced an ER stress led to the unfolded protein response in the renal tissue of experimental rats. Hampered mitochondrion functioning was detected with decreased mitochondrial membrane potential and upsurged mitochondria calcium. These changes in the mitochondria function and ER stress are preceded by apoptosis. The expression of Sigma-1 receptor protein found in the Mitochondria associated ER membranes, the connecting link between ER and mitochondria was found to decrease in the hyperoxaluric rats. Inhibition of ER stress by 4-Phenylbutyric acid prevented the decrease in mitochondria membrane potential and increase in mitochondria calcium observed in hyperoxaluric rats. Also, it restored the protein expression of the sigma-1 receptor protein. On the other hand, N-acetyl cysteine had a nominal impact on the reduction of the ER stress-induced mitochondrial dysfunction. In conclusion, our data showed that hyperoxaluria induces renal ER stress which triggers mitochondria dysfunction, might be via alteration in the sigma-1 receptor protein in the mitochondria-associated ER membranes, which leads to apoptosis, renal injury, and calcium oxalate crystal deposition.

Melatonin Improves Endoplasmic Reticulum Stress-Mediated IRE1α Pathway in Zücker Diabetic Fatty Rat.

Obesity and diabetes are linked to an increased prevalence of kidney disease. Endoplasmic reticulum stress has recently gained growing importance in the pathogenesis of obesity and diabetes-related kidney disease. Melatonin, is an important anti-obesogenic natural bioactive compound. Previously, our research group showed that the renoprotective effect of melatonin administration was associated with restoring mitochondrial fission/fusion balance and function in a rat model of diabesity-induced kidney injury. This study was carried out to further investigate whether melatonin could suppress renal endoplasmic reticulum (ER) stress response and the downstream unfolded protein response activation under obese and diabetic conditions. Zücker diabetic fatty (ZDF) rats and lean littermates (ZL) were orally supplemented either with melatonin (10 mg/kg body weight (BW)/day) (M–ZDF and M–ZL) or vehicle (C–ZDF and C–ZL) for 17 weeks. Western blot analysis of ER stress-related markers and renal morphology were assessed. Compared to C–ZL rats, higher ER stress response associated with impaired renal morphology was observed in C–ZDF rats. Melatonin supplementation alleviated renal ER stress response in ZDF rats, by decreasing glucose-regulated protein 78 (GRP78), phosphoinositol-requiring enzyme1α (IRE1α), and ATF6 levels but had no effect on phospho–protein kinase RNA–like endoplasmic reticulum kinase (PERK) level. In addition, melatonin supplementation also restrained the ER stress-mediated apoptotic pathway, as indicated by decreased pro-apoptotic proteins phospho–c–jun amino terminal kinase (JNK), Bax, and cleaved caspase-3, as well as by upregulation of B cell lymphoma (Bcl)-2 protein. These improvements were associated with renal structural recovery. Taken together, our findings revealed that melatonin play a renoprotective role, at least in part, by suppressing ER stress and related pro-apoptotic IRE1α/JNK signaling pathway.

The Role of the Unfolded Protein Response on Renal Lipogenesis in C57BL/6 Mice.

Renal injury observed in several pathologies has been associated with lipid accumulation in the kidney. While it has been suggested that the accumulation of renal lipids depends on free fatty acids released from adipose tissue, it is not known whether in situ renal lipogenesis due to endoplasmic reticulum (ER) stress contributes to kidney injury. The aim of the present study was to elucidate the role of pharmacological ER stress in renal structure and function and its effect on renal lipid metabolism of C57BL/6 mice. ER stress increased serum creatinine and induced kidney structural abnormalities. Tunicamycin-administered mice developed hyperinsulinemia, augmented lipolysis and increased circulating leptin and adiponectin. Renal unfolded protein response (UPR) gene expression markers, the lipogenic transcription factor SREBP1 and the phosphorylation of eIF2α increased 8 h after tunicamycin administration. At 24 h, an increase in BiP protein content was accompanied by a reduction in p-eIF2α and increased SREBP-1 and FASn protein content, in addition to a significant increase in triglyceride content and a reduction in AMPK. Thus, ER stress induces in situ lipid synthesis, leading to renal lipid accumulation and functional alterations. Future pharmacological and/or dietary strategies must target renal ER stress to prevent kidney damage and the progression of metabolic diseases.

TMEM33 regulates intracellular calcium homeostasis in renal tubular epithelial cells

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.

Dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, slows the progression of renal complications through the suppression of renal inflammation, endoplasmic reticulum stress and apoptosis in prediabetic rats.

To evaluate the renoprotective roles of dapagliflozin in prediabetic rats in order to elucidate the effects of this sodium-glucose co-transporter-2 (SGLT2) inhibitor on the renal complications associated with metabolic dysfunction in diet-induced obesity. Obesity was induced by feeding a high-fat diet (HFD) to male Wistar rats for 16 weeks. HFD-fed rats were treated with dapagliflozin (1 mg/kg/d) or metformin (30 mg/kg/d) by oral gavage for 4 weeks after insulin resistance had been established. The metabolic characteristics and renal function associated with lipid accumulation, inflammation, fibrosis, endoplasmic reticulum (ER) stress and apoptosis in the renal tissue were examined. The results showed that HFD-fed rats developed both obesity and impaired renal function, along with increased renal triglyceride accumulation. Importantly, dapagliflozin had greater efficacy in improving renal function and reducing both body weight and visceral fat accumulation than metformin treatment. Dapagliflozin and metformin were found to have similar effects regarding the suppression of renal triglycerides, superoxide dismutase (SOD) expression and malondialdehyde (MDA) levels, subsequently leading to a decrease in renal inflammation and fibrosis. Renal ER stress and apoptosis were increased in HFD-fed rats and were effectively reduced after administration of dapagliflozin. The expression of renal SGLT2 was not affected by administration of dapagliflozin or metformin. Collectively, these findings indicate that dapagliflozin exerts renoprotective effects by alleviating obesity-induced renal inflammation, fibrosis, ER stress, apoptosis and lipid accumulation in the prediabetic condition.

Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.

Progress has been made in our understanding of the mechanisms of endoplasmic reticulum (ER) proteostasis, ER stress and the unfolded protein response (UPR), as well as ER stress-induced autophagy, in the kidney. Experimental models have revealed that disruption of the UPR, including a protein that senses misfolded proteins (namely, inositol-requiring enzyme 1α) in mouse podocytes causes podocyte injury and albuminuria as mice age. Protein misfolding and ER stress are evident in various renal diseases, including primary glomerulonephritides, glomerulopathies associated with genetic mutations, diabetic nephropathy, acute kidney injury, chronic kidney disease and renal fibrosis. The induction of ER stress may be cytoprotective, or it may be cytotoxic by activating apoptosis. The UPR may interact in a coordinated manner with autophagy to alleviate protein misfolding and its consequences. Monitoring the excretion of ER chaperones into the urine can potentially serve as a biomarker of renal ER stress. In specific kidney diseases, the treatment of experimental animals with chemical chaperones that improve protein folding or with chaperone inducers has alleviated kidney injury. Given the limited availability of mechanism-based therapies for kidney diseases, normalization of ER stress using pharmacological agents represents a promising therapeutic approach towards preventing or arresting the progression of kidney disease.

Renal endoplasmic reticulum stress is coupled to impaired autophagy in a mouse model of GSD Ia.

GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia.

The superoxide dismutase mimetic tempol blunts diabetes-induced upregulation of NADPH oxidase and endoplasmic reticulum stress in a rat model of diabetic nephropathy

Endoplasmic reticulum (ER) stress contributes to progression of diabetic nephropathy, which promotes end-stage renal failure in diabetic patients. This study was undertaken to investigate the actions of tempol and ramipril, pharmacological agents that target the consequences of NADPH oxidase, on diabetic nephropathy in a rat model of type 1 diabetes, with an emphasis on markers of ER stress. Male Sprague-Dawley rats were injected intravenously with a single bolus of streptozotocin (55mg/kg) to induce type 1 diabetes. An additional age-matched group of rats was administered with citrate vehicle as controls. After 4 weeks of untreated diabetes, rats received tempol (1.5mM/kg/day subcutaneously, n=8), ramipril (1mg/kg/day in drinking water, n=8) or remained untreated for an additional 4 weeks (n=7). After 8 weeks of diabetes in total, kidneys were collected for histological analysis, gene expression and protein abundance. Tempol and ramipril blunted diabetes-induced upregulation of NADPH oxidase isoforms (Nox4, Nox2, p47phox), accompanied by an amelioration of diabetes-induced glomerular injury (podocin, nephrin, Kim-1), tubulo-interstitial fibrosis (TGFβ1, TGFβ-R2, pSMAD3, α-SMA) and pro-inflammatory cytokines (TNFα, MCP-1, ANX-A1, FPR2) expression. In addition, the diabetes-induced renal ER stress, evidenced by increased expression of GRP-78 chaperone and stress-associated markers ATF4, TRB3, as well as XBP1s, phospho-p38 mitogen-activated protein kinase (MAPK) and 3-nitrotyrosination, were all attenuated by tempol and ramipril. These observations suggest that antioxidant approaches that blunt NADPH upregulation may attenuate diabetic nephropathy, at least in part by negatively regulating ER stress and inflammation, and hence ameliorating kidney damage.

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

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.

Abstract P185: Role of Ucp2 in the Oxidative/nitrosative Stress-mediated Hypertension Associated with Dj-1 Depletion

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.

Abstract P231: High Fat Diet-induced Er Stress and Inflammation in the Kidney: Role of Trpv1-positive Afferent Renal Nerves

Chronic high fat diet (HFD) intake may lead to enhancement in endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production, and inflammation in the kidney, but the mechanisms of HD effects are largely unknown. This study tests the hypothesis that HFD impairs afferent renal nerves expressing the transient receptor potential vanilloid 1 (TRPV1) channels, and that stimulating/preserving TRPV1-positive afferent renal nerves prevents against HFD-induced increases in ER stress, ROS production, and inflammation in the kidney. HFD decreased levels of TRPV1 in the kidney, levels of urinary calcitonin-gene related peptide (CGRP) and substance P (SP), and responses of afferent renal nerve activity (ARNA) to capsaicin, a TRPV1 agonist, perfused into the renal pelvis. HFD increased ER stress (XBP-1, CREB2, GRP78, p-JNK), ROS production (urinary 8-isoprostane), and inflammation (TNF-α, IL-1β) in the kidney. N-oleoyldopamine (OLDA, a TRPV1 agonist, 1 ng/kg, daily) or vehicle was given intrathecally (i.t.) via indwelled catheters to segments (T8-L3) supplying the kidneys of rats fed a HFD or normal fat diet (Con) for 8 weeks. OLDA prevented HFD-induced decreases in the levels of renal TRPV1, urinary CGRP and SP, and ARNA responses to capsaicin, as well as HFD-induced increases in renal ER stress, ROS production, and inflammation. OLDA-induced protection during HFD was abolished by degeneration of TRPV1-positive afferent renal nerves by topical application of resiniferatoxin (RTX, 2 μg/ml x 2 times), a potent TRPV1 agonist, on renal nerves (TNF-α, Con: 0.17±0.02, HFD: 0.41±0.03, HFD+OLDA: 0.21±0.02, HFD+OLDA+RTX: 0.46±0.04, p<0.05; 8-isoprostane, Con: 8.8±1.2, HFD: 17±2, HFD+OLDA: 11±1, HFD+OLDA+RTX: 22±3 ng/day, p<0.05). Thus, our data show that HFD impairs TRPV1-positive afferent renal nerves, and chronic stimulation of TRPV1 via intrathecal injection of OLDA to T8-L3 segments preserves TRPV1-positive afferent renal nerves to against HFD-induced ER stress, ROS production, and inflammation in the kidney given that impairment of this population of afferent renal nerves abolishes OLDA effects. These data indicate that TRPV1-positive afferent renal nerves play a counteractive role against HFD-induced renal injury.

Evidence that Vascular Endothelial Derived Endothelin‐1 Promotes Development of Tunicamycin‐Induced Endoplasmic Reticulum Stress in Renal Vessels

Levels of the vasoconstrictor peptide endothelin-1 (ET-1) are elevated in cardiovascular disease. Endoplasmic reticulum (ER) stress is one of the known mechanisms for cellular apoptosis. Our group has recently implicated the ET-1 system in the activation of renal ER stress pathways, although it is unclear which cells are involved. To clarify the role of ET-1 in the development of renal vascular ER stress, we hypothesized that lack of ET-1 in endothelial cells attenuates tunicamycin (TM)-induced ER stress in renal vessels. Female mice lacking ET-1 in vascular endothelial cells (VEET KO) and the corresponding flox controls (n=5-9/group) received a single i.p. injection of either TM (1.5 mg/kg) or saline. 24 hours post-injection, renal vessels were isolated, ER stress genes determined by qRT-PCR, and renal apoptosis detected by TUNEL staining. TM significantly increased the expression of ER stress markers in renal vessels from flox mice (saline vs. TM, fold change/flox+saline, p<0.05; GRP94: 1.0±0.3 vs. 16.5±6.6, ATF-6: 1.0±0.5 vs. 6.8±3.5, and CHOP: 1.4±0.4 vs. 12.5±1.9). TM treatment led to increased outer medullary, non-vascular TUNEL staining in flox mice as well. TM failed to increase renal vascular ER stress markers or renal apoptosis in VEET KO mice. These results indicate that ET-1 is critical for the development of renal vascular ER stress and apoptosis in response to TM, and highlight the possibility of targeting the ET-1 system as a therapeutic approach against ER stress-induced renal vascular damage. Funded by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP.

TUDCA Attenuates High Salt‐Induced Renal Injury in ET B Deficient sl/sl Rats

Endothelin (ET-1) mediates sodium excretion via the ETB receptor pathway, however the ET-1/ETA receptor pathway plays a role in salt-dependent hypertension. The mechanisms by which ET-1 leads to high salt-induced renal injury are unclear. Reports have associated endoplasmic reticulum (ER) stress with renal injury, thus we hypothesized that the loss of functional ETB receptors increases high salt-induced renal injury via activation of ER stress. ETB deficient (sl/sl) rats and transgenic controls were placed on normal (1% NaCl,NSD) or high salt (8%, HSD) diet for three weeks (n=5-7/group) with daily i.p. injections of saline or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 400 mg/kg/day). Histological injury assessments, urinary injury biomarkers, renal immune cell infiltration, renal apoptosis by TUNEL staining, and renal ER stress gene expression by qRT-PCR were studied. In sl/sl rats, HSD slightly increased glomerulosclerosis and significantly increased urinary markers of renal injury (KIM-1: NSD 14.2±3.4 to HSD 104.1±20.6 pg/day; NGAL: NSD 43.6±17.6 to HSD 215.6±27.7 pg/day, p<0.001) with no difference in control rats. TUDCA significantly decreased renal tubular damage in sl/sl rats (KIM-1: 55.7±13.8 pg/day; NGAL: 114.2±18.0 pg/day, p<0.05 vs. HSD alone). Renal ER stress markers, immune cell infiltration, and apoptosis were not different amongst groups. Thus, our data suggest that HSD contributes to renal tubular injury via cellular stress pathways and ETB receptor activation is reno-protective. Further investigation in the HSD-induced renal tubular stress pathways is warranted. Supported by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP.