Renal Reactive Oxygen Species Production Research Articles

Atorvastatin ameliorates diabetic nephropathy through inhibiting oxidative stress and ferroptosis signaling.

Clinically, statins have long been used for the prevention and treatment of chronic renal diseases, however, the underlying mechanisms are not fully elucidated. The present study investigated the effects of atorvastatin on diabetes renal injury and ferroptosis signaling. A mouse model of diabetes was established by the intraperitoneal injection of streptozotocin (50 mg/kg/day) plus a high fat diet with or without atorvastatin treatment. Diabetes mice manifested increased plasma glucose and lipid profile, proteinuria, renal injury and fibrosis, atorvastatin significantly lowered plasma lipid profile, proteinuria, renal injury in diabetes mice. Atorvastatin reduced renal reactive oxygen species (ROS), iron accumulation and renal expression of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), transferrin receptor 1 (TFR1), and increased renal expression of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2) and ferritin heavy chain (FTH) in diabetes mice. Consistent with the findings in vivo, atorvastatin prevented high glucose-induced ROS formation and Fe2+ accumulation, an increase in the expression of 4-HNE, MDA and TFR1, and a decrease in cell viability and the expression of NRF2, GPX4 and FTH in HK2 cells. Atorvastatin also reversed ferroptosis inducer erastin-induced ROS production, intracellular Fe2+ accumulation and the changes in the expression of above-mentioned ferroptosis signaling molecules in HK2 cells. In addition, atorvastatin alleviated high glucose- or erastin-induced mitochondria injury. Ferroptosis inhibitor ferrostatin-1 and antioxidant N-acetylcysteine (NAC) equally reversed the expression of high glucose-induced ferroptosis signaling molecules. Our data support the notion that statins can inhibit diabetes-induced renal oxidative stress and ferroptosis, which may contribute to statins protection of diabetic nephropathy.

Ovariectomy Via 12/15-lipoxygenase Augments Angiotensin II-Induced Hypertension and Its Pathogenesis in Female Mice.

Ang II (angiotensin II) releases arachidonic acid from tissue phospholipids that are metabolized by 12/15-lipoxygenase (ALOX15), generating 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE), which have been implicated in cardiovascular and renal diseases. In this study, we tested the hypothesis that ovariectomy augments Ang II-induced hypertension and renal pathophysiological changes via ALOX15 activation in female mice. Ang II (700 ng/kg/min) was infused subcutaneously by osmotic pumps for 2 weeks in intact and ovariectomized wild-type and Alox15 knockout (ALOX15KO) female mice for evaluation of hypertension and associated pathogenesis. Ang II increased blood pressure, impaired autonomic function, and increased renal reactive oxygen species production and plasma 12(S)-HETE level without altering renal function in intact wild-type mice. However, in OVX-wild-type mice with depleted plasma 17β-estradiol, the effects of Ang II on blood pressure, autonomic impairment, renal reactive oxygen species production, and plasma 12(S)- but not 15(S)-HETE was markedly enhanced. In OVX-wild-type mice, Ang II also increased renal alox15 mRNA, urine 12(S)-HETE, water intake, urine output, decreased osmolality, increased urinary excretion of vasopressin prosegment copeptin, protein/creatinine ratio, and caused renal hypertrophy, fibrosis, and inflammation. These effects of Ang II were attenuated in ALOX15KO mice. These data suggest that 17β-estradiol protects against Ang II-induced hypertension and associated pathogenesis in female mice, most likely via inhibition of ALOX15-arachidonic acid derived production of 12(S)-HETE. Therefore, the selective inhibitors of ALOX15 or 12(S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women, or females with ovarian failure.

Mitochondria-Targeted Antioxidant Mitoquinone Maintains Mitochondrial Homeostasis through the Sirt3-Dependent Pathway to Mitigate Oxidative Damage Caused by Renal Ischemia/Reperfusion.

Mitochondrial dysfunction is a critical factor contributing to oxidative stress and apoptosis in ischemia-reperfusion (I/R) diseases. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant whose potent anti-I/R injury capacity has been demonstrated in organs such as the heart and the intestine. In the present study, we explored the role of MitoQ in maintaining mitochondrial homeostasis and attenuating oxidative damage in renal I/R injury. We discovered that the decreased renal function and pathological damage caused by renal I/R injury were significantly ameliorated by MitoQ. MitoQ markedly reversed mitochondrial damage after I/R injury and inhibited renal reactive oxygen species production. In vitro, hypoxia/reoxygenation resulted in increased mitochondrial fission and decreased mitochondrial fusion in human renal tubular epithelial cells (HK-2), which were partially prevented by MitoQ. MitoQ treatment inhibited oxidative stress and reduced apoptosis in HK-2 cells by restoring mitochondrial membrane potential, promoting ATP production, and facilitating mitochondrial fusion. Deeply, renal I/R injury led to a decreased expression of sirtuin-3 (Sirt3), which was recovered by MitoQ. Moreover, the inhibition of Sirt3 partially eliminated the protective effect of MitoQ on mitochondria and increased oxidative damage. Overall, our data demonstrate a mitochondrial protective effect of MitoQ, which raises the possibility of MitoQ as a novel therapy for renal I/R.

MP07-03 THE PROTECTIVE ROLE OF ESTROGEN RECEPTOR Β IN RENAL CALCIUM OXALATE FORMATION VIA REDUCING THE LIVER OXALATE BIOSYNTHESIS AND RENAL CELL INJURY

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology (MP07)1 Sep 2021MP07-03 THE PROTECTIVE ROLE OF ESTROGEN RECEPTOR Β IN RENAL CALCIUM OXALATE FORMATION VIA REDUCING THE LIVER OXALATE BIOSYNTHESIS AND RENAL CELL INJURY Wei Zhu, Wenbiao Ren, Chawnshang Chang, Edward M. Messing, Jean V. Joseph, Rajat Jain, David Levy, David Bushinsky, Guohua Zeng, and Shuyuan Yeh Wei ZhuWei Zhu More articles by this author , Wenbiao RenWenbiao Ren More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , Rajat JainRajat Jain More articles by this author , David LevyDavid Levy More articles by this author , David BushinskyDavid Bushinsky More articles by this author , Guohua ZengGuohua Zeng More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001980.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney stone is a common disorder that poses a significant health care burden. The incidence of kidney stone disease is lower in premenopausal women than age-matched men, as well as in female animal models with renal calcium oxalate (CaOx) crystal deposition. However, it is unclear if this gender difference is related to altered estrogen/estrogen receptor (ER) signaling. METHODS: Kidney and liver cell lines, HK-2, HKC-8, HepG2, and HEK-293, were used. The shRNA and cDNA of NOX2 and ERβ were applied to study their functions. Hydrogen Peroxide Assay and DHE Staining were used to detect Oxalate treatment effects and ROS in renal cells. The q-PCR and western blot assays were applied to assess the RNA and protein expressions, respectively. ERβ knockout (ERβKO,) and WT female mice were used to compare CaOx formation. Effects of ERβ antagonist was tested in hydroxyl-L-proline (HLP) diet induced CaOx crystal deposition rat model. RESULTS: CaOx is one major composition accounts for majority of kidney stones. We found that ERβ signals could suppress hepatic oxalate biosynthesis via transcriptional up-regulation of the glyoxylate aminotransferase (AGT1) expression in liver cells. Results from renal cell lines also showed that ERβ could function via suppressing the oxalate-induced injury to reduce the reactive oxygen species (ROS) production and decrease the renal calcium oxalate (CaOx) deposition. Mechanism studies showed that ERβ could suppress the oxalate-induced oxidative stress via transcriptional suppression of the NADPH oxidase subunit 2 (NOX2) via binding to the NOX2 5′ promoter. We further applied two in vivo animal models rat fed with 5% HLP diet-induced renal CaOx deposition. Our data demonstrated that mice lacking ERβ (ERβKO) as well as mice or rats treated with ERβ antagonist PHTPP had increased urinary oxalate excretion, renal ROS production as well as renal CaOx crystal deposition. Importantly, targeting ERβ-regulated NOX2 with the NADPH oxidase inhibitor, apocynin, can reduce the renal CaOx crystal deposition in the in vivo animal model. CONCLUSIONS: Results from multiple in vitro cell lines and in vivo mouse/rat models all demonstrate that ERβ may protect against renal CaOx deposition via reducing the hepatic oxalate biosynthesis and oxidative stress-induced renal injury. Source of Funding: This work was partially supported by URMC Urology Research fund, National Natural Science Foundation of China, and George Whipple Professorship Endowment at U of Rochester © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e140-e140 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei Zhu More articles by this author Wenbiao Ren More articles by this author Chawnshang Chang More articles by this author Edward M. Messing More articles by this author Jean V. Joseph More articles by this author Rajat Jain More articles by this author David Levy More articles by this author David Bushinsky More articles by this author Guohua Zeng More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Loading ...

Clozapine Worsens Glucose Intolerance, Nonalcoholic Fatty Liver Disease, Kidney Damage, and Retinal Injury and Increases Renal Reactive Oxygen Species Production and Chromium Loss in Obese Mice.

Clozapine is widely employed in the treatment of schizophrenia. Compared with that of atypical first-generation antipsychotics, atypical second-generation antipsychotics such as clozapine have less severe side effects and may positively affect obesity and blood glucose level. However, no systematic study of clozapine’s adverse metabolic effects—such as changes in kidney and liver function, body weight, glucose and triglyceride levels, and retinopathy—was conducted. This research investigated how clozapine affects weight, the bodily distribution of chromium, liver damage, fatty liver scores, glucose homeostasis, renal impairment, and retinopathy in mice fed a high fat diet (HFD). We discovered that obese mice treated with clozapine gained more weight and had greater kidney, liver, and retroperitoneal and epididymal fat pad masses; higher daily food efficiency; higher serum or hepatic triglyceride, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine levels; and higher hepatic lipid regulation marker expression than did the HFD-fed control mice. Furthermore, the clozapine group mice exhibited insulin resistance, poorer insulin sensitivity, greater glucose intolerance, and less Akt phosphorylation; their GLUT4 expression was lower, they had renal damage, more reactive oxygen species, and IL-1 expression, and, finally, their levels of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and catalase) were lower. Moreover, clozapine reduced the thickness of retinal cell layers and increased iNOS and NF-κB expression; a net negative chromium balance occurred because more chromium was excreted through urine, and this influenced chromium mobilization, which did not help overcome the hyperglycemia. Our clozapine group had considerably higher fatty liver scores, which was supported by the findings of lowered adiponectin protein levels and increased FASN protein, PNPLA3 protein, FABP4 mRNA, and SREBP1 mRNA levels. We conclude that clozapine can worsen nonalcoholic fatty liver disease, diabetes, and kidney and retinal injury. Therefore, long-term administration of clozapine warrants higher attention.

Impact of Inhaled Oxygen on Reactive Oxygen Species Production and Oxidative Damage during Spontaneous Ventilation in a Murine Model of Acute Renal Ischemia and Reperfusion.

Acute kidney injury (AKI) affects 10% of patients following major surgery and is independently associated with extra-renal organ injury, development of chronic kidney disease, and death. Perioperative renal ischemia and reperfusion (IR) contributes to AKI by, in part, increasing production of reactive oxygen species (ROS) and leading to oxidative damage. Variations in inhaled oxygen may mediate some aspects of IR injury by affecting tissue oxygenation, ROS production, and oxidative damage. We tested the hypothesis that provision of air (normoxia) compared to 100% oxygen (hyperoxia) during murine renal IR affects renal ROS production and oxidative damage. We administered 100% oxygen or 21% oxygen (air) to 8-9 week-old FVB/N mice and performed dorsal unilateral nephrectomy with contralateral renal ischemia/reperfusion surgery while mice spontaneously ventilated. We subjected mice to 30 minutes of ischemia and 30 minutes of reperfusion prior to sacrifice. We obtained an arterial blood gas (ABG) by performing sternotomy and left cardiac puncture. We stained the kidney with pimonidazole, a marker of tissue hypoxia; 4-HNE, a marker of ROS-production; and we measured F2-isoprostanes in homogenized tissue to quantify oxidative damage. Hyperoxia during IR increased arterial oxygen content compared to normoxia, but both groups of mice were hypoventilating at the time of ABG sampling. Renal tissue hypoxia following reperfusion was similar in both treatment groups. ROS production was similar in the cortex of mice (3.8% area in hyperoxia vs. 3.1% in normoxia, P=0.19) but increased in the medulla of hyperoxia-treated animals (6.3% area in hyperoxia vs. 4.5% in nomoxia, P=0.02). Renal F2-isoprostanes were similar in treatment groups (2.2 pg/mg kidney in hyperoxia vs. 2.1 pg/mg in normoxia, P=0.40). Hyperoxia during spontaneous ventilation in murine renal IR did not appear to affect renal hypoxia following reperfusion, but hyperoxia increased medullary ROS production compared to normoxia.

Endothelial or vascular smooth muscle cell-specific expression of human NOX5 exacerbates renal inflammation, fibrosis and albuminuria in the Akita mouse.

Excessive production of reactive oxygen species (ROS) plays a detrimental role in the progression of diabetic kidney disease (DKD). Renal oxidative stress activates proinflammatory cytokines, chemokines and profibrotic factors in DKD. Increased expression of the prooxidant enzyme NADPH oxidase (NOX) 5 in kidneys of diabetic individuals has been hypothesised to correlate with renal injury and progression of DKD. Since the gene encoding NOX5 is not expressed in the mouse genome, we examined the effect of inducible human NOX5 expression in renal cells, selectively in either endothelial cells or vascular smooth muscle cells (VSMCs)/mesangial cells in a model of insulin-deficient diabetes, the Akita mouse. Renal structural injury, including glomerulosclerosis, mesangial expansion and extracellular matrix protein accumulation, as well as renal inflammation, ROS formation and albuminuria, were examined in the NOX5 transgenic Akita mouse model of DKD. Expression of NOX5 in either endothelial cells or VSMCs/mesangial cells in diabetic Akita mice was associated with increased renal inflammation (monocyte chemoattractant protein-1, NF-κB and toll-like receptor-4) and glomerulosclerosis, as well as upregulation of protein kinase C-α and increased expression of extracellular matrix genes (encoding collagen III, fibronectin and α-smooth muscle actin) and proteins (collagen IV), most likely mediated via enhanced renal ROS production. The effect of VSMC/mesangial cell-specific NOX5 expression resulted in more pronounced renal fibrosis in comparison with endothelial cell-specific NOX5 expression in diabetic mice. In addition, albuminuria was significantly increased in diabetic VEcad+NOX5+ mice (1192 ± 194μg/24h) when compared with diabetic VEcad+NOX5- mice (770 ± 98μg/24h). Furthermore, the regulatory components of NOX5 activation, including heat shock protein 90 and transient receptor potential cation channel subfamily C member 6, were upregulated only in the presence of both NOX5 and diabetes. The findings from this study highlight the importance of NOX5 in promoting diabetes-related renal injury and provide the rationale for the development of a selective NOX5 inhibitor for the prevention and/or treatment of DKD.

The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury.

Females develop kidney stones less frequently than males do. However, it is unclear if this gender difference is related to altered estrogen/estrogen receptor (ER) signaling. Here, we found that ER beta (ERβ) signals could suppress hepatic oxalate biosynthesis via transcriptional upregulation of the glyoxylate aminotransferase (AGT1) expression. Results from multiple in vitro renal cell lines also found that ERβ could function via suppressing the oxalate-induced injury through increasing the reactive oxygen species (ROS) production that led to a decrease of the renal calcium oxalate (CaOx) crystal deposition. Mechanism study results showed that ERβ suppressed oxalate-induced oxidative stress via transcriptional suppression of the NADPH oxidase subunit 2 (NOX2) through direct binding to the estrogen response elements (EREs) on the NOX2 5′ promoter. We further applied two in vivo mouse models with glyoxylate-induced renal CaOx crystal deposition and one rat model with 5% hydroxyl-L-proline-induced renal CaOx crystal deposition. Our data demonstrated that mice lacking ERβ (ERβKO) as well as mice or rats treated with ERβ antagonist PHTPP had increased renal CaOx crystal deposition with increased urinary oxalate excretion and renal ROS production. Importantly, targeting ERβ-regulated NOX2 with the NADPH oxidase inhibitor, apocynin, can suppress the renal CaOx crystal deposition in the in vivo mouse model. Together, results from multiple in vitro cell lines and in vivo mouse/rat models all demonstrate that ERβ may protect against renal CaOx crystal deposition via inhibiting the hepatic oxalate biosynthesis and oxidative stress-induced renal injury.

Role of Nox4 in Angiotensin II‐Mediated Changes in Volume Dynamics and Nitric Oxide Production in Podocytes

Nitric oxide (NO) is a powerful vasodilator that serves important functions in the kidney such as regulation of renal hemodynamics, pressure natriuresis, and diuresis. Significant reduction in NO production has been found to play a role in the pathogenesis of hypertension. Our recent studies revealed that Angiotensin II (Ang II), in addition to causing a release of calcium, stimulates NO production in the glomerular podocytes. This signaling pathway can be also triggered by extracellular application of hydrogen peroxide (H2O2) and is significantly reduced with the development of salt‐sensitive hypertension and kidney injury in Dahl salt‐sensitive (SS) rats (Palygin et al., Nitric Oxide, 2018). Recently, using a knock out of NADPH oxidase 4 (Nox4) in SS rats (SSNox4−/−), one of the primary source of renal reactive oxygen species (ROS) and specifically H2O2 we described the reduction in intracellular calcium activation in podocytes as well as H2O2 production in isolated glomeruli in response to Ang II (Ilatovskaya et al., J Am Soc Nephrol. 2018). Overall, SSNox4−/− rats displayed a protective phenotype with a significant reduction of albuminuria, glomeruli injury and reduction in the elevation of blood pressure under high salt challenge (Cowley et al., Hypertension, 2016). Here we used this model to characterize the effects of alterations in the balance in the relationship between ROS and NO production in podocytes and establish the relation between Nox4 and glomerular/podocyte volume dynamics. The in vivo concentration of H2O2, measured by biosensors amperometry as catalase sensitive current, was significantly reduced in both cortex and medullar parts of the SSNox4−/− rat kidney compared to wild type SS rats, indicating a direct association between Nox4 and the production of renal ROS. We found that acute application of Ang II promoted opposite changes in glomeruli and podocyte volumes, where glomeruli volume decreased, and podocyte volume increased approximately by 18% in SS rats. Overall, SSNox4−/− rats exhibited an increase in total NO production (by 16% to compare with SS), calculated using confocal microscopy as a total integral of NO fluorescent transient (DAF‐FM dye) in the population of podocytes within a freshly isolated glomerulus. In SSNox4−/− rats the elevation in NO together with declining in H2O2 levels directly correlated with an increase of podocyte volume in response to Ang II (13% vs. SS control). We anticipate a rapid podocyte volume change dependent on intracellular NO production and reflecting the ability of these cells to dynamically regulate the permeability of glomeruli filtration barrier. Thus far, we can conclude that in the absence of Nox4 the podocytes shift the ROS production in response to Ang II towards the increase of intracellular NO bioavailability and a decrease of renal H2O2 production. This mechanism may play a key role in the protection of glomeruli form oxidative stress and elevation of blood pressure during the development of salt‐sensitive hypertension.Support or Funding InformationNHLBI R35 HL135749; P01 HL116264; AHA 18PRE34030127; ADA 1‐15‐BS‐172; DK‐020595 Pilot & Feasibility projectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diabetes and Kidney Disease: Role of Oxidative Stress.

Intrarenal oxidative stress plays a critical role in the initiation and progression of diabetic kidney disease (DKD). Enhanced oxidative stress results from overproduction of reactive oxygen species (ROS) in the context of concomitant, insufficient antioxidant pathways. Renal ROS production in diabetes is predominantly mediated by various NADPH oxidases (NOXs), but a defective antioxidant system as well as mitochondrial dysfunction may also contribute. Recent Advances: Effective agents targeting the source of ROS generation hold the promise to rescue the kidney from oxidative damage and prevent subsequent progression of DKD. Critical Issues and Future Directions: In the present review, we summarize and critically analyze molecular and cellular mechanisms that have been demonstrated to be involved in NOX-induced renal injury in diabetes, with particular focus on the role of increased glomerular injury, the development of albuminuria, and tubulointerstitial fibrosis, as well as mitochondrial dysfunction. Furthermore, novel agents targeting NOX isoforms are discussed. Antioxid. Redox Signal. 25, 657-684.

Pretreatment of Sialic Acid Efficiently Prevents Lipopolysaccharide-Induced Acute Renal Failure and Suppresses TLR4/gp91-Mediated Apoptotic Signaling

Background/Aims: Lipopolysaccharides (LPS) binding to Toll-like receptor 4 (TLR4) activate NADPH oxidase gp91 subunit-mediated inflammation and oxidative damage. Recognizing the high binding affinity of sialic acid (SA) with LPS, we further explored the preventive potential of SA pretreatment on LPS-evoked acute renal failure (ARF). Methods: We determined the effect of intravenous SA 30 min before LPS-induced injury in urethane-anesthetized female Wistar rats by evaluating kidney reactive oxygen species (ROS) responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. Results: LPS time-dependently reduced arterial blood pressure, renal microcirculation, and increased blood urea nitrogen and creatinine in the rats. LPS enhanced monocyte/macrophage infiltration and ROS production, and subsequently impaired kidneys with the enhancement of TLR4/NADPH oxidase gp91/Caspase 3/poly-(ADP-ribose)-polymerase (PARP)-mediated apoptosis in the kidneys. SA pretreatment effectively alleviated LPS-induced ARF. The levels of LPS-increased ED-1 infiltration and ROS production in the kidney were significantly depressed by SA pretreatment. Furthermore, SA pretreatment significantly depressed TLR4 activation, gp91 expression, and Caspase 3/PARP induced apoptosis in the kidneys. Conclusion: We suggest that pretreatment of SA significantly and preventively attenuated LPS-induced detrimental effects on systemic and renal hemodynamics, renal ROS production and renal function, as well as, LPS-activated TLR4/gp91/Caspase3 mediated apoptosis signaling.

Dopamine treatment attenuates acute kidney injury in a rat model of deep hypothermia and rewarming – The role of renal H2S-producing enzymes

Hypothermia and rewarming produces organ injury through the production of reactive oxygen species. We previously found that dopamine prevents hypothermia and rewarming-induced apoptosis in cultured cells through increased expression of the H2S-producing enzyme cystathionine β-Synthase (CBS). Here, we investigate whether dopamine protects the kidney in deep body cooling and explore the role of H2S-producing enzymes in an in vivo rat model of deep hypothermia and rewarming. In anesthetized Wistar rats, body temperature was decreased to 15°C for 3h, followed by rewarming for 1h. Rats (n≥5 per group) were treated throughout the procedure with vehicle or dopamine infusion, and in the presence or absence of a non-specific inhibitor of H2S-producing enzymes, amino-oxyacetic acid (AOAA). Kidney damage and renal expression of three H2S-producing enzymes (CBS, CSE and 3-MST) was quantified and serum H2S level measured. Hypothermia and rewarming induced renal damage, evidenced by increased serum creatinine, renal reactive oxygen species production, KIM-1 expression and influx of immune cells, which was accompanied by substantially lowered renal expression of CBS, CSE, and 3-MST and lowered serum H2S levels. Infusion of dopamine fully attenuated renal damage and maintained expression of H2S-producing enzymes, while normalizing serum H2S. AOAA further decreased the expression of H2S-producing enzymes and serum H2S level, and aggravated renal damage. Hence, dopamine preserves renal integrity during deep hypothermia and rewarming likely by maintaining the expression of renal H2S-producing enzymes and serum H2S.

Sestrin2 Decreases Renal Oxidative Stress, Lowers Blood Pressure, and Mediates Dopamine D 2 Receptor–Induced Inhibition of Reactive Oxygen Species Production

The dopamine D 2 receptor (D 2 R) decreases renal reactive oxygen species (ROS) production and regulates blood pressure, in part, via positive regulation of paraoxonase 2. Sestrin2, a highly conserved antioxidant protein, regulates intracellular ROS level by regenerating hyperoxidized peroxiredoxins. We hypothesized that sestrin2 may be involved in preventing excessive renal ROS production and thus contribute to the maintenance of normal blood pressure. Moreover, the D 2 R may decrease ROS production, in part, through the regulation of sestrin2. Renal sestrin2 expression was lower (−62±13%) in D 2 R −/− than in D 2 R +/+ mice. Silencing D 2 R in human renal proximal tubule cells decreased sestrin2 expression (−53±3%) and increased hyperoxidized peroxiredoxins (2.9-fold). Stimulation of D 2 R in renal proximal tubule cells increased sestrin2 expression (1.6-fold), decreased hyperoxidized peroxiredoxins (−61±3%), and reduced ROS production (−31±4%). Silencing sestrin2 in renal proximal tubule cells increased hyperoxidized peroxiredoxins (2.1-fold) and ROS production (1.3-fold). Silencing sestrin2 also abolished D 2 R-induced decrease in peroxiredoxin hyperoxidation and partially prevented the inhibitory effect of D 2 R stimulation on ROS production. Silencing paraoxonase 2 increased sestrin2 ubiquitinylation (2.8-fold), decreased sestrin2 expression (−30±3%), and increased ROS production (1.3-fold), peroxiredoxin hyperoxidation (2.9-fold), and lipid peroxidation (2.3-fold), and blocked the increase in sestrin2 that occurs with D 2 R stimulation. In vivo renal selective silencing of sestrin2 by the renal subcapsular infusion of sestrin2 small interfering RNA (3 μg/day; 7 days) in mice increased renal oxidative stress (1.3-fold) and blood pressure. These results suggest that the D 2 R, via paraoxonase 2 and sestrin2, keeps normal renal redox balance, which contributes to the maintenance of normal blood pressure.

Osthole Mitigates Progressive IgA Nephropathy by Inhibiting Reactive Oxygen Species Generation and NF-κB/NLRP3 Pathway

Renal reactive oxygen species (ROS) and mononuclear leukocyte infiltration are involved in the progressive stage (exacerbation) of IgA nephropathy (IgAN), which is characterized by glomerular proliferation and renal inflammation. The identification of the mechanism responsible for this critical stage of IgAN and the development of a therapeutic strategy remain a challenge. Osthole is a pure compound isolated from Cnidiummonnieri (L.) Cusson seeds, which are used as a traditional Chinese medicine, and is anti-inflammatory, anti-apoptotic, and anti-fibrotic both in vitro and in vivo. Recently, we showed that osthole acts as an anti-inflammatory agent by reducing nuclear factor-kappa B (NF-κB) activation in and ROS release by activated macrophages. In this study, we examined whether osthole could prevent the progression of IgAN using a progressive IgAN (Prg-IgAN) model in mice. Our results showed that osthole administration resulted in prevention of albuminuria, improved renal function, and blocking of renal progressive lesions, including glomerular proliferation, glomerular sclerosis, and periglomerular mononuclear leukocyte infiltration. These findings were associated with (1) reduced renal superoxide anion levels and increased Nrf2 nuclear translocation, (2) inhibited renal activation of NF-κB and the NLRP3 inflammasome, (3) decreased renal MCP-1 expression and mononuclear leukocyte infiltration, (4) inhibited ROS production and NLRP3 inflammasome activation in cultured, activated macrophages, and (5) inhibited ROS production and MCP-1 protein levels in cultured, activated mesangial cells. The results suggest that osthole exerts its reno-protective effects on the progression of IgAN by inhibiting ROS production and activation of NF-κB and the NLRP3 inflammasome in the kidney. Our data also confirm that ROS generation and activation of NF-κB and the NLRP3 inflammasome are crucial mechanistic events involved in the progression of the renal disorder.

Abstract 75: Nrf2 Mediates the Antioxidant Effect of D2r Via Dj-1 in the Kidney

Renal dopamine D2 receptor (D2R) dysfunction is associated with increased oxidative stress and high blood pressure. We have reported that DJ-1 is regulated by D2R in the kidney. Nrf2 is a transcription factor that regulates the expression of several antioxidant genes. We hypothesize that Nrf2 is involved in the DJ-1-mediated regulation of reactive oxygen species (ROS) production in the kidney. DJ-1 and Nrf2 co-inmunoprecipitate in mouse kidney. Selective renal silencing of D2R in mice by the renal subcapsular infusion of D2R siRNA (~50%) decreases Nrf2 expression (63.4%±3.4, n=7, P<0.05), and silencing of D2R expression in mouse proximal tubule cells (mRPTCs) decrease Nrf2 promoter activity (53±2%, n=5, P<0.05). Silencing DJ-1 expression in mRPTCs, via siRNA, decreases the expression of DJ-1 (75±4.5%, n=4, P<0.05), Nrf2 (63.2±6%, n=3, P<0.05), NQO1 (71±1%, n=3, P<0.05), and GST Yc-2 (42±1%, n=4, P<0.05) and Nrf2 promoter activity (58±1%, n=5, P<0.05). Selective renal silencing of DJ-1 in mice by renal subcapsular infusion of DJ-1 siRNA decreases expression of DJ-1 (31±2.6%, n=3, P<0.05), Nrf2 (57±7.9%,n=3, P<0.05), NQO1 (46±9.3%, n=3, P<0.05) and GST Yc2 (28±2.2%, n=3, P<0.05) and increases blood pressure (BP). DJ-1 -/- mice also have increased systolic BP (30.7±1%, P<0.01, n=5 under anesthesia) and renal expression of nitro-tyrosine (76.8±13.5%, P<0.05, n=5), and decreases the expression of Nrf2 (46.8±6.8%, n=5, P<0.05) and NQO1 (19.4±2.7%, n=5, P<0.05). Silencing Nrf2 expression mRPTCs via siRNA decreases the expression of Nrf2 (34±1.8%, n=4, P<0.05), NQO1 (29.1±0.5%, n=4, P<0.05), and increases Nox2 (137±23.7%, n=4, P<0.05), but the expression of Nox4 and DJ-1 are not affected. Quinpirole a D2R agonist, does not modify the expression of Nrf2, NQO1, GST Yc-2, and Nrf2 promoter activity in MPCTS. However, in the presence of H 2 O 2 , quinpirole increases Nrf2 promoter activity (220±58.4%, n=5, P<0.05) and an effect that is blocked by pretreatment with D2R antagonist L741,626. Therefore, the inhibitory effect of DJ-1 on renal ROS production is, in part, mediated by positive regulation of Nrf2. Moreover, D2R and DJ-1 are necessary for normal Nrf2 activity to keep a normal redox balance in the kidney.

Maternally derived mitochondria in SHR exhibit significant reduction in oxidative phosphorylation gene expression

Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease including hypertension. We hypothesize that renal Mt‐genes derived from female SHR are linked to high blood pressure in offspring of SHR and normotensive (NT) Brown Norway (BN) rat crosses. After breeding a female Okamoto‐Aoki SHR (MAP=160mmHg) with a BN male (MAP=91mmHg), hypertensive female progeny were backcrossed with the founder BN for 5 consecutive generations. Mt‐protein coding genes (13 total) and nuclear transcription factors mediating Mt‐transcription were evaluated in kidney, heart and liver of NT(n=20) vs. HT(n=20) SHR/BN using quantitative real‐time PCR. GAPDH was used as reference. Renal but not liver or heart Mt‐gene expression was decreased ~2–5 fold in 12 of 13 protein coding genes of HT SHR/BN. Renal but not liver mRNA expression of nuclear transcription factors for Tfam, NRF1, NRF2 and Pgc1α were decreased in HT SHR/BN. This renal specific reduction of nuclear and Mt‐gene expression may down‐regulate oxidative metabolism and increase renal reactive oxygen species production in SHR leading to hypertension and cardiovascular disease.

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase

The dopamine D2 receptor (D2R) regulates renal reactive oxygen species (ROS) production, and impaired D2R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D2R may decrease ROS production, in part, through regulation of PON2. D2R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (−33±6%) in D2−/− relative to D2+/+ mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (−55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D2R interaction in humans, we studied human renal proximal tubule cells. Both D2R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D2R/D3R agonist quinpirole (1μM, 24h) decreased ROS production (−35±6%), associated with decreased NADPH oxidase activity (−32±3%) and expression of Nox2 (−41±7%) and Nox4 (−47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D2R and may, in part, mediate the inhibitory effect of renal D2R on NADPH oxidase activity and ROS production.

Role of Renal DJ-1 in the Pathogenesis of Hypertension Associated With Increased Reactive Oxygen Species Production

The D(2) dopamine receptor (D(2)R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D(2)R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D(2)R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D(2)R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D(2)Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D(2)(+/-) mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D(2)R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D(2)R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D(2)R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D(2)R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.

Dopamine D2 receptor regulation of reactive oxygen species production via paraoxonase 2

An intact dopaminergic system is necessary to maintain normal blood pressure. We have reported that D2 dopamine receptor (D2R) regulates renal reactive oxygen species (ROS) production and that altered D2R function results in ROS‐dependent hypertension. Paraoxonase 2 (PON2) belongs to the paraoxonase gene family that is expressed in various tissues including the kidney and protects against cellular oxidative stress.We hypothesized that PON2 may be involved in counter‐regulating excessive renal ROS production and thus may contribute to maintain normal blood pressure. Moreover, D2R may regulate ROS production in part through regulation of PON2. ROS production decreased (42%; P<0.05; n=4) in human renal proximal tubular (iNT) cells treated with the D2R agonist, quinpirole (24h, 1μM). This effect was associated with increased PON2 expression of mRNA and protein and enzymatic activity. PON‐2 silencing (siRNA, 10nM, 48h) down‐regulated PON2 expression (50%; n=3) and activity and increased ROS (34.9%, P<0.05). Furthermore, renal PON2 expression was down‐regulated in D2−/− mice (35.6%, P<0.05).These results show that PON2 expression and/or function are closely related to ROS production and suggest that the D2R is involved in its regulation. The increased oxidative stress due to D2R disruption could be related to the failure of its stimulatory effect on the expression or function of PON2 in the kidney.This work was supported in part by grants from the National Institutes of Health, HL68686, HL23081, HL074940, HL092196 and DK39308

Ioxitalamate Induces Renal Tubular Apoptosis via Activation of Renal Efferent Nerve–Mediated Adrenergic Signaling, Renin Activity, and Reactive Oxygen Species Production in Rats

To investigate the unrecognized role of renal efferent nerve activity (RENA) in iodinated contrast media (CM)-induced acute kidney injury, we explored the effects of CM on RENA, renal hemodynamics, plasma renin activity (PRA), reactive oxygen species (ROS) production, and renal injury in rats. Four types of CM including ioxitalamate (high osmolar and ionic), ioxaglate (low osmolar and ionic), iohexol (low osmolar and nonionic), and iodixanol (iso-osmolar and nonionic) were given iv (1600 mg I/kg body weight) to urethane-anesthetized female Wistar rats. We measured RENA by electrophysiologic recording techniques, renal blood flow with Doppler ultrasound, PRA by radioimmunoassay, and ROS by an in vivo chemiluminescence method. We graded the severity of CM-induced vacuoles in cortical tubular cells stained by hematoxylin and eosin and apoptosis production in outer medulla by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Besides, the effects of pretreatment with iv beta-adrenoceptor antagonist propranolol (10 mg/kg body weight), antioxidant N-acetylcysteine (100 mg/kg body weight), and renal denervation on CM-induced pathophysiologic parameters were determined. Ioxitalamate significantly increased RENA and renal vascular resistance, PRA, renal ROS production within 1 h, and formation of vacuoles and TUNEL apoptosis in renal tubular cells 2 h later; other CM had less effect on these parameters. On the other hand, propranolol, N-acetylcysteine, or renal denervation partially attenuated the ioxitalamate-aggravated responses on RENA, PRA, ROS production, and vacuole and TUNEL apoptosis formation in renal tubular cells. In conclusion, we suggest that ioxitalamate may induce acute tubular injury via aggravation of RENA, adrenergic signaling, PRA, and ROS production.