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Since the interfacial binding strength and structural integrity have a strong influence on the active sites of nanocomposites, this study focused on exploring the structural and electronic properties at the interface between the implanted metal ion and host support. For this, nanocomposites of gold embedded in CeO

Novel mesoporous ferrite nanoparticles of ruthenium (RuFeO3) and cerium (CeFeO3) with large surface area were successfully fabricated by hydrothermal method and applied for the removal of fluoxetine from aqueous solution. The morphology and physicochemical properties of meso-porous metal ferrites were characterized by different analytical techniques. The obtained results showed that the particle size of 20–30 nm for meso-CeFeO3, and 80–100 nm for meso-RuFeO3 respectively. Study of the adsorption of fluoxetine over metal ferrite nanoparticles was explored using UV–vis spectroscopy at different experimental conditions such as temperature, pH, initial fluoxetine concentration and time. The adsorption data were fitted and analyzed with kinetic and different adsorption isotherm models, and the results showed that both the metal ferrites effectively adsorbed fluoxetine (>99%). The maximum adsorption capacity of 729.6 mg g−1 for CeFeO3, and 683.5 mg g−1 for RuFeO3 at neutral pH and at 25 °C were observed. The amount adsorbed over the surface of metal ferrite nanoparticles increased with increasing initial fluoxetine concentration, reaction time, and temperature, and the adsorption performance improved under neutral conditions (pH 7 and 8). The pseudo-second order kinetic and Langmuir isotherm models satisfactorily fitted the adsorption data, showing that the adsorption of fluoxetine involved physical adsorption through intermolecular electrostatic force between the metal ferrites and fluoxetine which is further confirmed by intra-particle diffusion model. The observed wide band/gap, 3.815eV for CeFeO3, and 3.360eV for RuFeO3 respectively showed their stability towards visible light suggesting their real sample applicability, recovery and reuse capability involving physisorption with fluoxetine molecules. In addition, the feasible adsorption process of fluoxetine is analyzed with its geometry that is determined by theoretical energy calculations using DFT.

Significance: Kidney diseases remain a worldwide public health problem resulting in millions of deaths each year; they are characterized by progressive destruction of renal function by sustained inflammation. Pyroptosis is a lytic type of programmed cell death involved in inflammation, as well as a key fibrotic mechanism that is critical in the development of kidney pathology. Pyroptosis is induced by the cleavage of Gasdermins by various caspases and is executed by the insertion of the N-terminal fragment of cleaved Gasdermins into the plasma membrane, creating oligomeric pores and allowing the release of diverse proinflammatory products into the extracellular space. Inflammasomes are multiprotein complexes leading to the activation of caspase-1, which will cleave Gasdermin D, releasing several proinflammatory cytokines; this results in the initiation and amplification of the inflammatory response. Recent Advances: The efficacy of Gasdermin D cleavage is reduced by a change in the redox balance. Recently, several studies have shown that the attenuation of reactive oxygen species (ROS) production induced by antioxidant pathways results in a reduction of renal pyroptosis. In this review, we discuss the role of pyroptosis in the pathogenesis of chronic kidney disease (CKD) and acute kidney disease; summarize the clinical outcomes and different molecular mechanisms leading to Gasdermin activation; and examine studies about the capacity of antioxidants, particularly Nrf2 activators, to ameliorate Gasdermin activity. Future Directions: We illustrate the potential influence of the deregulation of redox balance on inflammasome activity and pyroptosis as a novel therapeutic approach for the treatment of kidney diseases. Antioxid. Redox Signal. 35, 40-60.

Effective receptor signaling is anchored on the preferential localization of the receptor in lipid rafts, which are plasma membrane platforms replete with cholesterol and sphingolipids. We hypothesized that the dopamine D(1) receptor (D(1)R) contains structural features that allow it to reside in lipid rafts for its activity. Mutation of C347 palmitoylation site and Y218 of a newly identified Cholesterol Recognition Amino Acid Consensus motif resulted in the exclusion of D(1)R from lipid rafts, blunted cAMP response, impaired sodium transport, and increased oxidative stress in renal proximal tubule cells. Kidney-restricted silencing of Drd1 in C57BL/6J mice increased blood pressure (BP) that was normalized by renal tubule-restricted rescue with D(1)R-wild-type but not the mutant D(1)R-347A that lacks a palmitoylation site. Kidney-restricted disruption of lipid rafts by β-MCD jettisoned the D(1)R from the brush border, decreased sodium excretion, and increased oxidative stress and BP in C57BL/6J mice. Deletion of the PX domain of the novel D(1)R-binding partner sorting nexin 19 (SNX19) resulted in D(1)R partitioning solely to non-raft domains, while silencing of SNX19 impaired D(1)R function in renal proximal tubule cells. Kidney-restricted silencing of Snx19 resulted in hypertension in C57BL/6J mice. Our results highlight the essential role of lipid rafts for effective D(1)R signaling.

Gastrin, secreted by stomach G cells in response to ingested sodium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodium-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with human gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra- or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1-like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates gastrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Dopamine, via the D1R, by interacting with PPAR-α, is involved in this process.

Photo-initiated oxidative degradation of 2-thiobenzimidazole (2-TBI) was studied using different semiconducting metal complexes derived from 8-hydroxyquinoline with the general formula, [MQ3C2H5OH] [Q = 8-quinolinolato anion; M = Cr3+, (CrQ3); Co3+, (CoQ3); Fe3+, (FeQ3)]. The degradation kinetics was monitored via UV-vis spectroscopy perceiving the changes in absorbance to subtract. The effect of pH and visible light irradiation on degradation was studied observing a significant increment in the oxidation rate with the presence of visible light whenever CoQ3 is used as a catalyst. By using density functional theory (DFT), it was found that the transition state energy of the adduct formed between CoQ3 and H2O2 is very low compared with other studied catalysts, which activates the formation of ˙OH radicals from H2O2 increasing the 2-TBI degradation rate. In addition, a thermodynamically feasible degradation mechanism was proposed by calculating energy barriers involved in the 2-TBI oxidation pathway.

Hypertension is the most prevalent cause of cardiovascular disease and kidney failure, but only about 50% of patients achieve adequate blood pressure control, in part, due to inter-individual genetic variations in the response to antihypertensive medication. Significant strides have been made toward the understanding of the role of reactive oxygen species (ROS) in the regulation of the cardiovascular system. However, the role of ROS in human hypertension is still unclear. Polymorphisms of some genes involved in the regulation of ROS production are associated with hypertension, suggesting their potential influence on blood pressure control and response to antihypertensive medication. This review provides an update on the genes associated with the regulation of ROS production in hypertension and discusses the controversies on the use of antioxidants in the treatment of hypertension, including the antioxidant effects of antihypertensive drugs.

Gastrin, secreted by G-cells, and glucagon-like peptide-1 (GLP-1), secreted by L-cells, may participate in the regulation of sodium balance. We studied the effect of sodium in mice in vivo and mouse ileum and human L-cells, on GLP-1 secretion, and the role of NFAT5 and gastrin-releasing peptide receptor (GRPR) in this process. A high-sodium diet increases serum GLP-1 levels in mice. Increasing sodium concentration stimulates GLP-1 secretion from mouse ileum and L-cells. GRP enhances the high sodium-induced increase in GLP-1 secretion. High sodium increases cellular GLP-1 expression, while low and high sodium concentrations increase NFAT5 and GRPR expression. Silencing NFAT5 in L-cells abrogates the stimulatory effect of GRP on the high sodium-induced GLP-1 secretion and protein expression, and the sodium-induced increase in GRPR expression. GLP-1 and gastrin decrease the expression of Na+-K+/ATPase and increase the phosphorylation of sodium/hydrogen exchanger type 3 (NHE3) in human renal proximal tubule cells (hRPTCs). This study gives a new perspective on the mechanisms of GLP-1 secretion, especially that engendered by ingested sodium, and the ability of GLP-1, with gastrin, to decrease Na+-K+/ATPase expression and NHE3 function in hRPTCs. These results may contribute to the better utilization of current and future GLP-1-based drugs in the treatment of hypertension.

Inflammasomes are components of the innate immune response that have recently emerged as crucial controllers of tissue homeostasis. In particular, the nucleotide-binding domain, leucine-rich-containing (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is a complex platform involved in the activation of caspase-1 and the maturation of interleukin (IL)-1β and IL-18, which are mainly released via pyroptosis. Pyroptosis is a caspase-1-dependent type of cell death that is mediated by the cleavage of gasdermin D and the subsequent formation of structurally stable pores in the cell membrane. Through these pores formed by gasdermin proteins cytosolic contents are released into the extracellular space and act as damage-associated molecular patterns, which are pro-inflammatory signals. Inflammation is a main contributor to the development of hypertension and it also is known to stimulate fibrosis and end-organ damage. Patients with essential hypertension and animal models of hypertension exhibit elevated levels of circulating IL-1β. Downregulation of the expression of key components of the NLRP3 inflammasome delays the development of hypertension and pharmacological inhibition of this inflammasome leads to reduced blood pressure in animal models and humans. Although the relationship between pyroptosis and hypertension is not well established yet, pyroptosis has been associated with renal and cardiovascular diseases, instances where high blood pressure is a critical risk factor. In this review, we summarize the recent literature addressing the role of pyroptosis and the inflammasome in the development of hypertension and discuss the potential use of approaches targeting this pathway as future anti-hypertensive strategies.

DJ-1 is a redox-sensitive chaperone with reported antioxidant and anti-inflammatory properties in the kidney. The 20 amino acid (aa) peptide ND-13 consists of 13 highly conserved aas from the DJ-1 sequence and a TAT-derived 7 aa sequence that helps in cell penetration. This study aimed to determine if ND-13 treatment prevents the renal damage and inflammation associated with unilateral ureter obstruction (UUO). Male C57Bl/6 and DJ-1&minus, /&minus, mice underwent UUO and were treated with ND-13 or vehicle for 14 days. ND-13 attenuated the renal expression of fibrotic markers TGF-&beta, and collagen1a1 (Col1a1) and inflammatory markers TNF-&alpha, and IL-6 in C57Bl/6 mice. DJ-1&minus, mice treated with ND-13 presented similar decreased expression of TNF-&alpha, IL-6 and TGF-&beta, However, in contrast to C57Bl/6 mice, ND-13 failed to prevent renal fibrosis or to ameliorate the expression of Col1a1 in this genotype. Further, UUO led to elevated urinary levels of the proximal tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL) in DJ-1&minus, mice, which were blunted by ND-13. Our results suggest that ND-13 protects against UUO-induced renal injury, inflammation and fibrosis. These are all crucial mechanisms in the pathogenesis of kidney injury. Thus, ND-13 may be a new therapeutic approach to prevent renal diseases.

Acute renal depletion of sorting nexin 1 (SNX1) in mice results in blunted natriuretic response and hypertension due to impaired dopamine D(5) receptor (D(5)R) activity. We elucidated the molecular mechanisms for these phenotypes in Snx1(−/−) mice. These mice had increased renal expressions of angiotensin II type 1 receptor (AT(1)R), NADPH oxidase (NOX) subunits, D(5)R, and NaCl cotransporter. Basal reactive oxygen species (ROS), NOX activity, and blood pressure (BP) were also higher in Snx1(−/−) mice, which were normalized by apocynin, a drug that prevents NOX assembly. Renal proximal tubule (RPT) cells from hypertensive (HT) Euro-American males had deficient SNX1 activity, impaired D(5)R endocytosis, and increased ROS compared with cells from normotensive (NT) Euro-American males. siRNA-mediated depletion of SNX1 in RPT cells from NT subjects led to a blunting of D(5)R agonist-induced increase in cAMP production and decrease in Na(+) transport, effects that were normalized by over-expression of SNX1. Among HT African-Americans, three of the 12 single nucleotide polymorphisms interrogated for the SNX1 gene were associated with a decrease in systolic BP in response to hydrochlorothiazide (HCTZ). The results illustrate a new paradigm for the development of hypertension and imply that the trafficking protein SNX1 may be a crucial determinant for hypertension and response to antihypertensive therapy.

Background The regulation of sodium excretion is important in the pathogenesis of hypertension and salt sensitivity is predictive of cardiovascular events and mortality. C57Bl/6 and BALB /c mice have different blood pressure sensitivities to salt intake. High salt intake increases blood pressure in some C57Bl/6J mouse strains but not in any BALB /c mouse strain. Methods and Results We determined the cause of the difference in salt sensitivity between C57Bl/6 and BALB /c mice. Basal levels of superoxide and H 2 O 2 were higher in renal proximal tubule cells ( RPTC s) from BALB /c than C57Bl/6J mice. High salt diet increased H 2 O 2 production in kidneys from BALB /c but C57Bl/6J mice. High sodium concentration (170 mmol/L) in the incubation medium increased H 2 O 2 levels in BALB /c‐ RPTC s but not in C57Bl/6J‐ RPTC s. H 2 O 2 (10 μmol/L) treatment decreased sodium transport in RPTC s from BALB /c but not C57Bl/6J mice. Overexpression of catalase in the mouse kidney predisposed BALB /c mice to salt‐sensitive hypertension. Conclusions Our data show that the level of salt‐induced H 2 O 2 production negatively regulates RPTC sodium transport and determines the state of salt sensitivity in 2 strains of mice. High concentrations of antioxidants could prevent H 2 O 2 production in renal proximal tubules, which would result in sodium retention and increased blood pressure.

Abnormalities of the D(2)R gene (DRD2) play a role in the pathogenesis of human essential hypertension; variants of the DRD2 have been reported to be associated with hypertension. Disruption of Drd2 (D(2)(−/−)) in mice increases blood pressure and may cause salt sensitivity. The hypertension of D(2)(−/−) mice has been related, in part, to increased sympathetic activity and renal oxidative stress and endothelin B receptor expression. We tested in D(2)(−/−) mice the effect of etamicastat, a reversible peripheral inhibitor of dopamine-β-hydroxylase that reduces the biosynthesis of norepinephrine from dopamine and decreases sympathetic nerve activity. Blood pressure was measured in anesthetized D(2)(−/−) mice treated with etamicastat by gavage, (10 mg/kg), conscious D(2)(−/−) mice and D(2)(+/+) littermates, with the D(2)R selectively silenced in the kidney, treated with etamiscastat in the drinking water (10 mg/kg/day). Tissue and urinary catecholamines and renal expression of selected G protein-coupled receptors, enzymes related to the production of reactive oxygen species, and sodium transporters were also measured. Etamicastat decreased blood pressure both in anesthetized and conscious D(2)(−/−) mice and mice with renal-selective silencing of D(2)R to levels similar or close to those measured in D(2)(+/+) littermates. Etamicastat decreased cardiac norepinephrine and increased cardiac and urinary dopamine levels in D(2)(−/−) mice. It also normalized the increased renal protein expressions of ETB, NADPH oxidase isoenzymes, NHE3, and NCC, and increased the renal expression of D(1)R but not D(5)R in D(2)(−/−) mice. In conclusion, etamicastat is effective in normalizing the increased blood pressure and some of the abnormal renal biochemical alterations of D(2)(−/−) mice.

We tested the hypothesis that salt-sensitive hypertension is caused by renal oxidative stress by measuring the blood pressure and reactive oxygen species-related proteins in the kidneys of human G protein-coupled receptor kinase 4γ (hGRK4γ) 486V transgenic mice and non-transgenic (Non-T) littermates on normal and high salt diets. High salt diet increased the blood pressure, associated with impaired sodium excretion, in hGRK4γ486V mice. Renal expressions of NOX isoforms were similar in both strains on normal salt diet but NOX2 was decreased by high salt diet to a greater extent in Non-T than hGRK4γ486V mice. Renal HO-2, but not HO-1, protein was greater in hGRK4γ486V than Non-T mice on normal salt diet and normalized by high salt diet. On normal salt diet, renal CuZnSOD and ECSOD proteins were similar but renal MnSOD was lower in hGRK4γ486V than Non-T mice and remained low on high salt diet. High salt diet decreased renal CuZnSOD in hGRK4γ486V but not Non-T mice and decreased renal ECSOD to a greater extent in hGRK4γ486V than Non-T mice. Renal SOD activity, superoxide production, and NOS3 protein were similar in two strains on normal salt diet. However, high salt diet decreased SOD activity and NOS3 protein and increased superoxide production in hGRK4γ486V mice but not in Non-T mice. High salt diet also increased urinary 8-isoprostane and 8-hydroxydeoxyguanosine to a greater extent in hGRK4γ486V than Non-T mice. hGRK4γwild-type mice were normotensive and hGRK4γ142V mice were hypertensive but both were salt-resistant and in normal redox balance. Chronic tempol treatment partially prevented the salt-sensitivity of hGRK4γ486V mice. Thus, hGRK4γ486V causes salt-sensitive hypertension due, in part, to defective renal antioxidant mechanisms.

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.

The causal role of dietary NaCl intake in the development of hypertension and salt sensitivity has long been recognized. The kidney sits at the fulcrum of maintaining an appropriate sodium balance ...

Lack or downregulation of the dopamine D2receptor (D2R) results in increased renal expression of injury markers and proinflammatory factors that is independent of a blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D2Rs. Silencing D2Rs in mouse renal proximal tubule cells increased the expression of the proinflammatory TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6. D2R downregulation also increased Akt phosphorylation and activity, and glycogen synthase kinase-3β (GSK3β) phosphorylation and cyclin D1 expression, downstream targets of Akt; however. phosphatidylinositol 3-kinase (PI3K) activity was not affected. Conversely, D2R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of protein phosphatase 2A (PP2A) with okadaic acid reproduced the effects of D2R downregulation on Akt, GSK3β, and cyclin D1. The PP2A catalytic subunit and regulatory subunit PPP2R2C coimmunoprecipitated with the D2R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D2R silencing that also blunted the increase in phosphatase activity induced by D2R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNF-α, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D2R effect on inflammatory factors. Indeed, the increase in NF-κB reporter activity induced by D2R silencing was blunted by increasing PP2A activity with protamine. Our results show that D2R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression.

Oxidative stress and inflammation are important players in the pathogenesis of cardiovascular and renal diseases. DJ-1 is a redox-sensitive chaperone that regulates the expression of several antioxidant genes. Activation of the DJ-1/Nrf2 pathway in the kidney inhibits the development and progression of several renal diseases. The 20 aa peptide ND-13 consists of 13 highly conserved aa from the DJ-1 sequence and a TAT- derived 7 aa sequence to help in cell penetration. ND-13 prevents neuronal degeneration in mice; however, its effects on kidney damage remain unknown. We hypothesized that treatment with ND-13 would prevent the renal damage and inflammation associated with unilateral ureter obstruction (UUO). C57Bl/6 mice and DJ1 -/- mice underwent UUO and were divided in 3 groups: control (no UUO), UUO+scrambled peptide (SP) or UUO+ND-13 (3 mg/kg, s.c. daily). After 14 days of treatment, urine and kidneys were collected for analysis of renal damage. ND-13 treatment prevented the development of fibrosis in C57Bl/6 mice (UUO+SP: 702±189% of control, UUO+ND-13: 264±8% of control, n=2-4/group, pTNF- α ( fold change from control: 101±46 in UUO+SP; 18±7 in UUO+ND-13, n=4-5/group, pIL-6 (6.7±2 in UUO+SP; 1.54±0.3 in UUO+ND-13, pTGF- β ( 3.3±1.12 UUO+SP; 1.4±0.03 UUO+ND-13, pColagen1 α 1 (79±16 UUO+SP; 33±3.8 in UUO +ND-13, p-/- mice, treatment with ND-13 similarly decreased expression of TNF-α, IL-6 and TGF-β, but, in contrast, failed to prevent renal fibrosis or kidney expression of col1 α 1 . UUO also led to elevated urinary NGAL, marker of proximal tubular injury, in DJ-1 -/- mice and ND-13 treatment prevented that increase (71±17% UUO vs control: -18±21% UUO+ND-13 vs control, n=5/group). Our results suggest that ND-13 has protective effects on renal injury, fibrosis and inflammation, crucial mechanisms in the pathogenesis of renal disease. Thus, ND-13 treatment may be a new therapeutic approach for the prevention of renal injury, fibrosis and inflammation in renal diseases. Funded by T32DK007545 to CDM and 5P01 HL074940-10, 7R01 DK039308-31 to PAJ.

C57Bl/6 and BALB/c are different in the sensitivity of their blood pressure (BP) to NaCl. High NaCl diet increases BP in C57Bl/6J but not BALB/c mice. However, C57Bl/6J mice are less prone to hypertension-induced renal injury than other mouse strains. The goal of this study was to determine the role of H 2 O 2 in salt-sensitive hypertension. Basal renal levels of reactive oxygen species, including H 2 O 2, were higher in BALB/c renal proximal tubule cells (RPTC-BALB/c) than RPTCs from C57Bl/6J mice (RPTC-C57Bl/6J) (60.8 ± 5 % vs 41.8 ± 12 %, n=8, P2 O 2 production in RPTC-BALB/c but not in RPTC-C57Bl/6J, compared with RPTCs incubated in low (90 mM) or normal (145 mM) NaCl (+68±43%, 90 vs 170 mM, n=7, P2 O 2 (10 μM) treatment of the basolateral side of RPTC-BALB/c in Transwells increased intracellular Na + 1.62-fold that of vehicle-treated cells (n=4, P2 O 2 -induced increase in intracellular Na + in RPTC-BALB/c. Over-expression of catalase in the kidneys of BALB/c mice on normal (0.9%) NaCl diet did not alter their SBP. However, on high (4%) NaCl diet, SBP was increased in catalase over-expressing mice, relative to vehicle-treated controls (98±1.1 vs 112±1.4 mm Hg, n=3, P2 O 2 by overexpression of catalase predisposes BALB/c mice to salt-sensitive hypertension, suggesting that high salt-induced H 2 O 2 negatively regulates renal sodium transport and provides resistance to salt-induced hypertension.

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na+/glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression.

To test if there is a sex difference in the salt sensitivity of C57Bl/6J mice, we studied blood pressure (BP), renal dopamine receptors, and sodium transporters in response to high salt diet (4% NaCl, 1 wk) and candesartan. Similar to males, the night-time systolic BP (SBP, telemetry, n=4) in females started to increase on day 1, peaked on day 2 (130±1 vs 117±1, mm Hg) and remained at high levels (126±1); the daytime SBP started to increase on day 2 that became significant on day 6 (124±1 vs 111±1). The high salt-diet induced-increase in SBP was prevented by candesartan (1 mg/kg, 1 wk, subcutaneously via osmotic mini-pump) (82±3 vs. 117±2 in males; 86±2 vs. 121±1 in females, under anesthesia, n=5/group). There were no sex differences in the SBP response to diet and candesartan, food and water intakes, urinary excretions of water and electrolytes, and serum concentrations of creatinine and electrolytes on high and normal salt diet. In females fed a high salt diet, candesartan increased renal D 1 R (151±12, immunoblotting, % of control, n=5/group) and D 5 R (156±5) but not D 2 R, D 3 R , or D 4 R protein expression, increased renal mRNA expression of D 1 R(160±17) but not D 5 R (real-time PCR), and decreased the renal protein expressions of sodium-hydrogen exchanger isoform 3 (36±6), sodium-potassium-2 chloride cotransporter (8±2), sodium-chloride cotransporter (30±3), and α (55±4), β (60±8) and γ (9±1) epithelial sodium channel, but not type 2 sodium phosphate cotransporter and α1Na + K + ATPase. Those changes were also seen in males except that renal D 1 R protein expression was not increased. Under normal salt intake, AT 1A R KO females (C57Bl/6J background) had increased renal protein expressions of D 1 R (152±8), D 5 R (131±6), and D 4 R (114±4), but not D 2 R and D 3 R; renal protein expressions of sodium-hydrogen exchanger isoform 3 (47±8), sodium-potassium-2 chloride cotransporter (52±11), and α (42±9) and γ (46±7) epithelial sodium channel were decreased. We conclude that the increase in BP caused by high salt diet and the effect of AT 1 R blockade on BP, renal dopamine receptors and sodium transporters are similar in male and female C57Bl/6J mice . The consequences of the sex-related differential regulation of D 1 R and D 5 R, in pathophysiology, other than BP, remain to be determined.

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.

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.