Renal T cell infiltration occurs despite attenuation of development of hypertension with hydralazine in Envigo's female Dahl rat maintained on a low-Na+ diet.

Many studies have suggested that renal T cell infiltration contributes to the pathogenesis of salt-sensitive hypertension. To investigate this mechanism further, we determined T cell profiles in the kidney and lymphoid tissues as a function of blood pressure in the female Envigo Dahl salt-sensitive (SS) rat maintained on low-Na+ (LS) diet. Mean arterial pressure and heart rate were measured by telemetry in SS rats from 1 mo old (juvenile) to 4 mo old. Normotensive salt-resistant (SR) rats were included as controls. Frequencies of T helper (CD4+) cells were greater in the kidney, lymph nodes, and spleen in 4-mo-old hypertensive SS rats compared with normotensive SR animals and SS juvenile rats, suggesting that renal T cell infiltration contributes to hypertension in the SS rat on a LS diet. At 1.5 mo, half of the SS rats were treated with vehicle (Veh), and the rest received hydralazine (HDZ; 25 mg·kg-1·day-1) for 11 wk. HDZ impeded the development of hypertension compared with Veh-treated control rats [mean arterial pressure: 157 ± 4 mmHg in the Veh-treated group (n = 6) vs. 133 ± 3 mmHg in the HDZ-treated group (n = 7), P < 0.001] without impacting T helper cell frequencies in the tissues, suggesting that HDZ can overcome mechanisms of hypertension driven by renal T cell infiltration under the LS diet. Renal frequencies of CD4+CD25+ and CD4+CD25+FoxP3+ regulatory T cells were significantly higher in 4-mo-old hypertensive rats compared with normotensive SR rats and SS juvenile rats, suggesting that these T cell subpopulations play a compensatory role in the development of hypertension. Greater understanding of these T cell populations could lead to new therapeutic targets for treating inflammatory diseases associated with hypertension.

Salt-sensitive (Rapp) rats from Envigo spontaneously develop accelerated hypertension independent of ovariectomy on a low-sodium diet.

Inbred salt-sensitive (SS) rats developed by John Rapp and distributed by Harlan (SS/JrHsd) were shown to model ovariectomy-induced hypertension because on a low-sodium (LS) diet, ovariectomized SS (SS-OVX) animals became hypertensive in contrast to their sham-operated (SS-SHAM) normotensive littermates. After Harlan merged with Envigo in 2015, inconsistencies in the LS normotensive phenotype were reported. To further investigate these inconsistencies, we studied the effects of ovariectomy on SS and salt-resistant (SR) rats purchased from Envigo (SS/JrHsd/Env) between 2015 and 2017. The mean arterial pressure (MAP) in SS rats on a LS diet exceeded 160 mmHg at 7 mo old. Ovariectomy at 3 mo had no detectable effect on MAP from 4 to 7 mo, nor did ovariectomy at 1.5 mo significantly affect MAP at 10 mo in either strain; only strain differences in MAP were observed [MAP: SR-SHAM ( n = 7 rats), 102 ± 3 mmHg; SR-OVX ( n = 6 rats), 114 ± 1 mmHg; SS-SHAM ( n = 7 rats), 177 ± 6 mmHg; SS-OVX ( n = 5 rats), 190 ± 12 mmHg; where P < 0.0001 vs. SR, same ovarian-status for SS-SHAM and SS-OVX, respectively]. Whole genome sequencing revealed more genomic variants of SS/JrHsd/Env, including single nucleotide and insertion deletion polymorphisms and higher heterozygous/homozygous ratios compared with the reference genome, than for SS/JrHsd/Mcwi and SS/Jr rats maintained in Milwaukee, WI and Toledo, OH, respectively, and which still exhibit normal blood pressure on a LS diet. These findings demonstrate that the female SS/JrHsd/Env rat has genetically diverged from the original phenotype, which was normotensive on a LS diet when the ovaries were intact but rapidly developed hypertension when the ovaries were removed. Nonetheless, the SS/JrHsd/Env rat could be a valuable model that complements other animal models of spontaneous hypertension used to investigate mechanisms of essential hypertension.

Renal and colonic potassium transporters in the pregnant rat.

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+-ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.

Relaxin-mediated renal vasodilation in the rat is associated with falls in glomerular blood pressure.

Relaxin (RLX) is a pleiotropic peptide hormone with marked renal vasodilatory actions that are physiologically important during pregnancy. RLX also has potent antifibrotic actions and is being tested therapeutically in various fibrotic diseases, including chronic kidney disease (CKD). Since renal vasodilation may expose the glomerulus to increased blood pressure [glomerular capillary pressure (PGC)], which exacerbates progression of CKD, we assessed the glomerular hemodynamic actions of acute (0.89 µg·100 g body wt-1·h-1 iv over 75 min) and chronic (1.5 µg·100 g body wt-1·h-1 sc) administration of RLX. Both acute and chronic RLX produced marked renal vasodilation and increased renal plasma flow (RPF) in euvolemic, anesthetized male rats. Glomerular filtration rate also increased with RLX, but the magnitude of the rise was much less than the increase in RPF due to concomitant decreases in filtration fraction. The fall in filtration fraction was the result of significant decreases in PGC, despite a slight increase in mean arterial blood pressure (MAP) with acute RLX and no net change in MAP with chronic RLX. This fall in PGC occurred because of the "in-series" arrangement of the afferent and efferent arteriolar resistance vessels, which can regulate PGC independently of MAP. With both acute and chronic RLX, efferent arteriolar resistance vessels relaxed to a greater extent than afferent arteriolar resistance vessels, thus producing falls in PGC. Based on this finding, RLX has a beneficial hemodynamic impact on the kidney, which, together with the antifibrotic actions of RLX, suggests a strong therapeutic potential for use in CKD.

The enigma of continual plasma volume expansion in pregnancy: critical role of the renin-angiotensin-aldosterone system.

Pregnancy is characterized by avid renal sodium retention and plasma volume expansion in the presence of decreased blood pressure. Decreased maternal blood pressure is a consequence of reduced systemic vascular tone, which results from an increased production of vasodilators [nitric oxide (NO), prostaglandins, and relaxin] and decreased vascular responsiveness to the potent vasoconstrictor (angiotensin II). The kidneys participate in this vasodilatory response, resulting in marked increases in renal plasma flow and glomerular filtration rate (GFR) during pregnancy. In women, sodium retention drives plasma volume expansion (∼40%) and is necessary for perfusion of the growing uterus and fetus. For there to be avid sodium retention in the presence of the potent natriuretic influences of increased NO and elevated GFR, there must be modifications of the tubules to prevent salt wasting. The purpose of this review is to summarize these adaptations.

Matrix Extension Study: Validation of the Compact Dry CF Method for Enumeration of Total Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" CF method, Performance Tested Method(SM) 110401, was originally certified for enumeration of coliform bacteria by the AOAC Research Institute Performance Tested Methods(SM) program for raw meat products. Compact Dry CF is a ready-to-use dry media sheet, containing a cold-soluble gelling agent, a chromogenic medium, and selective agents, which are rehydrated by adding 1 mL of diluted sample. Coliform bacteria produce blue/blue-green colonies on the Compact Dry CF, allowing a coliform colony count to be determined in the sample after 24 ± 2 h incubation. A validation study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom, to extend the method's claim to include cooked chicken, fresh bagged prewashed shredded iceberg lettuce, frozen fish, milk powder, and pasteurized 2% milk. Campden BRI collected single-laboratory data for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Thirteen laboratories participated in the interlaboratory study. The Compact Dry CF method was compared to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current version at the time this study was conducted. Each matrix was evaluated at either four or five contamination levels of coliform bacteria (including an uncontaminated level). After logarithmic transformation of counts at each level, the data for pasteurized whole milk were analyzed for sr, sR, RSDr, and RSDR. Regression analysis was also performed and r(2) was reported. Mean difference between methods with 95% confidence interval (CI) was calculated. A log10 range of -0.5 to 0.5 for the CI was used as the acceptance criterion to establish significant statistical difference between methods. In the single-laboratory evaluation (for cooked chicken, lettuce, frozen fish, and milk powder), sr and RSDr values were analyzed and r(2) was reported. Statistical differences were indicated between the Compact Dry CF and ISO 4832 methods in two of five contamination levels tested for lettuce, and in the low contamination levels for cooked chicken, frozen fish, and dry milk powder. For the low levels of cooked chicken, frozen fish, and milk powder, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. In most cases, mean differences between the Compact Dry CF and ISO 4832 methods were small (<0.5 log10), with CIs within the acceptance criterion. The sr and RSDr values were similar for both methods, and r(2) values were >0.94 for all matrixes. In the multilaboratory study, no statistical differences were indicated between methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller for the Compact Dry CF. The r(2) value was 0.99. The Compact Dry CF method offers comparable results to ISO 4832 in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry EC Method for Enumeration of Escherichia coli and non-E. coli Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" EC method, originally certified by the AOAC Research Institute Performance Test Method(SM) program for enumeration of Escherichia coli and non-E. coli coliforms in raw meat products (Performance Tested Method(SM) 110402), has undergone an evaluation to extend the method's claim to cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, instant nonfat dry milk powder, and pasteurized milk (2% fat). Compact Dry EC is a ready-to-use dry media sheet containing a cold-soluble gelling agent, selective agents, and a chromogenic medium, which are rehydrated by adding 1 mL diluted sample. E. coli form blue/blue-purple colonies, whereas other coliform bacteria form red/pink colonies. Users can obtain an E. coli count (blue/blue-purple colonies only) and a total coliform count (red/pink plus blue/blue-purple colonies) after 24 ± 2 h of incubation at 37 ± 1°C. The matrix extension study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom. Method comparison data for cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder were collected in a single-laboratory evaluation by Campden BRI. A multilaboratory study was conducted on pasteurized milk (2% fat), with 13 laboratories participating. The Compact Dry EC method was compared to ISO 16649-2:2001 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli-Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide" and to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current standards at the time of this study. Each matrix was evaluated separately for E. coli and non-E. coli coliforms at each contamination level (including an uncontaminated level). In the single-laboratory evaluation (cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder), colony counts were logarithmically transformed, and then the data were analyzed at each level for sr, RSDr, and mean difference between methods with 95% confidence intervals (CIs). A CI outside a range of -0.5 to 0.5 on the log10 mean difference between methods was used as the criterion to establish a significant statistical difference. In the multilaboratory study on pasteurized milk, after logarithmic transformation, the data were analyzed for sR and RSDR in addition to sr, RSDr, and mean difference with 95% CIs. Regression analysis was performed on all matrixes and reported as r(2). In the single-laboratory evaluation, statistical differences were indicated between the Compact Dry EC and ISO 16649-2 methods for the enumeration of E. coli in two of five contamination levels tested for lettuce, and in the low contamination level for cooked chicken. For the cooked chicken and lettuce at the low level, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. For the high contamination level of lettuce, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant difference. Statistical differences were also indicated between the Compact Dry EC and ISO 4832 methods for the enumeration of coliforms in two of five contamination levels tested for lettuce, two of five contamination levels of milk powder, and in the low contamination level for frozen fish. For the lowest levels of frozen fish and milk powder, only a few colonies were recovered for each method. For the lettuce and the other level of milk powder, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant differences indicated in the those contamination levels. In most cases, mean differences between the Compact Dry EC and International Organization of Standardization (ISO) methods were well below 0.5 log10, and the CIs were within the acceptance criterion (-0.5 to 0.5). The sr and RSDr values were similar for both methods, and r(2) values were >0.92 for all comparisons. In the multilaboratory study, no statistical differences were indicated between the methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller in most cases for the Compact Dry EC. The r(2) value was 0.97 in comparison to ISO 16649-2, and 0.99 in comparison to ISO 4832. The Compact Dry EC offers comparable results to the ISO standard plating methods in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry TC Method for Enumeration of Total Aerobic Bacteria in Selected Foods.

A validation study was conducted to extend the matrix claim for the Nissui Compact Dry Total Count (TC), Performance Tested Method(s)(SM) (PTM) Certification No. 010404, to cooked chicken, lettuce, frozen fish, milk powder, and pasteurized whole milk. The method was originally certified by the AOAC Research Institute Performance Tested Method(s)(SM) Program for raw meat products. The Compact Dry TC is a ready-to-use dry media sheet that is rehydrated by adding 1 mL of diluted sample. A total aerobic colony count can be determined in the sample following 48 h of incubation. Matrix extension studies were conducted by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology Limited), Chipping Campden, UK. Single-laboratory data were collected for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Fourteen laboratories participated in the collaborative study. The Compact Dry TC was tested at two time points, 48 ± 3 h and 72 ± 3 h and compared with the current International Organization for Standardization (ISO) method at the time of the study, ISO 4833:2003 (this standard is withdrawn and has been replaced by: ISO 4833-1:2013 and ISO 4833-2:2013), Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of microorganisms-Colony-count technique at 30°C. The data were logarithmically transformed and evaluated for repeatability (plus reproducibility for pasteurized milk), RSD of repeatability (plus RSD of reproducibility for milk), r(2), and mean difference between methods with 95% confidence interval (CI). A CI outside of (-0.5 to 0.5) on the log10 mean difference was used as the criterion to establish significant statistical difference between methods. No significant differences were found between the Compact Dry TC 48 and 72 h time points, with the exception of one contamination level of cooked chicken and one contamination level of dry milk powder. Mean differences were small at these levels (<0.5 log10), but the upper CIs were above 0.5. Statistical differences were indicated between the Compact Dry TC and ISO 4833 in two of five contamination levels tested each for lettuce and frozen fish. In each case, mean differences were >0.5 log10, and the total aerobic colony count was higher for the ISO method. In most cases, mean differences between the Compact Dry and ISO methods were small (<0.5 log10) with CIs within the acceptance criterion. Repeatability, reproducibility, and RSD were similar for both methods, and r(2) values were >0.97 for all matrixes. The Compact Dry TC, at 48 h, offers the advantage of a shorter time to results than ISO 4833 in an easy-to-use format.

The chloride-bicarbonate exchanger pendrin is increased in the kidney of the pregnant rat.

NEW FINDINGS: What is the central question of this study? Pregnancy requires a robust plasma volume expansion driven by renal sodium retention. In the late-pregnant kidney, the aldosterone-responsive epithelial Na(+) channel is increased, whereas the sodium-chloride cotransporter is decreased. Pendrin has been shown to support sodium reabsorption in the distal nephron and compensate for loss of the sodium-chloride cotransporter. We investigated the expression and abundance of pendrin in the pregnant kidney. What is the main finding and its importance? Pendrin protein, apical localization and thiazide sensitivity are increased in pregnancy. This implicates a possible role for pendrin in supporting the renal sodium chloride reabsorption and plasma volume expansion of pregnancy. Pregnancy is characterized by cumulative plasma volume expansion as a result of renal sodium retention, driven by activation of aldosterone. We previously reported that the abundance and activity of the aldosterone-responsive epithelial Na(+) channel is increased, whereas the sodium-chloride cotransporter (NCC) is decreased in the kidney of the late-pregnant rat. The chloride-bicarbonate exchanger pendrin is also aldosterone responsive and has been shown to support activity of the aldosterone-responsive epithelial Na(+) channel and compensate for the loss of NCC. Additionally, pendrin coupled to the sodium-dependent chloride-bicarbonate exchanger (NDCBE) mediates thiazide-sensitive sodium reabsorption in the cortical collecting duct. In this study, we investigated pendrin and NDCBE transcript expression, pendrin protein abundance, pendrin cellular localization and thiazide sensitivity in virgin, mid-pregnant and late-pregnant rats to test the hypothesis that increased pendrin activity might occur in pregnancy. By RT-PCR, NDCBE and pendrin mRNA expression was unchanged from virgins, whereas pendrin protein abundance determined by Western blotting was increased in both mid- and late-pregnant rats. The apical localization of pendrin was also increased in late-pregnant rats compared with virgins by immunohistochemistry. Pregnant rats displayed an increased natriuretic response to hydrochlorothiazide compared with virgins. Given that NCC expression is decreased in late pregnancy, an increased thiazide sensitivity may be due to inhibition of upregulated pendrin-NDCBE-coupled sodium reabsorption. Thus, increased pendrin in pregnant rats may compensate for the decreased NCC and aid in the renal sodium chloride reabsorption of pregnancy.

Renal NCC is unchanged in the midpregnant rat and decreased in the late pregnant rat despite avid renal Na+ retention.

Pregnancy is characterized by plasma volume expansion due to Na(+) retention, driven by aldosterone. The aldosterone-responsive epithelial Na(+) channel is activated in the kidney in pregnancy. In the present study, we investigated the aldosterone-responsive Na(+)-Cl(-) cotransporter (NCC) in mid- and late pregnant rats compared with virgin rats. We determined the abundance of total NCC, phosphorylated NCC (pNCC; pT53, pS71 and pS89), phosphorylated STE20/SPS-1-related proline-alanine-rich protein kinase (pSPAK; pS373), and phosphorylated oxidative stress-related kinase (pOSR1; pS325) in the kidney cortex. We also measured mRNA expression of NCC and members of the SPAK/NCC regulatory kinase network, serum and glucocorticoid-regulated kinase (SGK)1, total with no lysine kinase (WNK)1, WNK3, and WNK4. Additionally, we performed immunohistochemistry for NCC kidneys from virgin and pregnant rats. Total NCC, pNCC, and pSPAK/OSR1 abundance were unchanged in midpregnant versus virgin rats. In late pregnant versus virgin rats, total NCC and pNCC were decreased; however, pSPAK/OSR1 was unchanged. We detected no differences in mRNA expression of NCC, SGK1, total WNK1, WNK3, and WNK4. By immunohistochemistry, NCC was mainly localized to the apical region in virgin rats, and density in the apical region was reduced in late pregnancy. Therefore, despite high circulating aldosterone levels in pregnancy, the aldosterone-responsive transporter NCC is not increased in total or activated (phosphorylated) abundance or in apical localization in midpregnant rats, and all are reduced in late pregnancy. This contrasts to the mineralocorticoid-mediated activation of the epithelial Na(+) channel, which we have previously reported. Why and how NCC escapes aldosterone activation in pregnancy is not clear but may relate to regional differences in aldosterone sensitivity the increased K(+) intake or other undefined mechanisms.

Pregnant rats treated with a high-fat/prooxidant Western diet with ANG II and TNF-α are resistant to elevations in blood pressure and renal oxidative stress.

Oxidative stress and inflammation are risk factors for hypertension in pregnancy. Here, we examined the 24-h mean arterial pressure (MAP) via telemetry and the nitric oxide (NO) and redox systems in the kidney cortex, medulla, and aorta of virgin and pregnant rats treated with a high-fat/prooxidant Western diet (HFD), ANG II, and TNF-α. Female Sprague-Dawley rats were given a normal diet (ND) or a HFD for 8 wk before mating. Day 6 of pregnancy and age-matched virgins were implanted with minipumps infusing saline or ANG II (150 ng·kg(-1)·min(-1)) + TNF-α (75 ng/day) for 14 days. Groups consisted of Virgin + ND + Saline (V+ND) (n = 7), Virgin + HFD +ANG II and TNF-α (V+HFD) (n = 7), Pregnant + ND + Saline (P+ND) (n = 6), and Pregnant + HFD + ANG II and TNF-α (P+HFD) (n = 8). After day 6 of minipump implantation, V+HFD rats displayed an increase in MAP on days 7, 8, and 10-15 vs. V+ND rats. P+HFD rats, after day 6 of minipump implantation, showed an increase in MAP only on day 7 vs. P+ND rats. P+HFD rats had a normal fall in 24-h MAP, hematocrit, plasma protein concentration, and osmolality at late pregnancy. No change in kidney cortex, medulla, or aortic oxidative stress in P+HFD rats. P+HFD rats displayed a decrease in nNOSß abundance, but no change in kidney cortex NOx content vs. P+ND rats. Pregnant rats subjected to a chronic HFD and prooxidant and proinflammatory insults have a blunted increase in 24-h MAP and renal oxidative stress. Our data suggest renal NO bioavailability is not altered in pregnant rats treated with a HFD, ANG II, and TNF-α.

Serelaxin reduces oxidative stress and asymmetric dimethylarginine in angiotensin II-induced hypertension.

Recent findings suggest the therapeutic action of relaxin during hypertension is dependent on nitric oxide synthase (NOS) activation; however, the mechanisms underlying the beneficial effects of relaxin on the NOS system have not been fully elucidated. We hypothesized that the protective effects of relaxin include reducing both oxidative stress and the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). We examined the effect of Serelaxin [human recombinant relaxin-2 (RLX)] in male Sprague-Dawley rats given high-dose angiotensin (ANG) II (400 ng·kg(-1)·min(-1) sc) for 6 wk or shams. RLX was administered (4 µg/h sc) to half of the rats in each group after 2 wk of ANG II for the remaining 4 wk. ANG II induced hypertension and proteinuria, reduced NO oxidation products (NOx), and increased oxidative stress (NADPH oxidase activity, thiobarbituric acid-reactive substances, and 8-isoprostane excretion) and plasma ADMA. While RLX had no effect on sham rats, RLX attenuated the ANG II-dependent hypertension (165 ± 5 vs. 135 ± 13 mmHg, P < 0.05) and proteinuria at 6 wk (62 ± 6 vs. 41 ± 4 mg·day(-1)·100 g(-1), P < 0.05) and normalized oxidative stress and circulating ADMA, in association with restored NOx excretion and kidney cortex NOx. We found that RLX had no impact on the ADMA-regulatory enzymes protein arginine methyltransferase and dimethylarginine-dimethylaminohydrolase (DDAH). Furthermore, RLX treatment did not increase DDAH activity in kidney cortex or liver. These data suggest that benefits of RLX treatment include reduced ADMA levels and increased NO bioavailability, possibly due to its antioxidant effects.

Chronic vasodilation increases renal medullary PDE5A and α-ENaC through independent renin-angiotensin-aldosterone system pathways.

We have previously observed that many of the renal and hemodynamic adaptations seen in normal pregnancy can be induced in virgin female rats by chronic systemic vasodilation. Fourteen-day vasodilation with sodium nitrite or nifedipine (NIF) produced plasma volume expansion (PVE), hemodilution, and increased renal medullary phosphodiesterase 5A (PDE5A) protein. The present study examined the role of the renin-angiotensin-aldosterone system (RAAS) in this mechanism. Virgin females were treated for 14 days with NIF (10 mg·kg(-1)·day(-1) via diet), NIF with spironolactone [SPR; mineralocorticoid receptor (MR) blocker, 200-300 mg·kg(-1)·day(-1) via diet], NIF with losartan [LOS; angiotensin type 1 (AT1) receptor blocker, 20 mg·kg(-1)·day(-1) via diet], enalapril (ENAL; angiotensin-converting enzyme inhibitor, 62.5 mg/l via water), or vehicle (CON). Mean arterial pressure (MAP) was reduced 7.4 ± 0.5% with NIF, 6.33 ± 0.5% with NIF + SPR, 13.3 ± 0.9% with NIF + LOS, and 12.0 ± 0.4% with ENAL vs. baseline MAP. Compared with CON (3.6 ± 0.3%), plasma volume factored for body weight was increased by NIF (5.2 ± 0.4%) treatment but not by NIF + SPR (4.3 ± 0.3%), NIF + LOS (3.6 ± 0.1%), or ENAL (4.0 ± 0.3%). NIF increased PDE5A protein abundance in the renal inner medulla, and SPR did not prevent this increase (188 ± 16 and 204 ± 22% of CON, respectively). NIF increased the α-subunit of the epithelial sodium channel (α-ENaC) protein in renal outer (365 ± 44%) and inner (526 ± 83%) medulla, and SPR prevented these changes. There was no change in either PDE5A or α-ENaC abundance vs. CON in rats treated with NIF + LOS or ENAL. These data indicate that the PVE and renal medullary adaptations in response to chronic vasodilation result from RAAS signaling, with increases in PDE5A mediated through AT1 receptor and α-ENaC through the MR.

Renal nitric oxide synthase and antioxidant preservation in Cyp1a1-Ren-2 transgenic rats with inducible malignant hypertension.

BACKGROUND: Dietary administration of 0.30% indole-3-carbinol (I3C) to Cyp1a1-Ren2 transgenic rats (TGRs) generates angiotensin II (ANG II)-dependent malignant hypertension (HTN) and increased renal vascular resistance. However, TGRs with HTN maintain a normal or slightly reduced glomerular filtration rate. We tested the hypothesis that maintenance of renal function in hypertensive Cyp1a1-Ren2 TGRs is due to preservation of the intrarenal nitric oxide (NO) and antioxidant systems. METHODS: Kidney cortex, kidney medulla, aortic endothelial (e) and neuronal (n) nitric oxide synthase (NOS), superoxide dismutases (SODs), and p22phox (nicotinamide adenine dinucleotide phosphate-oxidase subunit) protein abundances were measured along with kidney cortex total antioxidant capacity (TAC) and NOx. TGRs were fed a normal diet that contained 0.3% I3C or 0.3% I3C + candesartan (AT1 receptor antagonist; 25mg/L in drinking water) (n = 5-6 per group) for 10 days. RESULTS: Blood pressure increased and body weight decreased in I3C-induced TGRs, while candesartan blunted these responses. Abundances of NOS, SOD, and p22phox as well as TAC were maintained in the kidney cortex of I3C-induced TGRs with and without candesartan, while kidney cortex NOx production increased in both groups. Kidney medulla eNOS and extracellular (EC) SOD decreased and nNOS were unchanged in both groups of I3C-induced TGRs. In addition, a compensatory increase occurred in kidney medulla Mn SOD in I3C-induced TGRs + candesartan. Aortic eNOS and nNOS∝ fell and p22phox and Mn SOD increased in hypertensive I3C-induced TGRs; all changes were reversed with candesartan. CONCLUSIONS: The preservation of renal cortical NO and antioxidant capacity is associated with preserved renal function in Cyp1a1-Ren2 TGRs with ANG II-dependent malignant HTN.

Renal redox response to normal pregnancy in the rat.

Normal pregnancy involves increased renal sodium reabsorption, metabolism, and oxygen consumption, which can cause increased oxidative stress (OS). OS can decrease nitric oxide (NO) bioavailability and cause pregnancy complications. In this study we examined the NO synthases (NOS) and redox state in the kidney cortex and aorta in early (E), mid (M), and late (L) pregnant (P) (days 3, 12, 20) and 2-4 days postpartum (PP) rats compared with virgin rats (V). Protein abundance of endothelial NOS (eNOS) was unchanged and neuronal NOS (nNOS)α fell at LP in the kidney cortex. Kidney cortex nNOSß was elevated at MP, LP, and PP. No changes in aortic NOS isoforms were observed. Kidney cortex nitrotyrosine (NT) abundance decreased in EP, MP, and PP, whereas aortic NT increased in EP, MP, and PP. The NADPH oxidase subunit p22phox decreased in the kidney cortex at EP while aortic p22phox increased in EP and LP. No changes in kidney cortex NADPH-dependent superoxide production or hydrogen peroxide levels were noted. Kidney cortex cytosolic (CuZn) superoxide dismutase (SOD) was unchanged, while mitochondrial SOD decreased at EP and extracellular SOD decreased at MP and LP in the kidney cortex. Despite falls in abundance of kidney cortex SODs, total antioxidant capacity (TAC) was elevated in EP, MP, and PP in the kidney cortex. Aortic CuZn SOD deceased at PP, while the other aortic SODs and aortic TAC did not change. Data from this study suggest that the kidney cortex is protected from OS during normal rat pregnancy via an increase in antioxidant activity.

Effects of voluntary wheel running on the kidney at baseline and after ischaemia-reperfusion-induced acute kidney injury: a strain difference comparison.

Exercise-induced vascular endothelial adaptations in the kidney are not well understood. Therefore, we investigated the impact of voluntary wheel running (VWR) on the abundance of endothelial nitric oxide synthase (eNOS) and extracellular superoxide dismutase (EC SOD), in kidney and lung, and other SOD isoforms and total antioxidant capacity (TAC), in kidney. We also determined whether VWR influences susceptibility to acute kidney injury (AKI). Male Sprague-Dawley and Fisher 344 rats, VWR or sedentary for 12 weeks, were subjected to AKI (uninephrectomy (UNX) and 35 min of left kidney ischaemia-24 h reperfusion, IR). We measured glomerular filtration rate (GFR) and renal plasma flow (RPF), and analysed renal structural injury. Running was comparable between strains and VWR reduced body weight. In Sprague-Dawley rats, VWR reduced eNOS and EC SOD, but increased Mn SOD in kidney. Similar changes were seen after 6 weeks of VWR in Sprague-Dawley rats. In Fisher 344 rats, VWR increased eNOS, all SOD isoforms and TAC in kidney. Both strains increased eNOS and EC SOD in lung with VWR. Compared to UNX alone, UNX-IR injury markedly reduced renal function for both strains; however, in the Sprague-Dawley rats, VWR exacerbated falls in GFR and RPF due to UNX-IR, whereas in the Fisher 344 rats, GFR was unaffected by VWR. Some indices of renal structural injury due to UNX-IR tended to be worse in SD vs. F344. Our study demonstrates that genetic background influences the effect of exercise on kidney eNOS and EC SOD, which in turn influence the susceptibility to AKI.

Sexual dimorphism: the aging kidney, involvement of nitric oxide deficiency, and angiotensin II overactivity.

Females develop less age-dependent loss of renal function, which may be in part due to cardiorenal protective effects of estrogens. The impact of androgen level on cardiovascular-renal health is controversial. Estrogen acts through multiple mechanisms, sometimes beneficial, sometimes damaging, which makes it difficult to predict the effect of hormone replacement therapy (HRT) in an aging population. Nitric oxide (NO) deficiency occurs in aging and contributes to age-dependent cardiovascular risk and kidney damage. The increased oxidative stress of aging has effects at multiple sites in the NO biosynthetic pathway to lower NO production/action. Loss of NO together with activated angiotensin promotes some of the decrements in cardiovascular-renal function seen with age, which may be related to actions of the sex steroids.

Sexual dimorphism in development of kidney damage in aging Fischer-344 rats.

BACKGROUND: Aging kidneys exhibit slowly developing injury and women are usually protected compared with men, in association with maintained renal nitric oxide. OBJECTIVES: Our purpose was to test 2 hypotheses: (1) that aging intact Fischer-344 (F344) female rats exhibit less glomerular damage than similarly aged males, and (2) that loss of female ovarian hormones would lead to greater structural injury and dysregulation of the nitric oxide synthase (NOS) system in aging F344 rat kidneys. METHODS: We compared renal injury in F344 rats in intact, ovariectomized, and ovariectomized with estrogen replaced young (6 month) and old (24 month) female rats with young and old intact male rats and measured renal protein abundance of NOS isoforms and oxidative stress. RESULTS: There was no difference in age-dependent glomerular damage between young or old intact male and female F344 rats, and neither ovariectomy nor estrogen replacement affected renal injury; however, tubulointerstitial injury was greater in old males than in old females. These data suggest that ovarian hormones do not influence these aspects of kidney aging in F344 rats and that the greater tubulointerstitial injury is caused by male sex. Old males had greater kidney cortex NOS3 abundance than females, and NOS1 abundance (alpha and beta isoforms) was increased in old males compared with both young males and old females. NOS abundance was preserved with age in intact females, ovariectomy did not reduce NOS1 or NOS3 protein abundance, and estrogen replacement did not uniformly elevate NOS proteins, suggesting that estrogens are not primary regulators of renal NOS abundance in this strain. Nicotinamide adenine dinucleotide phosphate oxidase-dependent superoxide production and nitrotyrosine immunoreactivity were increased in aging male rat kidneys compared with females, which could compromise renal nitric oxide production and/or bioavailability. CONCLUSIONS: The kidney damage expressed in aging F344 rats is fairly mild and is not related to loss of renal cortex NOS3 or NOS1 alpha.

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats.

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.