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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.

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.

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.

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.

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.

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.

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 nNOSβ abundance, but no change in kidney cortex NOxcontent 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.

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.

Angiotensin II (ANG II)–dependent hypertension (HTN) and renal vasoconstriction are induced in Cyp1a1-Ren2 (strain name: TGR (Cyp1a1Ren2)) transgenic rats (TGR) by dietary administration of indole-3-carbinol (I3C).1 Some of the damaging cardiovascular actions of ANG II are mediated by reactive oxygen species.2–4 Studies have shown that administration of the superoxide dismutase mimetic, tempol, reduces ANG II–dependent HTN and protects tissues from oxidative stress.3,5–7 When ANG II–dependent malignant HTN is induced in Cyp1a1-Ren2 TGRs, acute systemic administration of tempol decreases the elevated blood pressure (BP) and renal vascular resistance (RVR) but produces no effect in normotensive rats.8 Chronic administration of the angiotensin type 1 (AT1) receptor blocker (ARB), candesartan, normalizes BP and restores the elevated urinary excretion of 8-isoprostane to normal, supporting a role for AT1-stimulated production of superoxide anion in contributing to the malignant HTN in Cyp1a1-Ren2 rats.8 Increased levels of the superoxide anion also reduce the bioavailability of nitric oxide (NO), which further contributes to elevations in BP and RVR.9–11 In hypertensive Cyp1a1-Ren2 TGRs treated with tempol, acute NO synthase (NOS) inhibition with nitro-L-arginine (NLA) attenuates the tempol-induced fall in BP, thus BP increases; this also occurs in the normotensive rat.8 In addition, NLA administration increases RVR in both normotensive and hypertensive Cyp1a1-Ren2 TGRs treated with tempol and also elicits decreases in glomerular filtration rate (GFR) and renal plasma flow (RPF) in hypertensive rats.8 Collectively, this suggests that a superoxide anion-mediated decrease in NO bioavailability does not fully contribute to the increased RVR in hypertensive Cyp1a1-Ren2 TGRs and that maintained intrarenal actions of NO preserve renal hemodynamics.8 The activity of renal endothelial (e)NOS and neuronal (n)NOS plays a critical role in regulating normal renal hemodynamics and sodium excretion. While eNOS is abundant throughout the renal vasculature and controls blood flow, nNOS is abundant in the macula densa and regulates blood flow via tubuloglomerular feedback.12–15 Tubular NO derived from both eNOS (mainly thick ascending limb) and nNOS (predominantly collecting duct) inhibits sodium reabsorption.15–19 In renal injury, nNOSα decreases and nNOSβ increases; although the locations and significance of these changes remain to be determined.12,14,20. In this study, we hypothesized that the maintenance of renal hemodynamic function in hypertensive Cyp1a1-Ren2 TGRs is due to the preservation of the intrarenal NO and antioxidant systems in this form of severe ANG II–dependent HTN. To test this, we investigated the intrarenal NOS and antioxidant/oxidant systems in the kidney and aorta of Cyp1a1-Ren2 TGRs with ANG II–dependent malignant HTN. The aorta was chosen as a comparison tissue to the kidney to reflect systemic vascular vs. intrarenal alterations in the NOS and antioxidant/oxidant systems in Cyp1a1-Ren2 TGRs with elevated BP.

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.

The Compact Dry “Nissui” EC method, originally certified by the AOAC Research Institute Performance Test MethodSM program for enumeration of Escherichia coli and non-E. coli coliforms in raw meat products (Performance Tested MethodSM 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 r2. 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 r2 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 r2 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.

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.

Nitric oxide deficiency occurs by multiple mechanisms and contributes to the pathogenesis of progression of chronic kidney disease (CKD) and its cardiovascular complications. This article concentrates on recent developments on the regulation of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in CKD and on the importance of the nitric oxide synthases in kidney disease progression, particularly in diabetic nephropathy.The increased plasma ADMA seen in renal disease is generally predictive of severity of CKD progression and cardiovascular risk. However, some assumptions about the control of ADMA have been challenged: the primacy of the kidney as a metabolic organ for plasma ADMA regulation has come under scrutiny and the relative importance of the two isoforms of the ADMA-metabolizing enzymes dimethylarginine dimethylaminohydrolases (DDAHs) is being re-evaluated. Alterations in NOS also contribute to CKD progression with the endothelial isoform playing a major role in diabetic nephropathy.Improving our understanding of ADMA regulation is important since pharmacologic targeting of DDAH is underway. The major role of endothelial NOS-derived nitric oxide in diabetic nephropathy should lead to novel therapies. The beneficial actions of dietary nitrate supplementation on blood pressure and kidney disease are of considerable clinical relevance.

The ageing kidney exhibits slowly developing chronic kidney disease (CKD) and is associatedwithnitricoxide(NO)deficiencyandincreasedoxidativestress.Theimpactofexercise on the ageing kidney is not well understood. Here, we determined whether 12 weeks of treadmill exercise can influence age-dependent CKD in old (22-24 months) Fisher 344 (F344) male rats by comparing sedentary (SED) and exercise (EX) trained rats; young (3 months) rats were also studied.Inadditiontorenalstructureandfunction,weassessedproteinlevelsofvariousisoforms of the NO synthases (NOS) and superoxide dismutase (SOD) enzymes as well as markers of oxidativestress,inkidneycortexandmedulla.Renalfunctionasdeterminedbyplasmacreatinine, proteinuria, and glomerular structural injury worsened with age and was unaffected by exercise. Ageing also increased the protein abundance of neuronal NOSβ and p22phox while decreasing extracellular(EC)andcopper/zinc(CuZn)SOD,inkidneycortexandmedulla.H2O2 contentand nitrotyrosine abundance also increased in the kidney with age. None of these age-related changes were altered with exercise. However, exercise did increase renal cortical endothelial (e)NOS and EC SOD in young rats. Data indicate that exercise-induced increases in eNOS and EC SOD seen in young rats are lost with age. We conclude that chronic exercise is ineffective in reversing age-dependent CKD in the male F344 rat.

Aims Angiotensin II (Ang II) type AT1 receptors expressed on vascular smooth muscle cells (VSMCs) couple to the Jak2 signalling pathway. However, the importance of this tissue-specific coupling is poorly understood. The purpose of this investigation was to determine the importance of VSMC-derived Jak2 in angiotensin II-mediated hypertension. Methods and results The Cre- lox P system was used to conditionally eliminate Jak2 tyrosine kinase expression within the smooth muscle cells of mice. Following chronic Ang II infusion, the resulting increase in mean arterial pressure (MAP) was significantly attenuated in the Jak2 null mice when compared with littermate controls. The VSMC Jak2 null mice were also protected from the Ang II-induced vascular remodelling. Aortic rings from the VSMC Jak2 null mice exhibited reduced Ang II-induced contraction and enhanced endothelial-dependent relaxation via increased nitric oxide (NO) bioavailability. When compared with controls, the VSMC Jak2 nulls also had lower levels of hydrogen peroxide, Rho kinase activity, and intracellular Ca2+ in response to Ang II. Conclusions The data indicate that VSMC Jak2 expression is involved in the pathogenesis of Ang II-dependent hypertension due to the increased presence of reactive oxygen species (ROS). As such, VSMC-derived Jak2 tyrosine kinase modulates overall vascular tone via multiple, non-redundant mechanisms.

Plasma volume (PV) expansion is required for optimal pregnancy outcomes; however, the mechanisms responsible for sodium and water retention in pregnancy remain undefined. This study was designed to test the “arterial underfill hypothesis” of pregnancy which proposes that an enlarged vascular compartment (due to systemic vasodilation and shunting of blood to the placenta) results in renal sodium and water retention and PV expansion. We produced chronic vasodilation by 14 days administration of nifedipine (NIF; 10 mg·kg−1·day−1) or sodium nitrite (NaNO2; 70 mg·kg−1·day−1) to normal, nonpregnant female Sprague-Dawley rats. Mean arterial pressure, monitored by telemetry, was reduced by both NIF and NaNO2 but was unchanged in control rats. At day 14, vasodilator treatment lowered hematocrit and increased PV (determined by Evans blue dye dilution). Plasma osmolarity (Posm), sodium (PNa), and total protein concentrations all fell. These responses resemble the responses to normal pregnancy with hemodilution, marked PV expansion, and decreased Posmand PNa. Our previous work indicates a role of increased inner medullary phosphodiesterase-5 (PDE5) in the sodium retention of pregnancy. Here, we found that inner medullary PDE5A mRNA and protein expression were increased by both NIF and NaNO2 treatment vs. control; however, neither renal cortical nor aortic PDE5 expression was changed by vasodilator treatment. We suggest that a primary, persistent vasodilation drives increased inner medullary PDE5 expression which facilitates continual renal Na retention causing “refilling” of the vasculature and volume expansion.

Age-dependent renal damage is influenced by genetic background and the Fisher344xBrown Norway (F344xBN) rat is resistant to glomerular injury. In vulnerable strains, a fall in renal nitric oxide synthase (NOS) contributes to age-dependent renal damage. Here, we investigated renal NOS in young (3 months) and old (30 months) male F344xBN to test the hypothesis that renal NOS is maintained in “protected” strains. We also examined if 6 months of renin–angiotensin system (RAS) blockade using angiotensin converting enzyme inhibition (ACEI) and angiotensin receptor blockade (ARB) provides further benefit in these “protected” old rats. Aging increased tubulointerstitial injury but glomerular sclerosis was minimal and NOS and superoxide dismutase abundance increased. There was no change in the NOS inhibitor, ADMA (asymmetric dimethylarginine) or its regulatory enzymes. RAS blockade with ARB protected against tubulointerstitial injury and increased nNOSα, but ACEI, which also increased nNOSα, had no protective effect on the tubulointerstitium. We conclude that the glomerular sclerosis-resistant aged male F344xBN rat maintains renal NOS, thus reinforcing our hypothesis that progressive glomerular injury is related to renal NOS deficiency. The tubulointerstitial injury seen with aging is reversed with 6 months of ARB but not ACEI and is not associated with renal NOS.

Estradiol increases mRNA and/or protein expression of the nitric oxide synthase (NOS) isoforms in a variety of tissues including kidney. In this study we determined the relationship between cyclical variations in estradiol levels and renal function and total NO production in the virgin female rat. In addition, we used an aromatase inhibitor (Anastrozole), to inhibit synthesis of estradiol from testosterone. Estradiol levels were higher in proestrus vs. diestrus, and were markedly suppressed by 7 days treatment with aromatase inhibitor. There was no difference in total NO production (from urinary and plasma nitrate + nitrite = NOX) between proestrus and diestrus but aromatase inhibition resulted in increases in total NO production. The renal cortical NOS activity and protein abundance also increased in aromatase-inhibited female rats. There were no differences in blood pressure (BP) in any group but the renal vascular resistance (RVR) was low in proestrus, increased in diestrus and did not change further after aromatase inhibition. In summary, the cyclical changes in renal function correlate with estradiol but not NO levels. Pharmacologic castration with aromatase inhibition leads to a marked increase in total and renal NOS. This contrasts to earlier work where surgical castration causes decreased NOS.

Pregnancy is characterized by plasma volume expansion and renal sodium retention with loss of natriuretic response to atrial natriuretic peptide due to increased medullary phosphodiesterase-5 (PDE5). Here, we determined whether natriuretic responses to nitric oxide (NO) are also blunted in pregnancy due to increased PDE5. Anesthetized 16-day pregnant and virgin rats were studied at baseline and during intrarenal infusion of the NO donor spermine NONOate (2.5 nmol/min), the PDE5 inhibitor sildenafil (SILD; 0.5 μg/min), or a combination. The right (noninfused) kidney served as a control. Intrarenal NONOate had no effect on mean arterial pressure (MAP); however, SILD reduced MAP in virgin rats, and the combination of NONOate+SILD reduced MAP in both virgin and pregnant rats. Neither NONOate nor SILD altered glomerular filtration rate. NONOate and SILD each stimulated sodium excretion (UNaV) and fractional excretion of sodium (FENa) in virgin rats, but the combination did not result in an additional natriuretic response. However, NONOate infusion did not increase UNaV or FENain pregnant rats, but the natriuretic response to NONOate was restored with SILD, and SILD alone produced a natriuresis during pregnancy. Sodium nitroprusside (10−4mol/l)-stimulated cGMP accumulation from inner medullary collecting duct cells was blunted in cells from pregnant vs. virgin or postpartum rats and was restored by treatment with the PDE5 inhibitor DMPPO (10−7mol/l). Therefore, increased intrarenal PDE5 mediates the blunted natriuretic response to NO, and loss of responsiveness to the cGMP-dependent, natriuretic agents may contribute to volume expansion during pregnancy.

Background. The fawn-hooded hypertensive (FHH) rat develops spontaneous glomerulosclerosis that is ameliorated by inhibition of the angiotensin II type 1 receptor (AT1). Since kidney damage is associated with nitric oxide (NO) deficiency, we investigated how AT-1 antagonism influenced nitric oxide synthase (NOS), as well as NOS substrate [L-arginine (L-Arg)] and inhibitor [asymmetric dimethylarginine (ADMA)]. L-Arg is synthesized by renal argininosuccinate synthase/argininosuccinate lyase (ASS/ ASL) and then either consumed within the kidney by arginase II or NOS or released into the circulation. L-Arg is then taken up from plasma into cells where it can be utilized by NOS and other pathways. The competitive inhibitor of NOS, ADMA, is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Methods and results. Male FHH rats were put on a 40% casein diet for 13 weeks, and some received AT-1 antagonist which reduced blood pressure and kidney weight and prevented glomerulosclerosis and hyperfiltration. The AT1 antagonist reduced the expression of DDAH2, increased DDAH1 and increased total DDAH activity in the kidney cortex, although there was no change in plasma or kidney cortex ADMA levels. The AT-1 antagonist caused no change in the expression of renal ASS/ASL, but reduced renal and aortic arginase expression and renal arginase activity, which could explain the increased plasma L-Arg. In separate studies, 1 week of AT-1 blockade in young FHH rats had no effect on any of these parameters. Conclusion. Thus, the net result of AT-1 antagonist was an improved L-Arg to ADMA ratio due to the prevention of renal and vascular arginase activation which favours increased NO production. Since 1 week of AT-1 blockade in the absence of kidney damage was without effect on arginases, this suggests that the reduction in arginase activity is secondary to the prevention of structural damage rather than a direct immediate effect of AT-1 antagonism.

Recent studies have shown that asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, is increased in hypertension and chronic kidney disease. However, little is known about the effects of hypertension per se on ADMA metabolism. The purpose of this study was to test the hypothesis that ANG II-induced hypertension, in the absence of renal injury, is associated with increased oxidative stress and plasma and renal cortex ADMA levels in rats. Male Sprague-Dawley rats were treated with ANG II at 200 ng·kg−1·min−1 sc (by minipump) for 1 or 3 wk or at 400 ng·kg−1·min−1 for 6 wk. Mean arterial pressure was increased after 3 and 6 wk of ANG II; however, renal injury (proteinuria, glomerular sclerosis, and interstitial fibrosis) was only evident after 6 wk of treatment. Plasma thiobarbituric acid reactive substances concentration and renal cortex p22phox protein abundance were increased early (1 and 3 wk), but urinary excretion of isoprostane and H2O2 was only increased after 6 wk of ANG II. An increased in plasma ADMA after 6 wk of ANG II was associated with increased lung protein arginine methyltransferase-1 abundance and decreased renal cortex dimethylarginine dimethylaminohydrolase activity. No changes in renal cortex ADMA were observed. ANG II hypertension in the absence of renal injury is not associated with increased ADMA; however, when the severity and duration of the treatment were increased, plasma ADMA increased. These data suggest that elevated blood pressure alone, for up to 3 wk, in the absence of renal injury does not play an important role in the regulation of ADMA. However, the presence of renal injury and sustained hypertension for 6 wk increases ADMA levels and contributes to nitric oxide deficiency and cardiovascular disease.

Background/Aims: The C57Bl6 mouse is resistant to chronic kidney disease (CKD) induced by reduction of renal mass (RRM). Nitric oxide (NO) deficiency exacerbates CKD progression so this study investigated whether combination of RRM and NO deficiency would render the C57Bl6 mouse vulnerable to CKD. Methods: We used wild-type (WT) mice with RRM, chronic NO synthase (NOS) inhibition and a combination. Also, endothelial NOS (eNOS) knockout (KO) C57Bl6 mice were studied with and without RRM. Primary endpoints were albuminuria and structural damage. Results: Both nonselective (+L-NAME) and neuronal NOS ‘selective’ (+7NI) NOS inhibition greatly exacerbated the albuminuria and structural damage seen with RRM in the WT mice; NOS inhibition alone had little effect. The eNOS KO mice showed marked structural damage and significant albuminuria in the shams and RRM produced minimal exacerbation of structural damage although the albuminuria was massively amplified. Conclusion: These studies demonstrate that the C57Bl6 mouse is rendered vulnerable to RRM-induced CKD when concomitant NO deficiency is produced. This observation supports previous work in CKD-resistant rats and suggests that NO deficiency is required for progression of CKD.

The kidney is a major site of arginine synthesis where citrulline is converted to arginine via argininosuccinate synthase (ASS) and lyase (ASL). The rate-limiting step in arginine synthesis by the normal kidney is the rate of citrulline delivery and uptake to the renal cortex. We tested whether with chronic kidney disease (CKD) renal arginine synthesis may be compromised. Using the renal ablation/infarction (A/I) injury model, we measured renal citrulline delivery and uptake as well as arginine release at early, moderate, and severe stages of CKD vs. healthy controls. The renal plasma flow (RPF) and arterial-renal venous difference was measured at baseline and during citrulline infusion. Citrulline delivery was reduced at all stages of disease due to marked reductions in RPF and despite moderately increased plasma citrulline. Early after A/I, the kidney demonstrated a compensatory increase in citrulline uptake while at moderate and severe injury baseline citrulline uptake fell. At all stages of CKD, renal arginine release was markedly reduced. Citrulline infusion increased plasma citrulline in all groups, resulting in increased renal delivery vs. baseline. In healthy kidneys and early injury, citrulline uptake increased with the infusion, but only in the normal kidney did arginine production increase in parallel with the increased citrulline uptake. At moderate and severe injury, there was no increase in citrulline uptake or arginine production. The fall in arginine production in A/I was due to an early loss of ASS and ASL conversion of citrulline, which combined with a later reduction in citrulline uptake.

Females develop less age-dependent loss of renal function, in part because of cardiorenal protective effects of estrogens. The low androgen level in women also may be protective, although the animal and clinical data are controversial. Both estrogen and androgens act through multiple mechanisms, sometimes beneficial, sometimes damaging, which makes it difficult to predict the impact of hormone replacement therapy in an aging population. Nitric oxide (NO) deficiency contributes to age-dependent cardiovascular risk and kidney damage in animal models. The increased oxidative stress of aging impacts at multiple sites in the NO biosynthetic pathway to decrease NO production/action. Endothelial dysfunction develops with aging and is delayed in women, in association with a delayed increase in asymmetric dimethylarginine. Animal data suggest that the aging kidney develops NO deficiency because of changes in the neuronal NO synthase. Relative preservation of NO production in females contributes to the better cardiovascular and renal responses to aging.

A simple test for determining total aerobic viable counts in raw meat is presented. Homogenates of meat samples are prepared in full PBW medium, dispensed in the wells of 96-well plate together with the oxygen-sensing probe, Redlight, covered with oil and monitored on a fluorescent reader at 30 °C. The probe produces characteristic sigmoidal profiles of fluorescence reflecting depletion of sample dissolved oxygen, with onset time indicating the initial microbial load. The test provides rapid and accurate results (1 and 12 h for contamination levels of 10 8 and 10 3 cfu/g, respectively) and correlates well with the ISO:4833:2003 method ( r = 0.86), which make it useful as alternative to conventional culture methods for the quick, high throughput determination of TVC (30 °C) in meat samples.

Gunn rats, deficient in the enzyme uridine diphosphate glucuronyl transferase, were used to investigate the effects of unconjugated hyperbilirubinemia in cisplatin nephrotoxicity. The effect of bilirubin on the antineoplastic activity of cisplatin in osteosarcoma cell lines was also determined. The in vivo model involved three groups of rats (n =6 rats/group): homozygous Gunn rats (j/j), heterozygous Gunn rats (j/+), and congenic Wistar rats. On day 0, all rats were given 4 mg/kg cisplatin intraperitoneally. Blood was sampled on days 0, 3, and 5 for bilirubin, BUN, and creatinine and kidneys were taken on day 5. Cell culture was performed in four canine osteosarcoma cell lines using the average concentrations of bilirubin for homozygous Gunn rats at day 0 and 3. Bilirubin was added to cell lines alone and with cisplatin. Cell viability was assessed using the CellTiter Blue assay. Serum bilirubin levels were highly elevated in Gunn j/j, moderately elevated in Gunn j/+, and undetectable in Wistar rats at day 0. Bilirubin provided a nephroprotective effect, with significantly lower BUN and creatinine in Gunn j/j when compared with Wistar rats at day 5. Histological grading demonstrated preservation of the S3 segment in Gunn j/j when compared with Wistar rats (P < 0.05). Bilirubin had no significant effect on the antineoplastic effect of cisplatin at either concentration in the four cell lines (P < 0.001). Hyperbilirubinemia in the Gunn rat provided marked preservation of renal function and histology in a cisplatin nephrotoxicity model. Exogenous bilirubin did not interfere with the antineoplastic activity of cisplatin in vitro.

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes asymmetric dimethylarginine to generate L-citrulline and is present in large quantities in the kidney. We present a new study that optimizes the Prescott-Jones colorimetric assay to measure DDAH-dependent L-citrulline generation in kidney homogenates. We found that the removal of urea with urease is necessary since urea also produces a positive reaction. Deproteinization with sulfosalicylic acid was found to be optimal and that protease inhibitors were not necessary. All assays were conducted in phosphate buffer, since other common additives can create false positive and false negative reactions. Arginase or nitric oxide synthase isoenzymes were not found to influence L-citrulline production. Our optimized L-citrulline production assay to measure DDAH activity correlated closely with the direct measure of the rate of asymmetric dimethylarginine consumption. Using this assay, we found that both superoxide and nitric oxide inhibit renal cortical DDAH activity in vitro.

Hyperbaric oxygen therapy (HBOT) accelerates the healing of chronic wounds and is now primarily used as an adjunctive therapy in managing selected problem wound healing.1–4 Hyperoxia during HBOT is an important mediating factor for wound collagen deposition and cross-linking,5 neutrophil-dependent microbial killing,6 and neovascularization.7 Other HBOT-mediated processes in wound healing include the inhibition of integrin-mediated intravascular leukocyte adhesions and platelet aggregation,8 enhancement of cutaneous microvascular homeostasis after ischemia-reperfusion syndrome,9 increased oxygen capacitance and survival of ischemic flaps,10 and upregulation of platelet-derived growth factor (PDGF) receptor mRNA.11 Experimental normobaric oxygen activates gene expression in human dermal fibroblasts, supporting the concept of oxygen as a possible signal transducer.12 An additional signaling system modulated by oxygen availability is provided by the naturally occurring oxygen-free radicals (oxidants), or reactive oxygen species (ROS), found in the wound environment. ROS have been observed to increase cellular vascular endothelial growth factor (VEGF) expression and signal transduction that is considered to promote an overall enhancement of the wound healing process.13 Further, accelerated wound healing during HBOT may be mediated by the combined effects of hyperoxia and the increased local (wound) production of nitric oxide (NO), an important cellular signal for tissue repair.14,15 Ongoing experimental and clinical wound healing studies have established NO as a critical mediator of normal tissue repair.16 Angiogenesis, granulation tissue formation, epidermal migration, collagen deposition, and microvascular homeostasis are significant processes critical to normal wound repair that are regulated by NO production and bioactivity.17–20 Functional recovery of cutaneous vascular beds after ischemia-reperfusion injury21 and increased local random tissue flap survival22 have also been linked to increased vascular endothelial NOS activity in experimental models. Optimal NO activity is required for the full expression and receptor upregulation of VEGF and PDGF.17,23 A deficiency in NO bioactivity is associated with diabetes-impaired wound healing.24 Successful recombinant topical PDGF-BB (becaplermin) therapy for chronic lower-extremity ulcer (LEU) patients with diabetes may also depend on optimal NO production for successful wound treatment.25 Although HBOT and NO may mediate comparable mechanisms of cutaneous tissue repair, direct evidence of a relationship between enhanced wound healing, HBOT, and increased NO production has not been demonstrated. The purpose of this preliminary study was to document general somatic and local wound NO production during and after HBOT. This was accomplished by serially measuring NOx (nitrate and nitrite, which are the stable oxidation products of NO) in the fasting plasma and wound fluid of 6 patients receiving adjunctive HBOT who demonstrated enhanced healing of their chronic wounds with this therapy.

Chronic kidney disease is accompanied by nitric oxide (NO) deficiency and oxidative stress, which contribute to progression. We investigated whether the antioxidant vitamin E could preserve renal function and NO bioavailability and reduce oxidative stress in the 5/6th nephrectomy (NX) rat model. We studied the following three groups of male Sprague-Dawley rats: sham ( n = 6), 5/6 NX control ( n = 6), and 5/6 NX treated with vitamin E (5,000 IU/kg chow; n = 5). The 5/6 NX group showed increased severity of glomerulosclerosis vs. sham, and this was ameliorated by vitamin E therapy. Both 5/6 NX groups showed similar elevations in plasma creatinine and proteinuria and decreased 24-h creatinine clearance compared with sham. There was increased NADPH-dependent superoxide production in 5/6 NX rats vs. sham that was prevented by vitamin E. Total NO production was similarly reduced in both 5/6 NX groups. There was unchanged abundance of endothelial nitric oxide synthesis (NOS) in renal cortex and medulla and neuronal (n) NOS in medulla. However, in kidney cortex, 5/6 NX rats had lower nNOS abundance than sham, which was restored by vitamin E. An increased plasma asymmetric dimethylarginine occurred with 5/6 NX associated with decreased renal dimethylarginine dimethylaminohydrolase activity and increased type 1 protein arginine methyltransferase expression.

Normal rat pregnancy is characterized by plasma volume expansion due to renal sodium retention and is associated with a blunted response to natriuretic stimuli, such as atrial natriuretic peptide (ANP). ANP signals via cGMP, and phosphodiesterases (PDE) inactivate cGMP and terminate the natriuretic response. We previously reported that increased medullary PDE-5 activity occurs in rat pregnancy, which may be the mechanism of the blunted natriuretic effect of ANP. Here, we used anesthetized 16-day pregnant and virgin rats to investigate whether intrarenal infusion of a selective PDE-5 inhibitor, sildenafil, would reverse the blunted response to ANP in pregnancy. We measured blood pressure, renal clearances using inulin and p-aminohippuric acid, and electrolyte excretion at baseline and during an ANP infusion. ANP caused a fall in mean arterial pressure in all groups, and sildenafil induced a further reduction. We observed an increase in sodium excretion with ANP in all rats, but this was blunted in the vehicle-infused pregnant rats. This could not be explained by differences in renal hemodynamics and was of tubular origin, as reflected by the reduced rise in fractional excretion of sodium with ANP in the pregnant rat given vehicle (45 ± 11 vs. 204 ± 49%; P < 0.05). However, intrarenal sildenafil increased the natriuretic response and the rise in fractional excretion of sodium to the virgin value (226 ± 23 vs. 245 ± 73%; not significant), whereas the blunting persisted in the contralateral kidney. This demonstrates that increased intrarenal PDE-5 mediates the blunted natriuretic response to ANP during pregnancy and may contribute to the physiological volume expansion.

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.

BACKGROUND: The goal was to determine whether administration of the oxygen-therapeutic blood substitute HBOC-201 had any deleterious effect on renal function and/or structure in a rat model of chronic kidney disease (CKD). STUDY DESIGN AND METHODS: Longitudinal studies were conducted in the conscious chronically catheterized male Sprague-Dawley rat with rapidly progressing CKD produced by ablation-infarction of 5/6th of the functional renal mass. Experimental rats received a loading dose (after acute removal of 10% blood volume) and six subsequent daily maintenance doses of HBOC-201 at Weeks 4 to 5 after induction of CKD and were studied again at 6 weeks. Controls received isoosmotic human serum albumin (HSA, 13%) over the same period. Blood pressure (BP) and renal function were measured in acute experiments at Weeks 4, 5, and 6. Proteinuria and 24-hour creatinine clearance was measured longitudinally and renal pathology was assessed at killing. RESULTS: There were differences in the acute response to the infusions, with rats given HBOC-201 exhibiting an increase in BP and renal vascular resistance, whereas rats given HSA showed a decrease in BP. These changes did not persist, however, because mean BP, glomerular filtration rate, level of proteinuria, and glomerular pathology were all similar at the end of the study (6 weeks after induction of CKD) in rats given HBOC-201 and HSA. CONCLUSION: In this model of CKD, daily doses of HBOC-201 had no long-term damaging effects on renal function or structure, compared to rats given the osmotic control agent, 13 percent HSA.

Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) rat. We previously showed that the Wistar-Furth (WF) rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD rats, whereas low-dose NOSI caused progression of CKD in WF rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given NG-nitro-l-arginine methyl ester (l-NAME) (150 and 300 mg/l for 6–10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic l-NAME. SD given 150 mg/l l-NAME for 6 wk developed proteinuria and renal injury, whereas WF rats receiving 150 mg/l l-NAME for 6–10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD rat. There was little impact on renal nitric oxide synthase expression with l-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF rats, whereas WF rats showed a much smaller increment in glomerular blood pressure compared with SD rats. In conclusion, WF rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD rat. This protection may be associated with protection from glomerular hypertension.

There is clear evidence that chronic inhibition of nitric oxide synthase (NOS) in animals causes hypertension and also leads to progressive kidney damage. There is also evidence that nitric oxide (NO) deficiency occurs in man with chronic kidney disease (CRD) and this may contribute to further progression of CRD, to hypertension, and to other cardiovascular complications. There are multiple ways in which NO deficiency develops in CRD. At end stage there are uremic factors in plasma that inhibit L-arginine transport into cells and this may cause a “net” substrate deficiency. Also, increases occur in endogenous NOS inhibitors, in particular asymmetric dimethylarginine (ADMA). The increased oxidative stress of CRD is likely to be a primary cause of the increased plasma ADMA since the catabolic enzyme, dimethylarginine dimethylaminohydrolase (DDAH) is extremely sensitive to inhibition by oxidants. Animal studies demonstrate a decrease in abundance of the neuronal NOS within the injured kidney that correlates with extent of injury. Overall, there is substantial clinical, “in vitro,” and animal data to suggest that systemic, endothelial, and renal NO deficiency is a common feature of CRD irrespective of the primary genesis of the disease. This NO deficiency, which is multifactorial, contributes to the progressive nature of the CRD and the endothelial dysfunction and associated risk for cardiovascular events. Strategies that reverse NOS inhibition and/or can boost the ability of the damaged kidney to produce NO might help preserve residual renal function and/or slow down the rate of progression to end stage.

The Wistar-Furth (WF) rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) rat, and protection was associated with preserved renal nitric oxide (NO) production. This study examined CRD induced with repeated administration of puromycin aminonucleoside (PAN). SD PAN developed nephrotic range proteinuria (>1 g/24 h), and at 15 wk severe renal injury developed and the glomerular filtration rate (GFR) was reduced to ∼10% of sham. Total NO production, renal NO synthase (NOS) activity, and renal neuronal (n) and medullary endothelial (e)NOS abundance were reduced in the SD PAN. WF PAN exhibited less severe initial proteinuria (>400 mg/24 h), which abated within weeks, whereas GFR was normal and injury was minimal at 15 wk. Total NO production and renal NOS activity and abundance were significantly elevated compared with SD PAN. NOS mRNA (nNOS, eNOS, and inducible NOS) was not altered in WF, whereas SD showed significant increases in NOS gene expression with PAN. In conclusion, WF showed resistance to a second model of CRD with maintained renal NOS activity compared with SD.

Renal neuronal nitric oxide synthase protein expression as a marker of renal injury. Background Animal studies suggest that nitric oxide deficiency occurs in the remnant after 5/6 removal of renal mass. The present studies investigated the time course in relation to progression of renal disease, as well as the impact on individual renal nitric oxide synthase (NOS) isoforms. Methods Rats were studied from 2 to 11 weeks after 5/6 ablation/infarction (A/I) of renal mass, with acceleration of progression by high protein and salt intake, in some groups. Measurements were made before sacrifice of 24-hour protein and creatinine excretion, blood was taken for creatinine and blood urea nitrogen (BUN) determination and the kidneys were investigated histologically for structural damage, abundance of endothelial NOS (eNOS) and neuronal NOS (nNOS), and in some groups for in vitro NOS activity. Results A time-dependent fall in glomerular filtration rate (GFR) and rise in proteinuria and glomerular sclerosis developed after 5/6 A/I. The nNOS abundance in cortex and medulla was decreased relative to shams, in all but the mildest injury and there was a strong, steep correlation between level of glomerular sclerosis and the degree of reduction in renal nNOS. Where measured, cortical NOS activity correlated with the nNOS abundance. In contrast, the eNOS abundance was either increased or unchanged in rats post A/I. Conclusion Renal nNOS adundance was reduced in the 5/6 A/I model of renal disease when plasma creatinine>∼1mg/dL and when>∼20% of remaining glomeruli were sclerosed.

Wistar Furth (WF) rats do not develop renal injury after severe reduction of renal mass. Because clinical and animal studies suggested that nitric oxide (NO) deficiency occurs and may contribute to chronic renal disease (CRD), the status of the NO system in WF versus Sprague Dawley (SD) rats was examined with the 5/6 renal ablation/infarction (A/I) model of CRD. Eleven weeks after A/I, SD rats developed proteinuria, severe kidney damage, decreased renal function, and marked decreases in total and renal NO synthase (NOS), specifically neuronal NOS. In contrast, WF rats exhibited elevated baseline and maintained post-A/I total NO production, with no decrease in renal cortex NOS activity despite a decrease in remnant neuronal NOS abundance. When low-dose chronic Nomega-nitro-L-arginine methyl ester treatment was added for WF A/I-treated rats, rapid progression of CRD was observed. In conclusion, elevated NO production in WF rats was associated with protection from the progression of CRD after renal mass reduction. The protection might be attributable to greater total and renal NO-generating capacity and increased nephron number, compared with SD rats. NOS inhibition rendered WF rats susceptible to progression, suggesting a possible critical threshold for NO production, below which renal injury occurs.

The Nimbus Technology & Engineering e − Beam Mastering System was developed to gain a large improvement in optical disc and structured hard disc recording capacity, significantly more than is possible from deep UV and SIL mastering. The current electron beam recorder is essentially a production machine capable of making full-length exposures at capacities of up to 50 GB with a simple low-cost upgrade path to disc capacities of several hundred gigabytes and beyond and hard disk drives (HDD) with capacities of up to 1 tera bit per square inch.

It has been suggested that L-arginine availability declines with advanced age, which could contribute to the endothelial dysfunction and decreased nitric oxide (NO) production that are features of aging. L-Arginine is made in the kidney and since the aging kidney develops progressive injury there may be decreased synthesis limiting availability. In this study we investigated the impact of aging on the regulation, at the gene level, of the various enzymes that synthesize L-arginine in the kidney (argininosuccinate synthetase and argininosuccinate lyase) and citrulline, the precursor of L-arginine made in the small intestine (phosphate-dependent glutaminase, carbamyl phosphate synthetase-1 and ornithine transcarbamylase). Studies were in young (3-5 months), middle-aged (11-13 months) and old (18-22 months) male and female Sprague-Dawley rats aged under barrier conditions. The plasma, renal cortical and brain cerebellar levels of L-arginine are unchanged in the old male rat, and expression of the genes involved in renal arginine synthesis and small intestinal citrulline synthesis is unchanged or upregulated with age in both males and females. This study shows that the synthesis of L-arginine is maintained with aging despite developing kidney damage. Therefore, the reduced NO generating capacity that occurs in aging must be due to downstream changes in the NO biosynthesis pathway, such as reduced abundance of NO biosynthetic enzymes.

OBJECTIVE: To determine whether vasodilatory cyclooxygenase (COX) products are activated in kidney during chronic nitric oxide synthase inhibition (NOSI) in pregnant rats. METHODS: Chronically catheterized pregnant and nonpregnant Sprague-Dawley female rats were treated with daily NOSI ( L-NAME; 100 mg/L drinking water) for 14 days. Renal function experiments were conducted twice at 7-9 days of chronic NOSI (equivalent to midterm in pregnant rats) and after 14 days (equivalent to late pregnancy). Measurements were made of arterial blood pressure (BP) and renal hemodynamics, specifically glomerular filtration rate (GFR), renal plasma flow (RPF), and renal vascular resistance (RVR), in control and then during acute COX inhibition with indomethacin (2 mg/kg i.v.). RESULTS: Chronic NOSI increased BP and RVR in both virgin and pregnant rats. The normal midterm renal vasodilation was prevented and there was no BP or renal hemodynamic response to acute COX inhibition. By late pregnancy, RPF had fallen further and again, acute COX inhibition had no impact on renal hemodynamics, although a small reduction was seen in GFR in LV. CONCLUSIONS: Vasodilatory COX products cannot compensate for nitric oxide as the renal vasodilatory factor of pregnancy in the rat.

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.

We previously found that Jak2 kinase, expressed within vascular smooth muscle cells (VSMC), plays a critical role in angiotensin II (Ang II)-mediated hypertension. Given that Jak2 mediates both pressor-dependent and pressor-independent events, we sought to determine the role of blood pressure (BP), per se, on the deleterious effects of Jak2 within the kidney. To investigate this, three groups of mice were examined; i) wild type mice (Controls) that received Ang II infusion, ii) mice lacking Jak2 expression within the VSMC (VSMC Jak2 Null) that also received Ang II, and iii) Control mice that received Ang II plus an anti-hypertensive triple therapy (3Rx). After baseline BP recordings, Ang II was infused (1000 ng/kg/min, SC) to all groups and the 3Rx regimen (80 mg/L hydralazine, 5 mg/L reserpine, 30 mg/L hydrochlorothiazide in the drinking water) was initiated two days later to the 3Rx group, in order to maintain BP at similar levels to the VSMC Jak2 Null group. After 28 days of Ang II, mice were euthanized and the kidneys were assessed via histological, molecular, and functional approaches. Chronic Ang II infusion significantly increased the levels of intrarenal Ang II in all three groups; Control = 1,262±283 fmol/g, VSMC Jak2 Null = 1,655±666 fmol/g, and 3Rx = 2,174±588. While Ang II infusion significantly increased the mean BP in the Control group (152 ± 2 mm Hg), it was significantly, and similarly, lower in both the VSMC Jak2 Null and 3Rx groups (125 ± 5 mm Hg and 131 ± 5 mm Hg, respectively). Glomerular sclerosis was absent and interstitial fibrosis ranged from absent- mild- moderate, and was similar in all groups. The increases in i) perivascular infiltration of CD3+ lymphocytes, ii) CTGF gene expression, iii) tubule casts and iv) albuminuria that were observed in the Control mice, were significantly reduced in both the VSMC Jak2 Null and 3Rx groups. [CTGF mRNA Levels: Control = 100%±17, VSMC Jak2 Null = 70%±12*, 3Rx= 56%±17*. Urine Albumin (ng/day): Control = 414 ± 262, VSMC Jak2 Null = 138 ± 172*, 3Rx= 101 ± 89* (*, p

The fetus requires a large amount of potassium for normal development. To accommodate this need the normal pregnant rat accumulates ~6.3 mEq of potassium over the course of gestation, over 70% of which is retained during late pregnancy. However, the mechanism of potassium retention during pregnancy is largely unknown. The purpose of this study was to examine the mechanism permitting increased potassium reabsorption in the setting of high circulating aldosterone and enhanced distal nephron sodium reabsorption in the pregnant rat. Recent work on the WNK kinases has provided interesting targets for divergent modulation of sodium and potassium handling in the distal nephron. We measured the mRNA expression of WNK1, WNK3, WNK4, and ROMK in the renal cortex and outer medulla of virgin (V, n=5), mid pregnant (MP, n=5), and late pregnant (LP, n=7) rats using quantitative real-time PCR. We found increased WNK3 expression in the outer medulla of LP rats compared to V (156 ±15 vs 100±10 % Control). The expression le...

Increased oxidative stress and inflammation are risk factors for hypertension in pregnancy. Here we examined the systolic BP (SBP) via telemetry in virgin (V) and pregnant (P) rats treated with a HFD, ANG II, and TNFα. Methods: Female Sprague Dawley rats were pretreated with a normal diet (ND) or a HFD for 8 weeks before mating. Day 6 P and age-matched V rats were implanted with either a mini-pump infusion of saline or ANGII (150 ng/kg/min) + TNFα (75ng/day) for 14 days and sacrificed at gestational day 21. Groups consisted of: V Con (ND + Saline, n=7); V Treatment (HFD + ANGII/TNFα, n=7); P Con (ND + Saline, n=6); P Treatment (HFD + ANGII/TNFα, n=8). Results: The total pressor response (area under the curve [AUC]) increased in V Treatment rats compared to V Con rats (AUC: 120±32 vs. -35±29 mmHg X 20 days, P< 0.05) and in P Treatment rats compared to P Con rats (AUC: 61±28 vs. 21±30 mmHg X 20 days, P< 0.05). However the pressor response was greater in V Treatment rats vs. P Treatment rats (120±32 vs. 21±3...

The efficiency of calcium channel blockers (CCBs) in the treatment of chronic renal disease (CRD) is controversial. In this study, we investigated whether combined T- and L-type CCBs, using mibefradil (30 mg/kg/d), provided superior protection versus traditional L-type voltage-gated CCBs, using amlodipine (10 mg/kg/d), in the deoxycorticosterone acetate (DOCA)-salt model of high glomerular blood pressure (P(GC)) and rapidly developing kidney damage. After 4 to 5 weeks of DOCA-salt, amlodipine did not reduce proteinuria (protein, 341 +/- 90 versus 482 +/- 54 mg/24 h; P = not significant) or degree of glomerular damage (20% +/- 4% versus 28% +/- 6% damaged glomeruli; P = not significant) compared with untreated rats. Conversely, mibefradil reduced proteinuria and glomerular damage versus untreated DOCA-salt rats (protein, 244 +/- 75 mg/24 h; P < 0.02; damaged glomeruli, 11% +/- 3%; P < 0.05). Both CCBs had similar antihypertensive actions, returning blood pressure to the untreated sham value. Of note, P(GC) also was reduced by a similar extent (and to the sham value) with both mibefradil (58 +/- 2 mm Hg; P < 0.001) and amlodipine (61 +/- 2 mm Hg; P < 0.005) versus untreated DOCA-salt rats (70 +/- 1 mm Hg). This study shows that combined T- and L-type CCBs provide superior protection against CRD in the DOCA-salt model compared with L-type CCBs alone. However, this protection was not hemodynamic because similar systemic and glomerular antihypertensive responses occurred with both mibefradil and amlodipine. Although mibefradil was withdrawn from the market because of adverse drug interactions not associated with CCBs, other mixed channel blockers may provide an alternative or adjunctive therapy to angiotensin-converting enzyme inhibition in CRD.

Neutral endopeptidase 24.11 (NEP) inhibitors prevent breakdown of atrial natriuretic peptide (ANP), and may be useful therapeutically, in sodium overload states as often occurs in the aged. However, age-dependent changes in ANP/NEP may limit the activity of these agents in the elderly. To investigate this we conducted experiments in young, middle aged and old conscious male rats, studied in the baseline euvolemic state and during acute NEP inhibition (NEPI). NEPI produced a marked increase in sodium excretion (100%) in all groups, regardless of age. A selective, potassium sparing effect was also seen, only in the middle-aged and old rats. Although baseline hemodynamics were affected by age with mean blood pressure, BP, and renal vascular resistance (RVR) being higher in old versus young (131+/-5 vs. 115+/-3 mmHg; P0.05 and 29+/-3 vs. 20+/-1 mmHg/ml per min per 100 g body weight (BW); P0.02, respectively); NEPI produced similar mild pressor and significant renal vasoconstrictor effects in all age groups. Despite the tendency of NEPI to reduce renal perfusion, this is an effective method of increasing sodium excretion in all age groups while the potassium sparing actions seen selectively in the older rats may increase the usefulness of NEPI as a diuretic agent for the elderly.