Renal Afferent Arterioles Research Articles

Abstract P434: Role of Dusp5 in Hypertension Induced Chronic Renal Disease

We recently identified 4 single nucleotide polymorphisms in Dusp5 in FHH as compared with Brown-Norway (BN) rats, two of which alter CpG sites and another one causes G155R mutation. We then created a Dusp5 knockout (KO) rat in the FHH.1 BN genetic background, in which a small region in Chr. 1 of BN rats containing 15 genes, including Dusp5 into the FHH rats. We found that knockout of Dusp5 enhances the myogenic response and autoregulation of cerebral circulation. In the present study, we evaluated whether Dusp5 also plays a role in the regulation renal function. The expression of Dusp5 was lower in FHH. 1 BN . Dusp5 KO rats in comparison to the FHH.1 BN rats. The levels of p-PKC and p-ERK1/2 were elevated in Dusp5 KO vs. FHH.1 BN rats. The blood pressure and urinary protein excretion was similar in FHH. 1 BN . Dusp5 KO (n = 43) and FHH.1 BN rats (n = 30) when they were at 12-week of age (114 ± 2 vs. 115 ± 1 mmHg and 32 ± 2 vs. 29 ± 3 mg/day). The autoregulation of RBF was similar in FHH.1 BN (n=22) and Dusp5 KO (n = 9) rats in response to elevation of MAP from 100 to 140 mmHg, but was impaired in FHH rats (n=21) that increased by 37.3 ± 2.3% when they were at 12-week of age. In response to an elevation in pressure from 60 to 120 mm Hg, the renal afferent arterioles constricted by 29 ± 2% and 11 ± 1%, respectively in Dusp5 KO (n = 6) in comparison to FHH.1 BN rats (n = 17). Blood pressure was similar in Dusp5 KO (n = 11) vs. FHH.1 BN rats (n = 14) after 3-week DOCA-salt treatment (169 ± 5 vs. 177 ± 5 mmHg). However, proteinuria was significantly reduced in Dusp5 KO rats (258 ± 22 mg/day, n = 12) in comparison to FHH.1 BN rats (338 ± 30 mg/day, n = 12). The renal injury induced by DOCA-salt was markedly reduced in Dusp5 KO vs. FHH.1 BN rats as the glomerular injury scores were 2.49 ± 0.03 vs. 2.54 ± 0.01; the areas of renal fibrosis were 5.5 ± 0.5% vs. 8.3 ± 0.3%; the areas of renal protein casts were 0.5 ± 0.2% vs. 2.4 ± 1.0% and renal arteriolar media-to-lumen ratios were 0.64 ± 0.03 vs. 0.81 ± 0.04, respectively. These results indicate that KO of Dusp5 enhances renal vascular function to protect against DOCA-salt hypertension induced chronic renal injury via activation of PKC/ERK signaling pathway. In conclusion, an activating mutation in Dusp5 may contribute to the impaired myogenic response and promote the development of chronic renal injury in FHH rats.

Enhanced hemodynamic responses to angiotensin II in diabetes are associated with increased expression and activity of AT1 receptors in the afferent arteriole.

The prevalence of hypertension is about twofold higher in diabetic than in nondiabetic subjects. Hypertension aggravates the progression of diabetic complications, especially diabetic nephropathy. However, the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that enhanced constrictive responsiveness of renal afferent arterioles (Af-Art) to angiotensin II (ANG II) mediated by ANG II type 1 (AT1) receptors contributes to the development of hypertension in diabetes. In response to an acute bolus intravenous injection of ANG II, alloxan-induced diabetic mice exhibited a higher mean arterial pressure (MAP) (119.1 ± 3.8 vs. 106.2 ± 3.5 mmHg) and a lower renal blood flow (0.25 ± 0.07 vs. 0.52 ± 0.14 ml/min) compared with nondiabetic mice. In response to chronic ANG II infusion, the MAP measured with telemetry increased by 55.8 ± 6.5 mmHg in diabetic mice, but only by 32.3 ± 3.8 mmHg in nondiabetic mice. The mRNA level of AT1 receptor increased by ~10-fold in isolated Af-Art of diabetic mice compared with nondiabetic mice, whereas ANG II type 2 (AT2) receptor expression did not change. The ANG II dose-response curve of the Af-Art was significantly enhanced in diabetic mice. Moreover, the AT1 receptor antagonist, losartan, blocked the ANG II-induced vasoconstriction in both diabetic mice and nondiabetic mice. In conclusion, we found enhanced expression of the AT1 receptor and exaggerated response to ANG II of the Af-Art in diabetes, which may contribute to the increased prevalence of hypertension in diabetes.

Extravasal albumin concentration modulates contractile responses of renal afferent arterioles.

Afferent arterioles (AA) hold a key position in the regulation of renal blood flow and glomerular filtration rate. Being the effector site of tubuloglomerular feedback, the afferent arteriole contributes to the renal handling of sodium and fluid. Dehydration goes along with increased renal interstitial protein concentration. Here, the hypothesis was tested that extravasal protein concentration directly modulates afferent arteriolar tone, a mechanism which may contribute to body fluid volume control. The effect of increased extravasal albumin concentration on the vascular reactivity was investigated in renal AA and interlobar arteries of mice, in rat renal AA and in pancreatic islet arterioles. Albumin (2 and 4% in the bath solution) significantly potentiated the contractile response of renal afferent arterioles induced by angiotensin II and adenosine, as well as their combination, compared to the control situation (0.1% albumin). Albumin did not influence the contractility of larger renal vessels or pancreatic islet arterioles. Mimicking the increase in the osmolality induced by 4% albumin by applying mannitol to the bath solution also increased the response of renal arterioles to Ang II. However, the effect was smaller compared to that of albumin. The nitric oxide bioavailability, measured by DAF-FM fluorescence, was reduced in afferent arterioles exposed to 4% albumin. The protein-induced modulation of AA tone is mediated by the increased osmolality as well as by NO scavenging. The results suggest a possible contribution of these mechanisms to the control of extracellular fluid volume via adjustment of renal blood flow and glomerular filtration rate.

Superoxide and hydrogen peroxide counterregulate myogenic contractions in renal afferent arterioles from a mouse model of chronic kidney disease

Myogenic contractions protect kidneys from barotrauma butare impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6%sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2+/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions.

Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-arts and MCAs cultured for 36-h using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mmHg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to norepinephrine (10-7 M). Add3 DsiRNA had a similar effect on the attenuation of the myogenic response in the MCA. Peak potassium currents were threefold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in nontransfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in the renal and cerebral circulation.

TRPV4 participates in pressure-induced inhibition of renin secretion by juxtaglomerular cells.

Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+ ]i ) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin. Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+ ]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA-mediated repression of TRPV4. Inhibition of TRPV4 impairs pressure-induced decrease in renin secretion. Compared to wild-type mice, Trpv4-/- mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure-renin relationship. We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus. The renin-angiotensin system is a crucial blood pressure regulation system. It consists of a hormonal cascade where the rate-limiting enzyme is renin, which is secreted into the blood flow by renal juxtaglomerular (JG) cells in response to low pressure in the renal afferent arteriole. In contrast, an increase in blood pressure results in a decreased renin secretion. This is accompanied by a transitory increase in [Ca2+ ]i of JG cells. The inverse relationship between [Ca2+ ]i and renin secretion has been called the 'calcium paradox' of renin release. How increased pressure induces a [Ca2+ ]i transient in JG cells, is however, unknown. We observed that [Ca2+ ]i transients induced by mechanical stimuli in JG As4.1 cells were completely abolished by HC067047 and RN1734, two inhibitors of TRPV4. They were also reduced by half by siRNA-mediated repression of TRPV4 but not after repression or inhibition of TRPV2 or Piezo1 ion channels. Interestingly, the stimulation of renin secretion by the adenylate cyclase activator forskolin was totally inhibited by cyclic stretching of the cells. This effect was mimicked by stimulation with GSK1016790A and 4αPDD, two activators of TRPV4 and inhibited in the presence of HC067047. Moreover, in isolated perfused kidneys from Trpv4-/- mice, the pressure-renin relationship was significantly altered. In vivo, Trpv4-/- mice presented increased plasma levels of renin and aldosterone compared to wild-type mice. Altogether, our results suggest that TRPV4 is involved in the pressure-induced entry of Ca2+ in JG cells, which inhibits renin release and allows the negative feedback regulation on blood pressure.

How Do Antihypertensive Drugs Work? Insights from Studies of the Renal Regulation of Arterial Blood Pressure.

Though antihypertensive drugs have been in use for many decades, the mechanisms by which they act chronically to reduce blood pressure remain unclear. Over long periods, mean arterial blood pressure must match the perfusion pressure necessary for the kidney to achieve its role in eliminating the daily intake of salt and water. It follows that the kidney is the most likely target for the action of most effective antihypertensive agents used chronically in clinical practice today. Here we review the long-term renal actions of antihypertensive agents in human studies and find three different mechanisms of action for the drugs investigated. (i) Selective vasodilatation of the renal afferent arteriole (prazosin, indoramin, clonidine, moxonidine, α-methyldopa, some Ca++-channel blockers, angiotensin-receptor blockers, atenolol, metoprolol, bisoprolol, labetolol, hydrochlorothiazide, and furosemide). (ii) Inhibition of tubular solute reabsorption (propranolol, nadolol, oxprenolol, and indapamide). (iii) A combination of these first two mechanisms (amlodipine, nifedipine and ACE-inhibitors). These findings provide insights into the actions of antihypertensive drugs, and challenge misconceptions about the mechanisms underlying the therapeutic efficacy of many of the agents.

Increased hydrogen peroxide impairs angiotensin II contractions of afferent arterioles in mice after renal ischaemia-reperfusion injury.

Renal ischaemia-reperfusion injury (IRI) increases angiotensin II (Ang II) and reactive oxygen species (ROS) that are potent modulators of vascular function. However, the roles of individual ROS and their interaction with Ang II are not clear. Here we tested the hypothesis that IRI modulates renal afferent arteriolar responses to Ang II via increasing superoxide (O2-) or hydrogen peroxide (H2 O2 ). Renal afferent arterioles were isolated and perfused from C57BL/6 mice 24h after IRI or sham surgery. Responses to Ang II or noradrenaline were assessed by measuring arteriolar diameter. Production of H2 O2 and O2- was assessed in afferent arterioles and renal cortex. Activity of SOD and catalase, and mRNA expressions of Ang II receptors were assessed in pre-glomerular arterioles and renal cortex. Afferent arterioles from mice after IRI had a reduced maximal contraction to Ang II (-27±2 vs. -42±1%, P<0.001), but retained a normal contraction to noradrenaline. Arterioles after IRI had a 38% increase in H2 O2 (P<0.001) and a 45% decrease in catalase activity (P<0.01). Contractions were reduced in normal arterioles after incubation with H2 O2 (-22±2 vs. -42±1%, P<0.05) similar to the effects of IRI. However, the impaired contractions were normalized by incubation with PEG catalase despite a reduced AT1 R expression. Renal IRI in mice selectively impairs afferent arteriolar responses to Ang II because of H2 O2 accumulation that is caused by a reduced catalase activity. This could serve to buffer the effect of Ang II after IRI and may be a protective mechanism.

Commentary: Oxygen regulation of breathing through an olfactory receptor activated by lactate.

Commentary: Oxygen regulation of breathing through an olfactory receptor activated by lactate.

Mechanisms of caffeine-induced diuresis

Caffeine is an alkaloid which belongs to the family of methylxanthines and is present in beverages, food and drugs. Caffeine competitively antagonizes the adenosine receptors (AR), which are G protein-coupled receptors largely distributed throughout the body, including brain, heart, vessels and kidneys. Caffeine consumption has a well-known diuretic effect. The homeostasis of salt and water involves different segments of the nephron, in which adenosine plays complex roles depending on the differential expression of AR. Hence, caffeine increases glomerular filtration rate by opposing the vasoconstriction of renal afferent arteriole mediated by adenosine via type 1 AR during the tubuloglomerular feedback. Caffeine also inhibits Na(+) reabsorption at the level of renal proximal tubules. In addition, caffeine perturbs the hepatorenal reflex via sensory nerves in Mall's intrahepatic spaces. Here, we review the physiology of caffeine-induced natriuresis and diuresis, as well as the putative pathological implications.

Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles.

Myogenic contraction is the principal component of renal autoregulation that protects the kidney from hypertensive barotrauma. Contractions are initiated by a rise in perfusion pressure that signals a reduction in membrane potential (Em) of vascular smooth muscle cells to activate voltage-operated Ca(2+) channels. Since ROS have variable effects on myogenic tone, we investigated the hypothesis that superoxide (O2 (·-)) and H2O2 differentially impact myogenic contractions. The myogenic contractions of mouse isolated and perfused single afferent arterioles were assessed from changes in luminal diameter with increasing perfusion pressure (40-80 mmHg). O2 (·-), H2O2, and Em were assessed by fluorescence microscopy during incubation with paraquat to increase O2 (·-) or with H2O2 Paraquat enhanced O2 (·-) generation and myogenic contractions (-42 ± 4% vs. -19 ± 4%, P < 0.005) that were blocked by SOD but not by catalase and signaled via PKC. In contrast, H2O2 inhibited the effects of paraquat and reduced myogenic contractions (-10 ± 1% vs. -19 ± 2%, P < 0.005) and signaled via PKG. O2 (·-) activated Ca(2+)-activated Cl(-) channels that reduced Em, whereas H2O2 activated Ca(2+)-activated and voltage-gated K(+) channels that increased Em Blockade of voltage-operated Ca(2+) channels prevented the enhanced myogenic contractions with paraquat without preventing the reduction in Em Myogenic contractions were independent of the endothelium and largely independent of nitric oxide. We conclude that O2 (·-) and H2O2 activate different signaling pathways in vascular smooth muscle cells linked to discreet membrane channels with opposite effects on Em and voltage-operated Ca(2+) channels and therefore have opposite effects on myogenic contractions.

Knockdown of the Expression of Gamma‐Adducin in the Renal Afferent Arteriole Enhances BK Channel Activity

We recently identified a region on chromosome 1 containing 15 genes including Gamma‐Adducin (Add3) that causes the impaired myogenic response in the renal afferent arteriole (Af‐Art) and the development of renal vascular injury in Fawn Hood Hypertensive (FHH) rats. To determine which isoform of Add3 is expressed in the vasculature and whether Add3 is the viable candidate gene that plays a role in the modulation of myogenic response in renal microvessels, we knocked down the expression of Add3 with newly designed 27‐mer Dicer‐Substrate RNAi (DsiRNA) in rat Af‐art cultured for 36 hours. We then identified positively transfected vascular smooth muscle cells (VSMCs) using a red fluorescent indicator, and examined the large conductance calcium sensitive potassium (BK) current using patch clamp techniques. Both Add3 isoforms were detected in brain, kidney and liver, however, only isoform 2 was detected in the vasculature. The expression of Add3 was reduced in the renal microvessels and in the membrane fraction of the brain of FHH vs. FHH.1BN rats. Add3 DsiRNA completely blocked the expression of Add3 protein in 293 cells and reduced the expression of mRNA in a dose‐dependent manner up to 50% in the cultured Af‐Art. The inner diameters of the Af‐Art transfected with Add3 DsiRNA dilated by 4 ± 3 % in response to an elevation in transmural pressure from 60 to 120 mmHg. In contrast, these vessels still constricted normally by 10 ± 1 % in vessels transfected with scrambled siRNA. The myogenic response of Af‐Art isolated from our novel Add3 KO rats is impaired versus controls. BK current was 3‐fold higher in VSMC freshly isolated from Af‐Art that were transfected with Add3 DsiRNA as indicated by the appearance of siGLO red fluorescence compared with that seen in non‐transfected cells. Similarly, BK current was higher in 293BKα cells transfected with Add3 DsiRNA or in VSMC isolated from Af‐Art of FHH relative to control rats and was blocked by IBTX. BKα protein expression was greater in the membrane fraction in FHH vs. FHH.1BN rats. It appeared a stronger florescence at the membrane of 293BKα cells transfected with Add3 DsiRNA when staining with BKα implying an inhibition of endocytosis of BKα from the cell membrane. These results indicate that vasculature specifically expressed Add3 isoform 2, knockdown of the expression of Add3 impairs the myogenic response of Af‐Art that is similar as seen in FHH rats and associated with an elevation in BK channel activity. It supports the hypothesis that Add3 is a viable candidate gene that may play a causal role in the impaired myogenic response of the renal circulation in FHH rats and contributes to the development of renal injury.Support or Funding InformationThis study was supported by grants HL36279 and DK104184 (RJR) from the National Institutes of Health.

Immunohistochemical localization of renin-containing cells in two elasmobranch species.

Renin immunoreactivity was localized at the light and electron microscopic level in two elasmobranch fish species, the Atlantic stingray, Dasyatis sabina, and river ray, Potamotrygon humerosa. At the light microscopic level, the peroxidase-anti-peroxidase method showed a positive immunoreactivity in modified smooth muscle cells in kidney afferent arterioles as well as in arterioles of several organs: rectal gland, inter-renal gland, conus arteriosus, and gill. Electron microscopic renin-positive immunogold localization was confined to the contents of membrane bound granules in the modified smooth muscle cells of these arterioles. The presence of renin-containing granules in the modified smooth muscle, "granular cells," of the renal glomerular afferent arteriole of these two stingray species adds support to earlier studies which showed the structural components of a complete juxtaglomerular apparatus and some of the biochemical and molecular components of a renin-angiotensin system (RAS) as found in teleost fish, reptiles, birds, and mammals. A notable result, however, was the renin-positive immunoreaction in the arteriolar wall of all other organs studied here. The presence of this "diffuse renin system" in the connective tissue of various organs suggests that in these two stingray species in addition to local organ-specific functions, the RAS may act as a systemic mechanism to regulate blood pressure and blood flow in the body.

Highly sensitive myosin phosphorylation analysis in the renal afferent arteriole.

The regulation of smooth muscle contraction and relaxation involves phosphorylation and dephosphorylation of regulatory proteins, particularly myosin. To elucidate the regulatory mechanisms, analyzing the phosphorylation signal transduction is crucial. Although a pharmacological approach with selective inhibitors is sensitive and a useful technique, it leads to speculation regarding a signaling pathway but does not provide direct evidence of changes at a molecular level. We developed a highly sensitive biochemical technique to analyze phosphorylation by adapting Phos-tag SDS-PAGE. With this technique, we successfully analyzed myosin light chain (LC20) phosphorylation in tiny renal afferent arterioles. In the rat afferent arterioles, endothelin-1 (ET-1) induced diphosphorylation of LC20 at Ser19 and Thr18 as well as monophosphorylation at Ser19 via ETB receptor activation. Considering that LC20 diphosphorylation can decrease the rate of dephosphorylation and thus relaxation, we concluded that LC20 diphosphorylation contributes, at least in part, to the prolonged contraction induced by ET-1 in the renal afferent arteriole.

Assessment of chloride levels on renal function after cardiac arrest

Chloride may alter renal function by inducing vasoconstriction of renal afferent arterioles, thereby inducing cortical hypoperfusion. Increased chloride delivery to the distal tube may also induce tubular dysfunction. The effects of chloride balance on renal function after cardiac arrest (CA) remain unknown.

Abstract P102: Type I Diabetes Mellitus is More Sensitive to Ang II Due to Increased AT1 Receptor Expression in Afferent Arteriole

The prevalence of hypertension is about twice in diabetic subjects than that in non-diabetics. In the presence of hypertension, the progress of diabetic nephropathy has been shown to be more severe and rapid. But the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that angiotensin II receptor type 1 (AT1 receptor) expression level in renal afferent arteriole is elevated, which enhances the responses to angiotensin II (Ang II) stimulation and contributes to the development of hypertension in diabetes. First, we measured mean arterial pressure (MAP) with telemetry in diabetic and non-diabetic mice with Ang II infusion for 8 weeks. MAP was 26.7 mmHg higher in diabetic mice (157.4±11.3 mmHg) than in non-diabetic mice (130.7±6.4 mmHg). Next, we induced 2-kidney-1-clip Goldblatt hypertension in diabetic mice and non-diabetic mice and measured MAP with telemetry for 8 weeks. MAP was 17.6 mmHg higher in diabetic mice (151.1±9.5 mmHg) than in non-diabetic mice (133.5±4.2 mmHg), indicating blood pressure of diabetic mice is also more responsive to both exogenous and endogenous Ang II than no-diabetic mice. To investigate the mechanisms of the enhanced sensitivity to Ang II, we dissected afferent arterioles from non-diabetic mice and diabetic mice and measured the expression of AT1 receptor mRNA by real-time PCR. AT1 receptor mRNA level was 6 times higher in diabetic mice that in non-diabetic control. AT1 receptor protein abundance in renal cortex was also 5 times higher in diabetic mice than controls. In conclusion, the blood pressure of the diabetic mice are more sensitive to Ang II than non-diabetic controls. Increased AT1 receptor expression in the afferent arterioles may contribute to the development of hypertension in diabetes.

Abstract P090: Agonistic Autoantibodies to the Angiotensin II Type 1 Receptor Enhances ANG II Induced Renal Vascular Sensitivity and Reduces Renal Function During Pregnancy

Preeclamptic women produce agonistic autoantibodies to the Angiotensin II type 1 receptor (AT1-AA) and exhibit increased blood pressure (BP) and vascular sensitivity to angiotensin II (ANG II). Although, together AT1-AAs and ANGII increase the BP, renal artery resistant index, and vasoconstriction of renal afferent arterioles in pregnant rats; the renal hemodynamics in the presence of the AT1-AAs during pregnancy has not been examined. Thus the objective of this study was to examine the changes in the glomerular filtration rate (GFR) and renal blood flow (RBF) during pregnancy in the presence of AT1-AAs and/or ANGII. Methods: Pregnant Sprague Dawley rats were divided into 4 groups: Normal Pregnant (NP, n=6), Pregnant + ANG II (Preg + ANG II, n=6), Pregnant + AT1-AA (Preg + AT1-AA, n=8), and Pregnant + ANG II + AT1-AA (Preg + ANGII + AT1-AA, n= 6). On day 13 of pregnancy, rats were implanted with mini-pumps infusing ANG II (50 ng/kg/min) and/or AT1-AA (1:40 dilution). On day 19 of pregnancy, rats were subjected to terminal renal function surgeries using FITC labeled Inulin. During the surgery, the BP was recorded and a transonic flowmeter probe was placed on the left renal artery to measure RBF. Results: BP was elevated in all pregnant rats administered ANG II and/or the AT1-AA. Although GFR was reduced, it was not significant between Preg + ANG II and Preg + AT1-AA vs. NP rats (1.5 ±0.24, 1.60 ±0.17 vs. 1.90 ±0.16 ml/min). However, the GFR was further decreased in Preg + ANGII + AT1-AA rats (1.20 ±0.08). No difference was observed with the RBF between Preg + ANG II and Preg + AT1-AA vs NP rats (14.4 ±2.96, 14.4 ±1.48 vs. 15.4 ±1.75 ml/min). RBF was decreased in Preg + ANGII + AT1-AA vs NP rats (7.4 ±1.09 vs. 15.4 ±1.75 ml/min). No change in RVR between Preg + ANG II and Preg + AT1-AA vs. NP rats (9.7 ±2.69, 8.3 ±0.58 vs. 6.4 ±0.77). However, the RVR was drastically increased between Preg + ANGII + AT1-AA vs NP rats (18.4 ±2.91 vs. 6.4 ±0.77). Conclusion: Together ANG II and AT1-AA drastically decreases renal function by 37%, RBF by 50%, and caused a 3 fold increase in RVR vs NP rats. These data indicate the importance of AT1-AAs to drastically enhance ANG II induced renal vascular sensitivity and reduce renal function during preeclampsia. Research Supported by T32HL105324 and RO1HD067541

Remodeling of Afferent Arterioles From Mice With Oxidative Stress Does Not Account for Increased Contractility but Does Limit Excessive Wall Stress.

Because superoxide dismutase (SOD) knockout enhances arteriolar remodeling and contractility, we hypothesized that remodeling enhances contractility. In the isolated and perfused renal afferent arterioles from SOD wild type (+/+) and gene-deleted mice, contractility was assessed from reductions in luminal diameter with perfusion pressure from 40 to 80 mm Hg (myogenic responses) or angiotensin II (10(-6) mol/L), remodeling from media:lumen area ratio, superoxide (O2 (·-)) and hydrogen peroxide (H2O2) from fluorescence microscopy, and wall stress from wall tension/wall thickness. Compared with +/+ strains, arterioles from SOD1-/-, SOD2+/-, and SOD3-/- mice developed significantly (P<0.05) more O2 (·-) with perfusion pressure and angiotensin II and significantly increased myogenic responses (SOD1-/-: -20.7±2.2% versus -12.7±1.6%; SOD2+/-: -7.4±1.3% versus -12.6±1.4%; and SOD3-/-: -9.1±1.9% versus -15.8±2.2%) and angiotensin II contractions and ≈2-fold increased media:lumen ratios. Media:lumen ratios correlated with myogenic responses (r(2) =0.23; P<0.01), angiotensin II contractions (r(2)=0.57; P<0.0001), and active wall tension (r(2) =0.19; P<0.01), but not with active wall stress (r(2)=0.08; NS). Differences in myogenic responses among SOD3 mice were abolished by bath addition of SOD and were increased 3 days after inducing SOD3 knockout (-26.9±1.7% versus -20.1±0.7%; P<0.05), despite unchanged media:lumen ratios (2.01±0.09 versus 2.02±0.03; NS). We conclude that cytosolic, mitochondrial, or extracellular O2 (·-) enhance afferent arteriolar contractility and remodeling. Although remodeling does not enhance contractility, it does prevent the potentially damaging effects of increased wall stress.

Superoxide enhances Ca2+ entry through L-type channels in the renal afferent arteriole.

Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane, and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was mediated exclusively by L-type channels because it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314±68 nmol/L (P<0.001) and doubled the plateau response. These effects were abolished by tempol and nitroblue tetrazolium, but not by catalase, confirming actions of superoxide and not of hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store-operated entry or ryanodine receptor-mediated calcium mobilization.

Ex vivo use of a Rho-kinase inhibitor during renal preservation improves graft function upon reperfusion

BackgroundActivation of the Rho–Rho-kinase pathway has been shown to cause vasoconstriction in renal afferent arterioles. Vascular dysfunction plays a pivotal role in triggering reperfusion injury after kidney transplantation. Therefore, the effect of a Rho-kinase inhibitor, added to the preservation solution, on renal function after 18h of storage at 4°C was evaluated. MethodsPorcine kidneys were preserved with cold HTK-solution. During preservation, in the study group, HTK was supplemented with the Rho-kinase inhibitor HA1077, whereas the control group received no further treatment (n=6, respectively). Kidney function after 18h of storage at 4°C was evaluated by 90min of isolated reperfusion in vitro. ResultsRho-kinase inhibition (RKI) was associated with significantly higher renal perfusate flow compared to the control group. Endothelial function, as measured by perfusate levels of nitric oxide and gene expression of eNOS, was significantly increased in the study group. In our model, RKI also significantly improved glomerular function (clearance of creatinine) as well as tubular cell integrity as reflected by reduced fractional sodium excretion and release of fatty acid binding protein, a specific tubular cell marker. ConclusionOur results indicate that blocking the Rho-kinase pathway during cold preservation may lead to a better graft function upon reperfusion.