Renal Renin Activity Research Articles

Combination of AT2R Agonist and Neprilysin Inhibitor is Superior Than the Combination of AT1R Antagonist and Neprilysin Inhibitor in Protecting Kidney from Injury in High‐Salt Fed Obese Rats

Angiotensin II type 1 receptor (AT1R) antagonists, angiotensin II type 2 receptor (AT2R) agonists, and atrial natriuretic peptide (ANP) are natriuretics that provide reno- and cardiovascular protection and reduce blood pressure and volume load. An approved combination therapy (Entresto) of the AT1R blocker valsartan (VAL), and inhibitor of the ANP degrading enzyme neprilysin (NEP) sacubitril (SAC), for patients with heart failure and reduced ejection fraction was found to be associated with increased albuminuria, an indicator of kidney injury. Based on studies showing that AT2R activation affects various renin angiotensin system (RAS) components by decreasing activity of the classical arm components, and increasing expression and activity of those of the protective arm such as angiotensin converting enzyme 2 (ACE2), we hypothesized that AT2R agonist treatment in combination with SAC will provide better reno-protection compared to VAL + SAC. We tested this hypothesis in obese Zucker rats (OZR), in which kidney injury was induced by feeding a high salt diet. Male OZR were fed a 0.4% normal sodium (NSD) or 4% high sodium diet (HSD) and administered vehicle, AT2R agonist C21 (1 mg/kg/day) + SAC (10 mg/kg/day), or VAL (10 mg/kg/day) + SAC (10 mg/kg/day) (16 days, daily gavage). While total food intake was significantly decreased and water intake significantly increased by HSD feeding, neither treatment had an effect compared to the control group. Yet, C21 + SAC treatment, not VAL + SAC, prevented increases in body weight gain and kidney weight, a sign of hypertrophy. Analysis of 24-hr urine samples revealed that HSD caused a marked increase in albuminuria and proteinuria, which was significantly prevented by only C21 + SAC treatment. ANP was similarly preserved and associated with comparable inhibition of NEP activity in both treatment groups compared to HSD control. Furthermore, C21 + SAC treatment caused an increase in the renal expression of ACE2, and a decrease in renal renin activity, unlike VAL + SAC treatment. Moreover, HSD caused a reduction in plasma renin levels, which were increased 3-times by VAL + SAC, but not by C21 + SAC. Renal AT2R expression was unchanged in all treatment groups compared with HSD control group. On the other hand, renal AT1R expression was modestly increased by only VAL + SAC treatment. Taken together, this data suggests the combination of C21 + SAC provides strong reno-protection in obese animals fed HSD and is superior to the protection afforded by the combination of VAL + SAC. Differential regulation of the opposing arms of the RAS may be the basis of the superiority of C21 + SAC over VAL + SAC.

(Pro)renin receptor decoy peptide PRO20 protects against adriamycin-induced nephropathy by targeting the intrarenal renin-angiotensin system.

Adriamycin (ADR) administration in susceptible rodents such as the BALB/c mouse strain produces injury to the glomerulus mimicking human chronic kidney disease due to primary focal segmental glomerulosclerosis. The goal of the present study was to use this model to investigate antiproteinuric actions of the (pro)renin receptor decoy inhibitor PRO20. BALB/c mice were pretreated for 1 day with PRO20 at 500 μg·kg-1·day-1 via an osmotic minipump followed by a single injection of vehicle or ADR (10 mg/kg) via the tail vein. Albuminuria and renal function were analyzed at the fourth week post-ADR administration. ADR-treated mice exhibited severe proteinuria, hypoalbuminemia and hyperlipidemia, glomerulosclerosis, podocyte loss, tubulointerstitial fibrosis, and oxidative stress, accompanied by elevated urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, all of which were significantly attenuated by PRO20. Urinary and renal renin activity and angiotensin II were elevated by ADR and suppressed by PRO20. In parallel, urinary and renal H2O2 levels and renal NADPH oxidase 4 (Nox4) and transient receptor potential channel C6 (TRPC6) expression in response to ADR were all similarly suppressed. Taken together, the results of the present study provide the first evidence that PRO20 can protect against podocyte damage and interstitial fibrosis in ADR nephropathy by preventing activation of the intrarenal renin-angiotensin system and upregulation of Nox4 and TRPC6 expression. PRO20 may have a potential application in the treatment of ADR nephropathy.

PO-099 Effects of long-term high-intensity exercise training on renal local renin-angiotensin system in rats

Objective Exercise stimulation to the body of the first stress: blood changes, regulated by the renin-angiotensin system. Long-term high-intensity exercise training will lead to changes in kidney structure and function, resulting in renal injury. This will not only affect training and competition, but also affect the health of athletes, thus it is becoming an influential factor in the occurrence and development of excessive fatigue. At present, the mechanism of renal injury and proteinuria caused by exercise is not very clear. Therefore, this study conducted an in-depth study on the upstream mechanism of renal blood flow changes from the molecular level and explored the effects of 6-week high-intensity exercise training on the renal local renin-angiotensin system in rats.
 Methods 30 SD male rats (8 weeks old) were randomly divided into control group (10) and exercise group (20). Then according to different time, the exercise group (20) was divided into two groups on average, which are 0h group and 24h group. The rats in the control group did not do any exercise, and the rats in the exercise group were trained to run on the platform with high intensity for 6 weeks. At the beginning of the sixth week, the urine of rats from different group were selected randomly, and the urine NGAL, urinary microalbumin (mAlb), urinary creatinine (UCr) and total urine protein (TP) were tested, to determine the rat model of exercise-induced renal injury with proteinuria. After training on the 6th weekend, the renal tissue renin activity and angiotensin Ⅱ (Ang Ⅱ) content in right kidney were tested. At the same time, the left kidney of the rat was taken to make HE staining sections.
 Results (1) The mAlb, Alb/Cr and TP of the exercise group were higher than those of the control group, while the UCr content was lower than that of the control group. There was a significant difference between mAlb and TP in the exercise group and the control group(p<0.05). UCr, mAlb/Cr were significantly different from the control group(p<0.01). The urine NGAL concentration in the exercise group was higher than that in the control group, and the difference was significant(p<0.05). (2) The glomerulus of the rats in the 0h group showed obvious congestion, swelling and erythrocyte exudation. The tissue morphology of rats in the 24h group recovered slightly, but it was still different from the control group. (3) The renin activity and Ang Ⅱ were the lowest in the 0h group, and the renin activity and Ang Ⅱ were highest in the 24h group. There is a very significant difference between the groups(p<0.01).
 Conclusions (1) The 6-week high-intensity training used in this study increased the levels of NGAL, TP, and mAlb in the urine of rats, and successfully established a rat model of exercise-induced renal injury with proteinuria. (2) Long-term high-intensity exercise training can cause obvious congestion, swelling, erythrocyte exudation in rat glomeruli, which can’t return to the quiet level 24 hours after exercise. (3) Renal injury caused by exercise decreased the expression of local renin activity and angiotensin II in the kidney, and the recovery of renal renin activity and angiotensin II was increased 24h after exercise.

Superoxide via Sp3 mechanism increases renal renin activity, renal AT1 receptor function, and blood pressure in rats.

We tested a hypothesis that superoxide, by inducing Sp3, increases renal renin activity, renal angiotensin II type 1 receptor (AT1R) function, and blood pressure (BP) in rats. Group 1 rats were treated with vehicle, saline. Group 2 rats were treated with superoxide dismutase (SOD) inhibitor diethylthiocarbamate (DETC). Group 3 rats were treated with DETC and an SOD mimetic, tempol. Group 4 rats were treated with tempol only. All four groups of rats were treated for 2 wk then anesthetized, and BP was recorded. Thereafter, diuresis and natriuresis in response to AT1R blocker candesartan were determined. When compared with vehicle rats, BP increased in DETC rats. The increased BP in DETC rats decreased with tempol. Diuresis and natriuresis in response to candesartan increased in controls, and this further increased in DETC rats and decreased with tempol. A second set of four groups of rats underwent the same treatment as above and were anesthetized, and their kidneys were obtained for biochemical studies. The levels of superoxide but not hydrogen peroxide increased, whereas SOD activities decreased further in the renal cortical tissues of DETC rats than vehicle rats. These effects were attenuated with tempol in DETC rats. Moreover, tissue renin activity and abundance of membranous AT1R proteins increased more in DETC rats than vehicle rats, and decreased with tempol in DETC rats. Furthermore, the levels of lysine-acetylated, but not serine-phosphorylated, Sp3 increased more in the nuclei of DETC rats than vehicle rats. The increased levels of Sp3 lysine acetylation decreased in DETC rats with tempol. Taken together, our results suggest that superoxide activates renal Sp3 via lysine acetylation increasing renin activity, AT1R function, and BP in rats.

Abstract P608: Role Of Nitric Oxide In The Pathophysiology Of Hypertension Associated With Low Nephron Endowment.

A low nephron endowment is a strong predictor for future risk of hypertension, kidney and cardiovascular disease. Previously, we have shown that fetal uninephrectomy (uni-x) results in elevated arterial pressure, reduced renal function and low plasma renin activity in adulthood in sheep. However, the mechanisms via which a reduction in renal mass in early life contributes to the development of hypertension and renal disease are not well understood. Nitric oxide (NO) plays an important role in driving the fall in renal vascular resistance (RVR) and rise in glomerular filtration rate (GFR) that characterizes the normal postnatal maturation of renal function in the newborn. We hypothesized that the early onset of renal dysfunction in individuals with a congenital reduction in renal mass is due to a reduced renal contribution of NO. Fetal uni-x (n=9) or sham operation (n=7) was performed at 100d of gestation (term=150 days). In conscious female lambs at 6 months of age, mean arterial pressure (MAP) was measured via an indwelling carotid arterial catheter and GFR and renal blood flow (RBF) were measured via clearance methods before and after nitric oxide synthase inhibition (L-NAME; 20mg/kg/h iv). Basal MAP was elevated in uni-x as compared to the sham sheep (sham: 80 ± 2 mmHg, uni-x: 86 ± 1 mmHg; P < 0.01). In response to systemic administration of L-NAME MAP increased similarly in both groups (sham: 13 ± 3 mmHg, uni-x: 13 ± 4 mmHg). Basal RBF (sham: 933 ± 29 ml/kg/h; uni-x 757 ± 43 ml/kg/h; P<0.01) and GFR (sham: 144 ± 3 ml/kg/h; uni-x 107 ± 3 ml/kg/h; P<0.01) was lower in the uni-x as compared to the sham sheep. In response to L-NAME the decline in RBF (sham: 73 ± 3 %; uni-x: 47 ± 3%; P<0.001) and GFR (sham: 54 ± 4 %; uni-x: 43 ± 1%; P<0.03) was less in uni-x as compared to sham sheep. Filtration fraction decreased significantly in the sham (P<0.001) but not the uni-x sheep in response to L-NAME. Renal dysfunction and hypertension in young hypertensive sheep born with a low nephron endowment maybe underpinned by impaired nitric oxide bioavailability. Targeting the nitric oxide system may offer therapeutic potential in children born with a reduced renal mass, in terms of treatment interventions and as a biomarker of prognosis.

Abstract 478: Development Of A Novel Technique To Visualize In Vivo Renin Activity And Assess Its Use In The Study Of The Pathogenesis Of Diabetic Nephropathy

Introduction and aims: Although systemic renin activity is suppressed in diabetes, a renin-angiotensin system (RAS) inhibitor exerts renoprotective effects. Activation of local/tissue RAS, therefore, appears to be involved in the pathogenesis of diabetic nephropathy. We attempted to develop a technique to visualize tissue renin activity in vivo using the fluorescence resonance energy transfer (FRET) system and multiphoton laser microscopy (MP-LM). In the FRET peptide, the fluorescence of 5-fluorescein amidite (FAM) is intramolecularly quenched. After cleavage into two fragments by renin, the fluorescence of 5-FAM is recovered and can be visualized at excitation/emission wavelengths of 490/520 nm. Methods and results: Diabetes was induced with streptozotocin (STZ) in C57B6/ mice, followed by aliskiren (Alis: 25 mg/kg/day with osmotic pump) or valsartan (Val: 15 mg/kg/day by lavage) treatment for 4 weeks starting at 6 weeks after STZ injection. Diabetic mice (DM) showed higher urinary albumin excretion (UAE; control: 85±15 μg/day, DM: 220±25 μg/day, P<0.05). Alis or Val treatment significantly reduced UAE (Alis: 120±21 μg/day, Val: 135±18 μg/day). Renin activity in the kidney assessed by the FRET system was markedly enhanced in the glomeruli in DM (1.7±0.2-fold that in controls, P<0.05). Alis reduced plasma and renal renin activity to baseline levels. Production of reactive oxygen species (ROS) and nitric oxide (NO) was evaluated by MP-LM using dichlorofluorescin and diaminorhodamine-4M acetoxymethyl ester. Increased ROS production and diminished bioavailable NO were noted in the glomeruli in DM, but were significantly ameliorated with Alis or Val treatment. Glomerular permeability of macromolecules was visualized in vivo using MP-LM with 70-kDa dextran. Glomerular permeability was significantly greater in DM than in the controls and was ameliorated by Alis or Val treatment. Conclusions: We developed an in vivo imaging technique to visualize renin activity in living animals. Alis inhibited both plasma and tissue renin activity, ameliorated tissue oxidative stress, and attenuated UAE in a diabetes model. The in vivo imaging technique with the FRET technique will provide a novel approach to study the pathophysiology of kidney diseases.

Time- and Dose-dependent Cytotoxicities of Ioxitalamate and Indigocarmine in Human Nucleus Pulposus Cells

Introduction Ioxitalamate (Telebrix 300) is an ionic iodinated contrast medium (CM) commonly used for discography or PELD (percutaneous endoscopic lumbar discectomy), though it has side effects such as anaphylactic shock and renal toxicity. Indigocarmine is an organic compound dye with a distinctive blue color that is commonly used during PELD to stain the acidic, degenerated nucleus pulposus. Although ioxitalamate and indigocarmine are widely used in spinal surgery, there have been no reports on their effects on nucleus pulposus (NP) cells. We studied the toxicity of both ioxitalamate and indigocarmine to NP cells. Materials and Methods NP cells were obtained via discectomy from lumbar disc patients and isolated. NP cells were cultured in three-dimensional (3D) alginate beads with 0.001, 0.1, 10, and 100 mg/mL ioxitalamate, 0.00001, 0.001, 0.1 and 10 mg/mL indigocarmine, or a mixture of both for 1, 2, or 3 days. The living cells were analyzed with trypan blue staining. FACS analysis using Annexin V and propidium iodide (PI) and 3D alginate bead immunostaining was performed to identify live, apoptotic, and necrotic cells. Results Ioxitalamate, indigocarmine, and their combination induced statistically significant NP cell injury that was both time and dose dependent ( p < 0.05). Also, at the same concentration, ioxitalamate was more cytotoxic than was indigocarmine or the combination ( p < 0.05). All three treatments also showed dose-dependent cytotoxicity according to flow cytometry and immunostaining. Conclusion Ioxitalamate and indigocarmine are toxic to human NP cells in vitro in a time- and dose-dependent manner. We assume that ioxitalamate and indigocarmine may have similar effects in patients undergoing discography and PELD. Thus, we suggest that ioxitalamate and indigocarmine should be used carefully at low concentrations. Disclosure of Interest None declared References Stout A. Discography. Phys Med Rehabil Clin N Am 2010;21(4):859–867 Manchikanti L, Glaser SE, Wolfer L, Derby R, Cohen SP. Systematic review of lumbar discography as a diagnostic test for chronic low back pain. Pain Physician 2009;12(3):541–559 Carragee EJ, Tanner CM, Yang B, Brito JL, Truong T. False-positive findings on lumbar discography. Reliability of subjective concordance assessment during provocative disc injection. Spine 1999;24(23):2542–2547 Laslett M, Oberg B, Aprill CN, McDonald B. Centralization as a predictor of provocation discography results in chronic low back pain, and the influence of disability and distress on diagnostic power. Spine J 2005;5(4):370–380 Kim D, Wadley R. Variability in techniques and patient safety protocols in discography: a national multispecialty survey of International Spine Intervention Society members. J Spinal Disord Tech 2010;23(6):431–438 Carragee EJ, Tanner CM, Khurana S, et al. The rates of false-positive lumbar discography in select patients without low back symptoms. Spine 2000;25(11):1373-1380, discussion 1381 Paul MD, Tsou M. Posterolateral percutaneous endoscopic lumbar discectomy. Oper Tech Orthop 2003;13:207-213 Yeung AT. Selective endoscopic discectomy. In: Kambin P, ed. Arthroscopic and Endoscopic Spinal Surgery. Totowa, NJ: Humana Press; 2005:205–225 Mayer HM, Brock M. Percutaneous endoscopic discectomy: surgical technique and preliminary results compared to microsurgical discectomy. J Neurosurg 1993;78(2):216–225 Yeung AT, Tsou PM. Posterolateral endoscopic excision for lumbar disc herniation: Surgical technique, outcome, and complications in 307 consecutive cases. Spine 2002;27(7):722–731 Lee H, Sowa G, Vo N, et al. Effect of bupivacaine on intervertebral disc cell viability. Spine J 2010;10(2):159–166 Hsu SP, Tsai TJ, Chien CT. Ioxitalamate induces renal tubular apoptosis via activation of renal efferent nerve-mediated adrenergic signaling, renin activity, and reactive oxygen species production in rats. Toxicol Sci 2010;114(1):149–158 Lee HC, Chang JG, Yen HW, Liu IH, Lai WT, Sheu SH. Ionic contrast media induced more apoptosis in diabetic kidney than nonionic contrast media. J Nephrol 2011;24(3):376–380 Belhadjali H, Bouzgarrou L, Youssef M, Njim L, Zili J. DRESS syndrome induced by sodium meglumine ioxitalamate. Allergy 2008;63(6):786–787 Lacour B, Lacroix C, Jungers P, Bailly M. [Effects of intravenous urography using sodium and meglumine ioxitalamate (Telebrix 38) on blood creatinine and urinary excretion of N-acetyl-beta-D-glucosaminidase]. Ann Biol Clin (Paris) 1984;42(6):407–411

Liver Angiotensinogen Is the Primary Source of Renal Angiotensin II

Angiotensin II content in the kidney is much higher than in the plasma, and it increases more in kidney diseases through an uncertain mechanism. Because the kidney abundantly expresses angiotensinogen mRNA, transcriptional dysregulation of angiotensinogen within the kidney is one potential cause of increased renal angiotensin II in the setting of disease. Here, we observed that kidney-specific angiotensinogen knockout mice had levels of renal angiotensinogen protein and angiotensin II that were similar to those levels of control mice. In contrast, liver-specific knockout of angiotensinogen nearly abolished plasma and renal angiotensinogen protein and renal tissue angiotensin II. Immunohistochemical analysis in mosaic proximal tubules of megalin knockout mice revealed that angiotensinogen protein was incorporated selectively in megalin-intact cells of the proximal tubule, indicating that the proximal tubule reabsorbs filtered angiotensinogen through megalin. Disruption of the filtration barrier in a transgenic mouse model of podocyte-selective injury increased renal angiotensin II content and markedly increased both tubular and urinary angiotensinogen protein without an increase in renal renin activity, supporting the dependency of renal angiotensin II generation on filtered angiotensinogen. Taken together, these data suggest that liver-derived angiotensinogen is the primary source of renal angiotensinogen protein and angiotensin II. Furthermore, an abnormal increase in the permeability of the glomerular capillary wall to angiotensinogen, which characterizes proteinuric kidney diseases, enhances the synthesis of renal angiotensin II.

Metoprolol Ameliorates Cyclosporine A–induced Hypertension and Nephrotoxicity in Rats

Despite their clinical relevance as important cardiovascular modulators, there are few studies regarding the potential protective effect of β-blockers against immunosuppressive-induced cardiovascular side effects. This study investigated the possible ameliorating effect of β1-blocker, metoprolol (MTP), against both hypertensive and nephrotoxic effects of cyclosporine A (CSA). Compared with vehicle (olive oil)-treated rats, chronic treatment with CSA (20 mg·kg·d subcutaneous, for 14 days) increased systolic blood pressure, elevated renal function indices and plasma renin activity, impaired renovascular responsiveness of isolated perfused rat kidneys to endothelium-dependent vasodilations induced by carbachol. These effects were abolished upon concurrent administration of MTP (5 mg·kg·d for 14 days, intraperitoneal). The possibility that alterations in the antioxidant and/or circulating cytokine levels contributed to the CSA-MTP interaction was also investigated. MTP abrogated the oxidative (superoxide dismutase, catalase), lipid peroxidation (malondialdyde), and elevated the cytokine (TNF-α and TGF-β) effects of CSA. Histologically, CSA caused tubular brush border loss and isometric vacuolization clustered in the proximal tubule; this effect disappeared in rats cotreated with MTP. The use of a nonhypotensive dose of MTP (1.25 mg/kg) countered in part endothelium dysfunction altered oxidative stress parameters and cytokine levels in CSA-treated rats. Collectively, MTP abrogates both the hypertensive and the nephrotoxic effects of CSA via ameliorating endothelium dysfunction, oxidative stress, and upregulated cytokine levels caused by CSA. The demonstration of nephroprotection by the low dose of MTP suggests that its renal effect may be partially unrelated to its hemodynamic activity.

Pharmacological and genetic evidence that cathepsin B is not the physiological activator of rodent prorenin

Renin is the first enzyme in the renin-angiotensin-aldosterone system which is the principal regulator of blood pressure and hydroelectrolyte balance. Previous studies suggest that cathepsin B is the activator of the prorenin zymogen. Here, we show no difference in plasma renin activity, or mean arterial blood pressure between wild-type and cathepsin B knockout mice. To account for potential gene compensation, a potent, selective, reversible cathepsin B inhibitor was developed to determine the role of cathepsin B on prorenin processing in rats. Pharmacological inhibition of cathepsin B in spontaneously hypertensive and double transgenic rats did not result in a reduction in renal mature renin protein levels or plasma renin activity. We conclude that cathepsin B does not play a significant role in this process in rodents.

INFLUENCE OF MATERNAL HIGH SODIUM DIET ON SYSTEMIC AND RENAL RENIN ANGIOTENSIN ALDOSTERONE SYSTEM OF MALES AND FEMALES OFFSPRING WISTAR RATS

Objectiveto evaluate the renin angiotensin system (RAS) of male and female offspring, which mothers received high sodium diet during pregnancy.Methods and Results (mean±SEM p<0.05 n=8/group)Female Wistar rats received normal salt diet (NSD 1.3%) or high salt diet (HSD 8.0% NaCl) during pregnancy. The offsprings (offNSD, offHSD), males (M) and females (F) were fed NSD until 12 week old. The blood pressure (mmHg) was lower in F (112±2.4) than M offHSD (120±2.4). Aldosterone (pg/mL) was higher in F offNSD (1005±153.3) and offHSD (988±210.2) compared to M offNSD (410±35.8) and offHSD (302±24.9) respectively. The plasma renin activity (ng/mL/h) was lower in M offHSD (2.2±0.6) than M offNSD (4.2±0.4). The renal renin activity (ng/mg tissue/h) was higher in M offHSD (16.4±1.8) than M offNSD (9.0±0.8); and it was lower in F (6.7±1.2) than M offHSD (16.4±1.8). The renin granules (%) was lower in M offHSD (38.4±4.2) than M offNSD (60.7±5.0), and in F offHSD (46.4±4.4) than F offNSD (65.8±4.2). The renin gene expression of renin (IDVrenin/β‐actin) in renal cortex (1.0±0.1) and medulla (0.6±0.06) was lower in M offHSD than M offNSD (1.5±0.2; 1.5±0.2 respectively).ConclusionMaternal HSD decreases systemic RAS and gene expression of renal renin in males but not females. Probably there is influence of estrogen in female offspring.Financial support: FAPESP

Increased renal renin content in mice lacking the Na+/H+ exchanger NHE2

Macula densa (MD) cells express the Na(+)/H(+) exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2(+/+)) and NHE2 knockout (NHE2(-/-)) mice. Immunofluorescence studies indicated that NHE2(-/-) mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2(-/-) mice was increased approximately 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2(-/-) mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2(+/+) mice, but the response was blunted in NHE2(-/-) mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2(-/-) mice compared with wild type. NHE2(-/-) mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2(-/-) kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE(2)-renin release pathway.

Renal medullary renin activity but not NADPH oxidase activity depends on sympathetic innervation

Renal medullary renin activity but not NADPH oxidase activity depends on sympathetic innervation

Effects of Anesthesia on Plasma and Kidney ANG II Levels in Normotensive and ANG II-Dependent Hypertensive Rats

Background: Previous studies have implicated that normotensive rats with normal renal renin activity respond to anesthesia and surgery with greater increases in plasma and kidney angiotensin II (ANG II) concentrations than ANG II-dependent hypertensive rats with intrarenal renin depletion. In the present study, we therefore compared plasma and kidney ANG II levels in anesthetized and conscious normotensive and ANG II-dependent hypertensive rats. Methods: Salt-replete Hannover-Sprague-Dawley rats (HanSD) served as controls. As models of ANG II-dependent hypertension we used: 1st, transgenic rats harboring the Ren-2 renin gene (TGR); 2nd, two-kidney, one-clip (2K1C) Goldblatt hypertensive rats, and, 3rd, ANG II-infused hypertensive rats. As additional model with enhanced renin-angiotensin system (RAS) activity, salt-depleted HanSD and TGR were employed. Results: In anesthetized salt-repleted HanSD, plasma and kidney ANG II levels were higher than in salt-repleted TGR, ANG II-infused and 2K1C rats. Salt depletion caused marked increases in ANG II levels in HanSD but did not alter them in TGR. In contrast, in conscious animals immediately after decapitation plasma and kidney ANG II levels were similar in salt-repleted and salt-depleted TGR, in ANG II-infused rats, in the clipped kidney of 2K1C rats and in salt-depleted HanSD and in all these groups they were significantly higher than in salt-repleted HanSD. Conclusions: These findings indicate that anesthesia increases plasma and kidney ANG II levels in HanSD to a greater degree than in ANG II-dependent models of hypertension. Therefore, the results from studies employing anesthetized animals must be interpreted with caution.

Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero

In the rat, protein restriction during pregnancy increases offspring blood pressure by 20-30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo. Hence, we hypothesized that exposure to a maternal low-protein diet increases glomerular Ang II AT1 receptor expression and decreases AT2 receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT1 and AT2 receptor expression, 125I-Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT1 receptor expression was increased (62%) and AT2 expression was decreased (35%) in LP rats. Maximum 125I-Ang II (125I-Ang II) binding (Bmax) was increased in LP rats (control n = 9, 291.6 +/- 27.4 versus LP n = 7, 445.7 +/- 27.4 fmol (mg glomerular protein)(-1), P < 0.01), but affinity (KD) was not statistically different from controls (control 2.87 +/- 0.85 versus LP 0.84 +/- 0.20 pmol 125I-Ang II, P = 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT1 receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo. This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.

Cyclooxygenase-2 and the renal renin-angiotensin system.

In the kidney, cyclooxygenase-2 (COX-2) is expressed in the macula densa/cTALH and medullary interstitial cells. The macula densa is involved in regulating afferent arteriolar tone and renin release by sensing alterations in luminal chloride via changes in the rate of Na(+)/K(+)/2Cl(-) cotransport, and administration of non-specific cyclooxygenase inhibitors will blunt increases in renin release mediated by macula densa sensing of decreases in luminal NaCl. High renin states [salt deficiency, angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers, diuretic administration or experimental renovascular hypertension] are associated with increased macula densa/cTALH COX-2 expression. Furthermore, there is evidence that angiotensin II and/or aldosterone may inhibit COX-2 expression. In AT1 receptor knockout mice, COX-2 expression is increased similar to increases with ACE inhibitors or AT1 receptor blockers. Direct administration of angiotensin II inhibits macula densa COX-2 expression. Previous studies demonstrated that alterations in intraluminal chloride concentration are the signal for macula densa regulation of tubuloglomerular feedback and renin secretion, with high chloride stimulating tubuloglomerular feedback and low chloride stimulating renin release. When cultured cTALH or macula densa cells were incubated in media with selective substitution of chloride ions, COX-2 expression and prostaglandin production were significantly increased. A variety of studies have indicated a role for COX-2 in the macula densa mediation of renin release. In isolated perfused glomerular preparations, renin release induced by macula densa perfusion with a low chloride solution was inhibited by a COX-2 inhibitor but not a COX-1 inhibitor. In vivo studies in rats indicated that increased renin release in response to low-salt diet, ACE inhibitor, loop diuretics or aortic coarctation could be inhibited by administration of COX-2-selective inhibitors. In mice with genetic deletion of COX-2, ACE inhibitors or low-salt diet failed to increase renal renin expression, although renin significantly increased in wild type mice. In contrast, in COX-1 null mice there were no significant differences in either the basal or ACE inhibitor-stimulated level of renal renin activity from plasma or renal tissue compared with wild type mice. In summary, there is increasing evidence that COX-2 expression in the macula densa and surrounding cortical thick ascending limb cells is regulated by angiotensin II and is a modulator of renal renin production. These interactions of COX-2 derived prostaglandins and the renin-angiotensin system may underlie physiological and pathophysiological regulation of renal function.

Interactions between COX-2 and the renin-angiotensin system in the kidney.

In adult mammalian kidney, COX-2 expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb cells (cTALH) and medullary interstitial cells. Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. In the MD region, COX-2 expression increases in high renin states [salt restriction, angiotensin converting enzyme (ACE) inhibition, renovascular hypertension], and selective COX-2 inhibitors significantly decrease plasma renin levels and renal renin activity and mRNA expression. An important role for COX-2-derived prostanoids in regulation of renin expression and secretion has also been determined by using mice with selective genetic deletion of either the COX-1 or COX-2 gene. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids, suggesting that in the kidney, cortical COX-2 expression is regulated in part by alterations in activity of the renin-angiotensin system.

Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1.

It is well known that nonselective, nonsteroidal anti-inflammatory drugs inhibit renal renin production. Our previous studies indicated that angiotensin-converting enzyme inhibitor (ACEI)-mediated renin increases were absent in rats treated with a cyclooxygenase (COX)-2-selective inhibitor and in COX-2 -/- mice. The current study examined further whether COX-1 is also involved in mediating ACEI-induced renin production. Because renin increases are mediated by cAMP, we also examined whether increased renin is mediated by the prostaglandin E(2) receptor EP(2) subtype, which is coupled to G(s) and increases cAMP. Therefore, we investigated if genetic deletion of COX-1 or EP(2) prevents increased ACEI-induced renin expression. Age- and gender-matched wild-type (+/+) and homozygous null mice (-/-) were administered captopril for 7 days, and plasma and renal renin levels and renal renin mRNA expression were measured. There were no significant differences in the basal level of renal renin activity from plasma or renal tissue in COX-1 +/+ and -/- mice. Captopril administration increased renin equally [plasma renin activity (PRA): +/+ 9.3 +/- 2.2 vs. 50.1 +/- 10.9; -/- 13.7 +/- 1.5 vs. 43.9 +/- 6.6 ng ANG I x ml(-1) x h(-1); renal renin concentration: +/+ 11.8 +/- 1.7 vs. 35.3 +/- 3.9; -/- 13.0 +/- 3.0 vs. 27.8 +/- 2.7 ng ANG I x mg protein(-1) x h(-1); n = 6; P < 0.05 with or without captopril]. ACEI also increased renin mRNA expression (+/+ 2.4 +/- 0.2; -/- 2.1 +/- 0.2 fold control; n = 6-10; P < 0.05). Captopril led to similar increases in EP(2) -/- compared with +/+. The COX-2 inhibitor SC-58236 blocked ACEI-induced elevation in renal renin concentration in EP(2) null mice (+/+ 24.7 +/- 1.7 vs. 9.8 +/- 0.4; -/- 21.1 +/- 3.2 vs. 9.3 +/- 0.4 ng ANG I x mg protein(-1) x h(-1); n = 5) as well as in COX-1 -/- mice (SC-58236-treated PRA: +/+ 7.3 +/- 0.6; -/- 8.0 +/- 0.9 ng ANG I x ml(-1) x h(-1); renal renin: +/+ 9.1 +/- 0.9; -/- 9.6 +/- 0.5 ng ANG I x mg protein(-1) x h(-1); n = 6-7; P < 0.05 compared with no treatment). Immunohistochemical analysis of renin expression confirmed the above results. This study provides definitive evidence that metabolites of COX-2 rather than COX-1 mediate ACEI-induced renin increases. The persistent response in EP(2) nulls suggests involvement of prostaglandin E(2) receptor subtype 4 and/or prostacyclin receptor (IP).

Physiological regulation of cyclooxygenase-2 in the kidney.

In adult mammalian kidney, cyclooxygenase-2 (COX-2) expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb (cTALH) and medullary interstitial cells (MICs). Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. COX-2 expression increases in high-renin states (salt restriction, angiotensin-converting enzyme inhibition, renovascular hypertension), and selective COX-2 inhibitors significantly decrease plasma renin levels, renal renin activity, and mRNA expression. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids. Conversely, nitric oxide generated by neuronal nitric oxide synthase is a positive modulator of COX-2 expression. Decreased extracellular chloride increases COX-2 expression in cultured cTALH, an effect mediated by increased p38 mitogen-activated protein kinase activity, and, in vivo, a sodium-deficient diet increases expression of activated p38 in MD/cTALH. In contrast to COX-2 in MD/cTALH, COX-2 expression increases in MICs in response to a high-salt diet as well as water deprivation. Studies in cultured MICs have confirmed that expression is increased in response to hypertonicity and is mediated, at least in part, by nuclear factor-kappaB activation. COX-2 inhibition leads to apoptosis of MICs in response to hypertonicity in vitro and after water deprivation in vivo. In addition, COX-2 metabolites appear to be important mediators of medullary blood flow and renal salt handling. Therefore, there is increasing evidence that COX-2 is an important physiological mediator of kidney function.

Genetic deletion of COX-2 prevents increased renin expression in response to ACE inhibition.

Cyclooxygenase-2 (COX-2) is expressed in macula densa (MD) and surrounding cortical thick ascending limb of the loop of Henle (cTALH) and is involved in regulation of renin production. We and others have previously found that selective COX-2 inhibitors can inhibit renal renin production (Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, and Harris RC. J Clin Invest 103: 953-961, 1999; Harding P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, and Carretero OA. Hypertension 29: 297-302, 1997; Traynor TR, Smart A, Briggs JP, and Schnermann J. Am J Physiol Renal Physiol 277: F706-F710, 1999; Wang JL, Cheng HF, and Harris RC. Hypertension 34: 96-101, 1999). In the present studies, we utilized mice with genetic deletions of the COX-2 gene in order to investigate further the potential role of COX-2 in mediation of the renin-angiotensin system (RAS). Age-matched wild-type (+/+), heterozygotes (+/-), and homozygous null mice (-/-) were administered the angiotensin-converting enzyme inhibitor (ACEI), captopril, for 7 days. ACEI failed to significantly increase plasma renin activity, renal renin mRNA expression, and renal renin activity in (-/-) mice. ACEI increased the number of cells expressing immunoreactive renin in the (+/+) mice both by inducing more juxtaglomerular cells to express immunoreactive renin and by recruiting additional renin-expressing cells in the more proximal afferent arteriole. In contrast, there was minimal recruitment of renin-expressing cells in the more proximal afferent arteriole of the -/- mice. In summary, these results indicate that ACEI-mediated increases in renal renin production were defective in COX-2 knockout (K/O) mice and provide further indication that MD COX-2 is an important mediator of the renin-angiotensin system.