Clipped Kidneys Research Articles

OXGR1-Dependent (Pro)Renin Receptor Upregulation in Collecting Ducts of the Clipped Kidney Contributes to Na+ Balance in Goldblatt Hypertensive Mice.

The two-kidney, one-clip (2K1C) Goldblatt rodent model elicits a reduction in renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio-ability causes the accumulation of the tricarboxylic cycle intermediary, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, OXGR1 is abundantly expressed in intercalated cells (ICs) of the collecting duct (CD), thus contributing to sodium transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin system (RAS), is a key regulator of sodium reabsorption and blood pressure (BP) that is expressed in ICs. The PRR is upregulated in 2K1C rats. Here, we tested the hypothesis that chronic reduction in RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alters sodium balance and BP in 2K1C mice. To determine the role of OXGR1 in regulating the PRR in the CDs during renovascular hypertension, we performed 2K1C Goldblatt surgery (clip = 0.13 mm internal gap, 14 days) in two groups of male mice: (1) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day); (2) OXGR1-/- knockout mice. Wild-type and sham-operated mice were used as controls. After 14 days, 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p < 0.01) and a lower natriuretic response after the saline challenge test. The CK group showed upregulation of erythropoietin, augmented α-ketoglutarate, and increased PRR expression in the renal medulla. The CK of OXGR1 knockout mice and mice subjected to the OXGR1 antagonist elicited impaired PRR upregulation, attenuated SBP, and better natriuretic responses. In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.

P100 ALPHA KETOGLUTARATE RECEPTOR 1 REGULATES THE EXPRESSION OF THE (PRO)RENIN RECEPTOR IN MEDULLARY RENAL TISSUES, MODULATES SODIUM EXCRETION AND ATTENUATES TWO-KIDNEY, ONE-CLIP GOLDBLATT HYPERTENSION IN MICE

Background: The two-kidney, one-clip (2K1C) Goldblatt model elicits a reduction of renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio ability causes the accumulation of the intermediary of the tricarboxylic cycle, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, the OXGR1 is abundantly expressed in intercalated cells (IC) of the collecting duct (CD), which may contribute to Na+ transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin-aldosterone system is a key regulator of Na+ reabsorption, and blood pressure (BP) and is also expressed in IC. PRR is upregulated in 2K1C. Here, we tested the hypothesis that reduction of RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alterations of Na+ balance and BP in 2K1C mice. Methods: To determine the role of OXGR1 in regulating PRR in the CDs during renovascular hypertension, we used 2K1C Goldblatt surgery (clip= 0.13 mm internal gap) in two groups of male mice: 1) Oxgr1-/- knockout (n=6) mice; and 2) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day, n=6). WT and sham-operated mice were used as controls. Results: 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p&lt;0.01) and a lower natriuretic response after the saline challenge test. The CK showed augmented levels of α-ketoglutarate, erythropoietin mRNA levels, PRR (mRNA and protein abundances) in the renal medulla. Non-CK showed PRR upregulation in the CDs. However, the CK of Oxgr1-/- knockout mice, and from those subjected to OXGR1 antagonism, elicited impaired PRR upregulation, attenuated SBP (Oxgr1-/- 2K1C: 90 ± 8 mmHg, p=non-significant vs. control; Montelukast 2K1C: 95 ± 3 mmHg, p&lt;0.05 vs. control) and better natriuretic responses. Conclusion: In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.

S-07-2: DISCOVERY OF NEUROENDOTHELIAL CELLS AND THEIR ROLE IN HYPERTENSION AND AGING

Objective: Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow and in the development of renal and cardiovascular diseases. Tissue-specific EC dysfunction can contribute to several different diseases including hypertension. Recent transcriptomic studies identified many EC subtypes in multiple organs including the brain and the kidneys, however, their functions are incompletely understood. The present study aimed to explore physiological functional significance and cardiovascular disease and hypertension relevance of a newly discovered minority subtype of scattered ECs expressing neuronal nitric oxide synthase (Nos1). Design and method: A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, mTORgof/lof), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, two-kidney one-clip (2K1C) model of renovascular (Goldblatt) hypertension (RVHT), whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. Results: Our studies identified and characterized, for the first time, a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, therefore we named them neuroendothelial cells (NECs). NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. NECs are found only in the three organs that exhibit the best blood flow autoregulation capacity, with the highest density in the brain&gt;kidney&gt;heart (NEC/EC (%) 8.42+/-0.79, 3.21+/−0.33, 1.73+/−0.07, respectively). NEC density is reduced with aging in the brain and the kidney (NEC/EC (%) 4.675, and 1.850, respectively, p &lt; 0.01, 2.5 years old compared to 2-month-old). The number of NECs increased significantly in the hypo-perfused, hypoxic clipped (CK) kidney, but reduced in the hyperperfused non-clipped (NCK) kidney in RVHT. Intravital multiphoton microscopy (MPM) of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations. Newly established NEC gain-of-function mouse models exhibited increased endothelium-dependent vasodilation and diminished agonist-induced vascular contractility in brain and kidney resistance arterioles compared to controls. In addition, preliminary single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Conclusions: These new vascular anatomy and hemodynamic findings strongly suggest sensory, blood flow and/or baroreceptor functions of NECs as well as a role in the autoregulation of organ blood flow and hypertension pathogenesis.

Antihypertensive Action of Exosomes Released from the Renal Medulla after Unclipping of the Renal Artery of 2 Kidney‐1 Clip Hypertension Mice

The renal medulla has been long known to exert antihypertensive action by releasing different bioactive substances. However, so far it remains unknown what is the identity of such renal medullary antihypertensive substances. In the present study, we hypothesized that exosomes, but not a single molecule released from the renal medulla are importantly implicated in the renal medullary antihypertensive action. To test this hypothesis, we first produced the 2 kidney‐one clip (2K1C) hypertension mouse model by clipping the right renal artery (127.9 ± 1.40 mmHg vs. 94.8 ± 1.75 mmHg of sham mice). After 1 week, we unclipped the right renal artery and monitored the mean arterial blood pressure (MAP) for 1 hour and at the same time collected blood samples for analysis of exosomes. It was found that unclipping the right renal artery of 2K1C mice significanty reduced MAP by 21.2 ± 2.65 mmHg but increased the plasma exosome concentrations by more than 5 folds (before unclipping 2.9 ± 0.7 vs. 3.3±0.5, N.S., after unclipping 4±0.5 vs. 17±5× 108particles/mL, p&lt;0.05). Immunohistochemical (IHC) assessments showed that the clipped kidneys had dramatic increase in exosome markers (CD63 and Annexin II) in the renal medulla of 2K1C mice compared to contralateral kidneys or sham mouse kidneys. These functional and IHC changes in 2K1C mice were almost completely blocked by pretreatment of mice with an exosome biogenesis inhibitor, GW‐4869 (1 mg/kg/day, i.p., n=8). We also found that rapamycin, a compound that increases autophagy and lysosome trafficking (6 mg/kg/day, i.p., n=8) substantially attenuated exosome release from the renal medulla and associated antihypertensive action in 2K1C with unclipping the right renal artery, even though it had some effects to reduce MAP and increase exosome release before the unclipping. In another series of studies using 2K1C hypertensive mice, we extracted and purified exosomes from the blood of mice with unclipping of the right renal artery and then infused these exosomes intravenously to another group of 2K1C mice. It was found that plasma exosomes from 2K1C mice with unlipping of the right renal artery signicantly reduced MAP when they are infused into recipient 2K1C mice (at a concentration of 3 x 108 exosomes) compard to exosomes from sham mice. Urinary flow and sodium excretion of recipient mice also increased slightly by infusion of exosomes from 2K1C mice. These results suggest that exosomes are enriched in the renal medulla of the clipped kidney in 2K1C mice and they can be released into the blood if the renal artery clip is released. These exosomes have strong effects to lower arterial blood pressure.

Genistein alleviates renin-angiotensin system mediated vascular and kidney alterations in renovascular hypertensive rats

Genistein is a bioflavonoid mainly found in soybean. This study evaluated the effect of genistein on vascular dysfunction and kidney damage in two-kidney, one-clipped (2K1C) hypertensive rats. Male Sprague–Dawley-2K1C hypertensive rats were treated with genistein (40 or 80 mg/kg) or losartan 10 mg/kg (n = 8/group). Genistein reduced blood pressure, attenuated the increase in sympathetic nerve-mediated contractile response and endothelial dysfunction in the mesenteric vascular beds and aorta of 2K1C rats. Increases in the intensity of tyrosine hydroxylase (TH) in the mesentery and plasma norepinephrine (NE) were alleviated in the genistein-treated group. Genistein also improved renal dysfunction, hypertrophy of the non-clipped kidney (NCK) and atrophy of the clipped kidney (CK) in 2K1C rats. Upregulation of angiotensin II receptor type I (AT1R), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 4 (Nox4) and Bcl2-associated X protein (BAX) and downregulation of B-cell lymphoma 2 (Bcl2) protein found in CK were restored by genistein. It also suppressed the overexpression of AT1R, transforming growth factor beta I (TGF-β1), smad2/3 and p-smad3 in NCK. Genistein reduced serum angiotensin converting enzyme (ACE) activity and plasma angiotensin II (Ang II) in 2K1C rats. Low levels of catalase activity as well as high levels of superoxide generation and malondialdehyde (MDA) in 2K1C rats were restored by genistein treatment. In conclusion, genistein suppressed renin-angiotensin system-mediated sympathetic activation and oxidative stress in 2K1C rats. It alleviated renal atrophy in CK via modulation of AT1R/NADPH oxidase/Bcl-2/BAX pathways and hypertrophy in NCK via AT1R/TGF-β1/smad-dependent signalling pathways.

Renin-Angiotensin System Induced Secondary Hypertension: The Alteration of Kidney Function and Structure

Long-term hypertension is known as a major risk factor for cardiovascular and chronic kidney disease (CKD). The Renin-angiotensin system (RAS) plays a key role in hypertension pathogenesis. Angiotensin II (Ang II) enhancement in Ang II-dependent hypertension leads to progressive CKD and kidney fibrosis. In the two-kidney one-clip model (2K1C), more renin is synthesized in the principal cells of the collecting duct than juxtaglomerular cells (JGCs). An increase of renal Ang I and Ang II levels and a decrease of renal cortical and medullary Ang 1–7 occur in both kidneys of the 2K1C hypertensive rat model. In addition, the activity of the angiotensin-converting enzyme (ACE) increases, while ACE2's activity decreases in the medullary region of both kidneys in the 2K1C hypertensive model. Also, the renal prolyl carboxypeptidase (PrCP) expression and its activity reduce in the clipped kidneys. The imbalance in the production of renal ACE, ACE2, and PrCP expression causes the progression of renal injury. Intrarenal angiotensinogen (AGT) expression and urine AGT (uAGT) excretion rates in the unclipped kidney are greater than the clipped kidney in the 2K1C hypertensive rat model. The enhancement of Ang II in the clipped kidney is related to renin secretion, while the elevation of intrarenal Ang II in the unclipped kidney is related to stimulation of AGT mRNA and protein in proximal tubule cells by a direct effect of systemic Ang II level. Ang II-dependent hypertension enhances macrophages and T-cell infiltration into the kidney which increases cytokines, and AGT synthesis in proximal tubules is stimulated via cytokines. Accumulation of inflammatory cells in the kidney aggravates hypertension and renal damage. Moreover, Ang II-dependent hypertension alters renal Ang II type 1 & 2 receptors (AT1R & AT2R) and Mas receptor (MasR) expression, and the renal interstitial fluid bradykinin, nitric oxide, and cGMP response to AT1R, AT2R, or BK B2-receptor antagonists. Based on a variety of sources including PubMed, Google Scholar, Scopus, and Science-Direct, in the current review, we will discuss the role of RAS-induced secondary hypertension on the alteration of renal function.

Abstract 17: Genetic Deletion Of Angiotensin Ii AT 1a Receptors Selectively In The Proximal Tubules Of The Kidney Attenuates Two-kidney, One-clip Goldblatt Hypertension In Pt- Agtr1a -/- Mice

The activation of the renin-angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two-kidney, one-clip Goldblatt hypertension (2K1C), but the roles of angiotensin II (Ang II) and AT 1a receptors in the proximal tubules has not been determined previously. The present study tested the hypothesis that genetic deletion of AT 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms. To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.2 mm internal diameter, on the left renal artery for 4 weeks in adult male wild-type (WT), global AT 1a receptor knockout ( Agtr1a -/- ), proximal tubule (PT)-specific Agtr1a -/- (PT- Agtr1a -/- ), or PT- Nhe3 -/- mice, respectively. In WT mice, systolic blood pressure increased in a time-dependent manner reaching a maximal response by Week 3 (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n=12, P &lt;0.01). 2K1C Goldblatt hypertension in WT mice was associated with significant increases in renin mRNA expression in the clipped kidney (Control: 2366 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P &lt;0.01) and decreases in renin mRNA expression in the nonclipped kidney (1738 ± 341 copies/ng RNA, P &lt;0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C Goldblatt hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P &lt;0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney ( P &lt;0.01). In contrast to WT mice, the development of 2K1C hypertension was completely attenuated in Agtr1a -/- (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n=9, n.s .), PT- Agtr1a -/- mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 3 mmHg, n=12, n.s .) and PT- Nhe3 -/- mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n=12, n.s .). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a -/- mice, but it was decreased in the nonclipped kidney of PT- Agtr1a -/- mice ( P &lt;0.05). Taken together, these data suggest that genetic deletion of AT 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms.

Bone marrow-derived mesenchymal stem cells transplantation ameliorates renal injury through anti-fibrotic and anti-inflammatory effects in chronic experimental renovascular disease

BackgroundProgressive renal fibrosis is an underlying pathological process of chronic kidney disease (CKD) evolution. This study aimed to evaluate the roles of bone-marrow-derived mesenchymal stem cells (MSC) in the remodeling of fibrotic kidney parenchyma in the two kidneys-one clip (2K1C) CKD animal model. MethodsWistar rats were allocated into three groups: Sham, 2K1C, and 2K1C þ MSC. MSCs (106) were transplanted into the renal subcapsular region two weeks after clipping the left renal artery. Six weeks after clipping, left kidney samples were analyzed using histological and western blotting techniques. ANOVA tests were performed and differences between groups were considered statistically significant if p < 0.05. ResultsClipped kidneys of 2K1C rats displayed renal fibrosis, with excessive collagen deposition, glomerulosclerosis and renal basement membrane disruption. Clipped kidneys of 2K1C þ MSC rats showed preserved Bowman's capsule and tubular basement membranes, medullary tubules morphological reconstitution and reduced collagen deposits. Expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were elevated, whereas tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 levels were decreased in clipped kidneys of 2K1C rats. MSCs transplantation restored these expression levels. Moreover, MSCs suppressed macrophages and myofibroblasts accumulation, as well as TNF-a expression in clipped kidneys of 2K1C animals. MSCs transplantation significantly increased IL-10 expression. ConclusionsTransplanted MSCs orchestrate anti-fibrotic and anti-inflammatory events, which reverse renal fibrosis and promote renal morphological restoration. This study supports the notion that only one MSCs delivery into the renal subcapsular region represents a possible therapeutic strategy against renal fibrosis for CKD treatment.

Responsiveness of afferent renal nerve units in renovascular hypertension in rats

Previous data suggest that renal afferent nerve activity is increased in hypertension exerting sympathoexcitatory effects. Hence, we wanted to test the hypothesis that in renovascular hypertension, the activity of dorsal root ganglion (DRG) neurons with afferent projections from the kidneys is augmented depending on the degree of intrarenal inflammation. For comparison, a nonhypertensive model of mesangioproliferative nephritis was investigated. Renovascular hypertension (2-kidney, 1-clip [2K1C]) was induced by unilateral clipping of the left renal artery and mesangioproliferative glomerulonephritis (anti-Thy1.1) by IV injection of a 1.75-mg/kg BW OX-7 antibody. Neuronal labeling (dicarbocyanine dye [DiI]) in all rats allowed identification of renal afferent dorsal root ganglion (DRG) neurons. A current clamp was used to characterize neurons as tonic (sustained action potential [AP] firing) or phasic (1–4 AP) upon stimulation by current injection. All kidneys were investigated using standard morphological techniques. DRG neurons exhibited less often tonic response if in vivo axonal input from clipped kidneys was received (30.4% vs. 61.2% control, p < 0.05). However, if the nerves to the left clipped kidneys were cut 7 days prior to investigation, the number of tonic renal neurons completely recovered to well above control levels. Interestingly, electrophysiological properties of neurons that had in vivo axons from the right non-clipped kidneys were not distinguishable from controls. Renal DRG neurons from nephritic rats also showed less often tonic activity upon current injection (43.4% vs. 64.8% control, p < 0.05). Putative sympathoexcitatory and impaired sympathoinhibitory renal afferent nerve fibers probably contribute to increased sympathetic activity in 2K1C hypertension.

Peperomia pellucida (L.) Kunth as an angiotensin-converting enzyme inhibitor in two-kidney, one-clip Goldblatt hypertensive rats

BackgroudPeperomia pellucida (L.) Kunth has been used widely to treat headache, kidney disease, fever, and hypertension. Previous in vitro studies discovered that the flavonoid-rich extract of this plant has potential hypotensive effects, specifically angiotensin-converting enzyme (ACE)-inhibitory activity. However, there is insufficient scientific evidence to validate the result in vivo.PurposeThis study investigated the dose dependencies of the effects of the ethyl acetate fraction of the ethanolic extract of this plant on blood pressure and biomarkers associated with the renin–angiotensin–aldosterone systems (RAAS), such as angiotensin II (AII) and the plasma renin concentration (PRC).Study designIn total, 30 two-kidney, one-clip (2K1C) hypertensive model rats were divided into five groups (n = 6 each): model group, captopril 25 mg/kg BW group, and three different ethyl acetate groups (25, 50, and 100 mg/kg BW). Another six rats comprised the sham group.MethodsRenal hypertensive rats (RHRs) were generated using stainless steel modification clips. Drugs were administered via oral gavage for 2 consecutive weeks. Blood pressure was measured weekly prior to treatment. Blood samples were collected before treatment and after the last dose to measure AII and PRC. The left kidney was isolated for histopathological examination.ResultsBlood pressure, AII levels, and PRC were elevated after 6 weeks in RHRs. Treatment with captopril and the ethyl acetate fraction of P. pellucida (L.) Kunth decreased blood pressure, AII levels, and PRC. The ethyl acetate fraction at a dose of 50 mg/kg BW had similar ACE-inhibitory effects as captopril. Histopathological examination disclosed coagulative necrosis in clipped kidneys. Impairment was alleviated in a dose-dependent manner by P. pellucida (L.) Kunth, similarly as observed in the captopril group.ConclusionP. pellucida (L.) Kunth targets the renin–angiotensin–aldosterone system, which might explain its antihypertensive effects.

Abstract P031: Reduced Augmentation Of The Intrarenal Renin-angiotensin System (ras), Lower Systolic Blood Pressure, And Preserved Renal Function In Female Compared To Male Rats With Unilateral Renal Artery Stenosis

Despite growing evidence of sex differences in the progression of hypertension, there are no guidelines that differentiate treatment between men and women. Intrarenal renin-angiotensin system (RAS) activation and tissue injury in 2-kidney, 1-clip (2K1C) hypertensive rats have been characterized in previous studies of male but not female rats. To evaluate possible sex differences in response to renovascular hypertension, urinary angiotensinogen (uAGT) excretion, systolic blood pressure (BP), urinary protein excretion, and renal function were assessed in female rats.Female (n=8) and male (n=6) rats underwent placement of a 0.2 mm clip on the left renal artery to simulate unilateral renal artery stenosis. BP was measured by tail-cuff plethysmography, and clearance studies were conducted in anesthetized rats to assess renal function. Urine protein concentration was determined by pyrogallol red method. uAGT was measured by ELISA as an index of intrarenal RAS activity. Systolic BP increased from 120±1 to 176±8 mmHg, and urinary protein excretion reached 20.2±5.6 mg/day in female rats. Although uAGT excretion increased from 13.2±7.7 ng/day to 74.1±29.9 ng/day in female rats, male rats had a significantly higher uAGT excretion of 1572.6±750 ng/day. Nonclipped kidneys exhibited more uAGT excretion compared to clipped kidneys, consistent with previous findings in males. Although 2K1C female rats demonstrate significantly lower renal function than sham females, they show more preserved renal function than male rats. Female rats also demonstrate significantly lower increases in systolic BP and urinary protein excretion compared to male rats. The data support substantial sex-dependent differences in renal responses to unilateral renal artery stenosis. The results show substantial increases in systolic BP, uAGT, and urinary protein excretion and decreased renal function after renal artery clipping in females, but the magnitude of the changes is markedly lower than in males. Nonclipped kidneys of both sexes exhibit greater uAGT excretion than clipped kidneys. Notably, females show less augmentation of the intrarenal RAS compared to male rats in renovascular hypertension.

Sex Differences in Urinary Angiotensinogen (uAGT) Excretion, Renal Function, and Systolic Blood Pressure in 2‐Kidney, 1‐Clip Hypertensive Rats

Intrarenal renin‐angiotensin system (RAS) activation and tissue injury in nonclipped kidneys of 2‐kidney, 1‐clip (2K1C) hypertensive rats have been characterized in previous studies of male but not female rats. To evaluate possible sex differences in response to renovascular hypertension, urinary angiotensinogen (uAGT) excretion, arterial blood pressure, urinary protein excretion, and renal function were assessed in 2K1C female rats.Female (n=8) and male (n=6) Sprague Dawley rats underwent placement of a 0.2 mm silver clip on the left renal artery to simulate unilateral renal artery stenosis. BP was measured by tail‐cuff plethysmography, and 24‐hour urine volume and water intake were monitored via metabolic cages over 21 days. After this period, renal clearance studies were conducted in anesthetized rats, and urine was collected from each kidney separately over 30 minute intervals. Urine protein concentration was determined by pyrogallol red method, and uAGT was measured by ELISA as an index of intrarenal RAS activity. Inulin and PAH concentrations were measured by spectrophotometry for calculation of glomerular filtration rate (GFR), renal plasma flow (RPF), and renal blood flow (RBF).21 days after renal artery clipping, systolic BP had risen to 176±8 mmHg from a baseline pressure of 120±1 mmHg, and urinary protein excretion increased to 20±5 mg/day in females. uAGT excretion was significantly increased from the baseline of 13 ng/day to 74 ng/day (507±127% increase). The nonclipped kidney showed 90±29% higher uAGT excretion compared to the clipped kidney, consistent with our previous findings in males. GFR and RPF were higher in nonclipped kidneys than in clipped kidneys but were greater than in corresponding measurements in males. GFR, RPF, and RBF were significantly higher in control rats compared to both clipped and nonclipped kidneys. Male rats showed significantly higher increases in uAGT, BP, and protein excretion.The present study demonstrates that BP, uAGT, and urinary protein excretion substantially increase after renal artery clipping in females, but the magnitude of the changes is markedly lower than in males. Additionally, the nonclipped kidney showed higher uAGT excretion compared to the clipped kidney. Although GFR, RPF, and RBF were significantly lower than control values for both clipped and nonclipped kidneys, these values were higher for females than males. These results demonstrated that female rats may be protected against AngII‐mediated renal injury and hypertension.Support or Funding InformationSupported by the ASPiRE Program at Tulane Univ Sch Medicine, New Orleans, LA and the Warren R. Bourgeois III, MD and Usha Ramadhyani Bourgeois, MD Student Research Endowed Fund at Tulane Univ Sch Medicine, New Orleans, LA

Abstract P420: Sexual Difference of Tubular Renin Angiotensin System (RAS) in 2 Kidney 1 Clip Rats

Background: Premenopausal female manifests lower blood pressure compared with age-matched male. Intrarenal RAS is thought to be an important role in hypertension and hypertensive renal disease, while there is rare information about sexual difference in intrarenal RAS. This study was performed to evaluate sexual difference of tubular RAS in 2 kidney 1 clip(2K1C) rat model. Methods: A 2.5-mm clip was inserted into the left renal artery of male and female Spargue-Dawley rats and they were euthanized at 5 week following the operation. Systolic blood pressure(SBP) was measured via the tail-cuff method at 10 day interval. Medullary area for tubular RAS and cardiac tissue was collected for analysis of local RAS expression. Results: At 30 days after clipping operation, SBP and albuminuria was significantly increased in 2K1C male rats (SBP: male 176.75±5.58 vs. female 106.71±8.68 mmHg, p 0.004, albumin/Cr ratio: male 116.93±33.96 vs. female 56.40±13.94 mg/g, p 0.029). Also, left ventricular hypertrophy was observed only in male rats, however cardiac ACE2 and MasR mRNA did not show sexual difference. The clipped kidney(CK) of 2K1C male rat presented worse glomerulosclerosis and more macrophage infiltration. Renin mRNA was more expressed in female CK, but protein expression was rather decreased in female CK and non-clipped kidney(NCK). 2K1C female rats exhibited highly augmented ACE2 and Mas receptor(MasR) in mRNA and protein, but ACE was augmented in mRNA and reduced in protein. Immunohistochemistry showed that tubular renin and ACE was increased in 2K1C male rats, in contrast ACE2 and MasR were increased in 2K1C female rats. Medullary angiotensin II(AngII) was significantly lower in male CK, but angiotensin 1-7(Ang1-7) was higher in female CK but did not show significance (CK AngII: male 201.88±10.40 vs. female 137.01±19.33 fg/mg p 0.030, CK Ang1-7: male 22.21±4.51 vs. female 57.93±19.59 fg/mg p 0.190). Conclusions: Same insult of renal artery stenosis aroused different clinical outcome in 2K1C male and female. Differently activated tubular RAS might influence difference in clinical manifestation. Superiority of nonclassic tubular RAS in female rats could limit the adverse effect of classic RAS under renovascular hypertension.

Increased angiotensinogen expression, urinary angiotensinogen excretion, and tissue injury in nonclipped kidneys of two-kidney, one-clip hypertensive rats.

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

Sequential activation of the intrarenal renin-angiotensin system in the progression of hypertensive nephropathy in Goldblatt rats.

The intrarenal renin-angiotensin system (RAS) has an important role in generating and maintaining hypertension in two-kidney, one-clip (2K1C) rats. This study evaluated how various intrarenal RAS components contributed to hypertension not only in the maintenance period (5w; 5 wk after operation) but also earlier (2w; 2 wk after operation). We inserted a 2.5-mm clip into the left renal artery of Sprague-Dawley rats and euthanized them at 2w and 5w following the operation. Systolic blood pressure increased within 1 wk after the operation, and left ventricular hypertrophy occurred in 2K1C rats. At 2w, juxtaglomerular apparatus (JGA) and collecting duct (CD) renin increased in clipped kidney (CK) of 2K1C rats. The tubular angiotensin I-converting enzyme (ACE) was not changed, but peritubular ACE2 decreased in nonclipped kidney (NCK) and CK of 2K1C rats. At 5w, ACE and CD renin were enhanced, and ACE2 was still lessened in both kidneys of 2K1C rats. However, plasma renin activity (PRA) was not different from that in sham rats. In proximal tubules of CK, the ANG II type 1 receptor (AT1R) was not suppressed, but the Mas receptor (MasR) was reduced; thus the AT1R/MasR ratio was elevated. Although hypoxic change in CK could not be excluded, the JGA renin of CK and CD renin in both kidneys was highly expressed independent of time. Peritubular ACE2 changed in the earlier period, and uninhibited AT1R in proximal tubules of CK was presented in the maintenance period. In 2K1C rats, attenuated ACE2 seems to contribute to initiating hypertension while upregulated ACE in combination with unsuppressed AT1R may have a key role in maintaining hypertension.

Abstract P021: The Combined Effects of Elevated Intrarenal Ang Ii and Blood Pressure Causes Greater Renal Injury in the Non-clipped Kidneys in 2k1c Rats

In 2-kidney 1-clip (2K1C) hypertension, intrarenal angiotensin II (Ang II) levels are increased in both kidneys, which are often implicated in the further augmentation of the intrarenal renin-angiotensin system and the development of hypertension and kidney injury. We recently reported that angiotensinogen (AGT) expression and secretion are increased only in the non-clipped kidneys (NCK). These findings provide the basis for the hypothesis that elevated Ang II levels, augmented AGT, and high arteriolar pressure in NCK synergistically cause greater kidney injury. Accordingly, we compared the degrees of kidney injury between clipped kidneys (CK) and NCK using a 2K1C model that maintains normal renal blood flow and GFR in the CK. Left kidneys of male rats were clipped for 21 days. Histological and immunohistological analyses were performed on both the CK and NCK. Twenty glomeruli and 20 microscope fields in the cortex and the medulla were examined for each group. The PAS-positive area in the glomeruli was higher in NCK compared with sham (33.9 ± 0.89 vs. 12.4 ± 0.51%) and CK (vs. 15.1 ± 0.58%). Similarly, the Masson's trichrome-positive area in the medullary region was greater in NCK compared with sham (2.21 ± 0.10 vs. 1.32 ± 0.05%) and CK (vs. 1.67 ± 0.10%), but the changes were not observed in the interstitial tissue of the cortex. Immunoreactivity for CD68, a marker of the macrophage and monocyte levels, was higher in NCK compared with sham (0.72 ± 0.07 vs. 0.36 ± 0.04%), but similar to that in CK. In contrast, accumulation of the immune cells in the glomeruli was greater in NCK compared with sham (8.99 ± 0.69 vs. 3.46 ± 0.46%) and CK (vs. 3.08 ± 0.24%). The proliferating cell nuclear antigen levels, a marker of cell proliferation, were greater in NCK (3.49 ± 0.09 %) but not in the CK. Levels of vimentin, a cell transformation and regeneration marker, were also higher in NCK compared with CK and sham. Increased vascular wall thickness (α-SMA) was observed in both kidneys. These results indicate that pathological factors associated with the high blood pressure are required for the development of renal injury in the 2K1C model including glomerular expansion, medullary fibrosis, immune cell infiltration in glomeruli and cell proliferation/transformation.

Evidence that Combined Aliskiren and L‐arginine Treatment Reverses Renovascular Hypertension in an Animal Model

Renovascular hypertension is characterized by increased renal sympathetic activity, angiotensin II, and by endothelial dysfunction. The purpose of this study was to determine the role of renal sympathetic nerve activity (RSNA) in mediating the anti-hypertensive effects of aliskiren (ALSK) and L-arginine (L-ARG) in a rat renovascular hypertension model. Hypertension was induced by clipping the right renal artery, and the following 5 groups were divided: Sham operated; 2-kidney, 1-clip (2K1C); 2K1C plus ALSK; 2K1C plus L-ARG; and 2K1C plus ALSK+ L-ARG. The systolic blood pressure (SBP) of 2K1C rats increased from 114.4±5.2 to 204±12.7 mmHg, (P< 0.05) and was only reduced by ALSK+L-ARG treatment (138.4±4.37 mmHg). The 2K1C hypertension increased the baseline RSNA (SHAM: 62.4±6.39 vs 2K1C: 97.4±8.43%). L-ARG or ALSK+L-ARG treatment significantly decreased baseline RSNA (2K1C L-ARG:70.7±2.39; 2K1C ALSK+L-ARG: 69.3±4.23%), but ALSK treatment alone did not (2K1C ALSK: 84.2±2.5%). Urinary water, Na+, Cl- and urea excretion were similar in the 2K1C L-ARG, 2K1C ALSK+L-ARG and SHAM groups. The combination of ALSK+L-ARG restored urine flow and increased the glomerular filtration rate. The nNOS expression in the non clipped kidney was significantly increased in 2K1C ALSK+L-ARG rats. In conclusion, combined ALSK+L-ARG treatment normalizes SBP and prevents renal dysfunction in 2K1C hypertensive rats, an effect that can be attributed to reduced RSNA.

Combined Aliskiren and L-arginine treatment reverses renovascular hypertension in an animal model.

Renovascular hypertension is characterized by increased renal sympathetic activity, angiotensin II and by endothelial dysfunction. The purpose of this study was to determine the role of renal sympathetic nerve activity (RSNA) in mediating the anti-hypertensive effects of aliskiren (ALSK) and L-arginine (L-ARG) in a rat renovascular hypertension model. Hypertension was induced by clipping the right renal artery, and the following five groups were divided: SHAM operated; 2-kidney, 1-clip (2K1C); 2K1C plus ALSK; 2K1C plus L-ARG; and 2K1C plus ALSK+ L-ARG. The systolic blood pressure (SBP) of 2K1C rats increased from 114.4±5.2 to 204±12.7 mm Hg (P<0.05) and was only reduced by ALSK+L-ARG treatment (138.4±4.37 mm Hg). The 2K1C hypertension increased the baseline RSNA (SHAM: 62.4±6.39 vs. 2K1C: 97.4±8.43%). L-ARG or ALSK+L-ARG treatment significantly decreased baseline RSNA (2K1C L-ARG:70.7±2.39; 2K1C ALSK+L-ARG: 69.3±4.23%), but ALSK treatment alone did not (2K1C ALSK: 84.2±2.5%). Urinary water, Na(+), Cl(-) and urea excretion were similar in the 2K1C L-ARG, 2K1C ALSK+L-ARG and SHAM groups. The combination of ALSK+L-ARG restored urine flow and increased the glomerular filtration rate. The nNOS expression in the non clipped kidney was significantly increased in 2K1C ALSK+L-ARG rats. In conclusion, combined ALSK+L-ARG treatment normalizes SBP and prevents renal dysfunction in 2K1C hypertensive rats.

Loss of prolyl carboxypeptidase in two-kidney, one-clip goldblatt hypertensive mice.

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.

Multiple Mechanisms in Renal Artery Stenosis-Induced Renal Interstitial Fibrosis

Background/Aims: Renal artery stenosis (RAS), which may lead to renal fibrosis, is a common cause of end-stage renal disease in elderly patients. However, the potential mechanisms leading to the development of renal fibrosis and atrophy have not been clarified. Methods: A two-kidney, one-clip Goldblatt mouse model was established in the present study. Blood pressure, morphological and pathological alterations were examined on days 7, 14, and 28 after surgery. Peritubular capillary loss and pericyte changes after injury were evaluated. Inflammatory macrophage infiltration and Wnt/β-catenin signaling were also investigated. Results: A significant increase in blood pressure and obvious renal atrophy were observed on days 7, 14, and 28 after surgery. Following surgery, the clipped kidneys developed aggravated interstitial fibrosis and tubular epithelial injury over time. Moreover, RAS induced obvious peritubular capillary loss and inflammatory macrophage infiltration. Increased pericyte number was found in the clipped kidneys, but these cells detached from the endothelial cells and migrated to the interstitium. Wnt/β-catenin signaling was also significantly upregulated in the clipped kidneys after surgery. Conclusion: Our study provides a novel insight into the mechanisms linking peritubular capillary loss and pericyte changes in RAS-induced renal fibrosis. Our findings also suggest that inflammatory macrophages and Wnt/β-catenin signaling participate in these pathological processes. Therefore, multi-target therapeutic strategies may significantly contribute to the prevention of renal interstitial fibrosis and the preservation of renal function in patients with RAS.