Intrarenal Angiotensinogen Research Articles

Abstract 044: Stimulation Of Intrarenal Angiotensinogen Expression And The Development Of Hypertension And Kidney Injury In Angiotensin II-infused Hepatic Angiotensinogen Knockout Mice

Regulation of systemic and local angiotensinogen (AGT) levels is a key determinant of tissue angiotensin II (Ang II) levels and inappropriate AGT augmentation promotes the development of hypertension and tissue injury. Kidney and urinary AGT levels are increased in Ang II-mediated hypertension. Recent studies have demonstrated that circulating hepatocyte-derived AGT (hAGT) enters kidneys sustaining kidney and urinary AGT levels. However, roles of hAGT in blood pressure elevation and kidney injury in Ang II-mediated hypertension have not been delineated. This study tested if hAGT contributes to the development of the pathophysiological events in Ang II-infused mice. A low dose of Ang II (400 ng/kg/min) was infused to male wild type (WT) and hAGT gene knockout (KO) mice (N=9 and 13) for 4 weeks. The control group in each genotype received vehicle (Veh) infusion (N=5 and 6). Western blot confirmed non-detectable levels of hAGT in KO mice. hAGT KO markedly decreased plasma AGT levels (WT+Veh:12.2±0.6 vs. hAGT KO+Veh: 0.8±0.1 μg/ml). Ang II infusion did not elevate plasma AGT levels in either WT and hAGT KO mice. Although hAGT KO mice exhibited a lower baseline of systolic blood pressure (SBP) than WT mice, Ang II-mediated increases in SBP was not attenuated in hAGT KO mice (ΔSBP in WT+Ang II: 30.1±4.4 vs. hAGT KO+Ang II: 26.0±4.2 mmHg). Kidney AGT mRNA levels were increased by Ang II infusion to the same extent in both WT and hAGT mice (WT+Ang II: 1.30±0.04 vs. hAGT KO+Ang II: 1.34±0.06, ratio to control). Likewise, Ang II infusion increased IL-6 mRNA to the same magnitude in both WT and hAGT KO mice. Urinary AGT was sustained in hAGT KO+Veh mice (66±9%) compared to WT+Veh mice. Ang II infusion did not alter urinary AGT levels in both groups. Glomerular mesangial expansion and fibrosis by Ang II infusion were not observed. Ang II infusion developed tubulointerstitial fibrosis in renal cortex and medulla. hAGT KO prevented the fibrosis only in the medulla. These outcomes demonstrate that elevation of SBP, augmentation of intrarenal AGT and IL-6 expression, and the development of renal cortical fibrosis in Ang II-mediated hypertension do not require hAGT. In contrast, hAGT contributes to renal medullary fibrosis which may be due to the lower absolute levels of blood pressure.

Hepatic and proximal tubule angiotensinogen play distinct roles in kidney dysfunction, glomerular and tubular injury, and fibrosis progression.

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a major unmet medical need with no effective treatment. Current findings demonstrate that hepatic and proximal tubule angiotensinogen have distinct roles in tubular and glomerular injury, fibrogenesis, and renal dysfunction during CKD development. As renin-angiotensin system components, including angiotensinogen, are important targets for treating CKD in the clinic, the results from our study may be applied to developing better tissue-targeted treatment strategies for CKD and other fibroproliferative diseases.

Immunosuppression by Mycophenolate Mofetil Mitigates Intrarenal Angiotensinogen Augmentation in Angiotensin II-Dependent Hypertension

Augmentation of intrarenal angiotensinogen (AGT) leads to further formation of intrarenal angiotensin II (Ang II) and the development of hypertensive kidney injury. Recent studies demonstrated that macrophages and the enhanced production of pro-inflammatory cytokines can be crucial mediators of renal AGT augmentation in hypertension. Accordingly, this study investigated the effects of immunosuppression by mycophenolate mofetil (MMF) on intrarenal AGT augmentation. Ang II (80 ng/min) was infused with or without daily administration of MMF (50 mg/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7 ± 6.1 mmHg) than the Ang II + MMF group (154.7 ± 2.0 mmHg), but was not statistically different from the Ang II + MMF group. MMF treatment suppressed Ang II-induced renal macrophages and IL-6 elevation. Augmentation of urinary AGT by Ang II infusion was attenuated by MMF treatment (control: 89.3 ± 25.2, Ang II: 1194 ± 305.1, and Ang II + MMF: 389 ± 192.0 ng/day). The augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Elevated intrarenal AGT mRNA and protein levels in Ang II infused rats were also normalized by the MMF treatment (AGT mRNA, Ang II: 2.5 ± 0.2 and Ang II + MMF: 1.5 ± 0.1, ratio to control). Ang II-induced proteinuria, mesangial expansion and renal tubulointerstitial fibrosis were attenuated by MMF. Furthermore, MMF treatment attenuated the augmentation of intrarenal NLRP3 mRNA, a component of inflammasome. These results indicate that stimulated cytokine production in macrophages contributes to intrarenal AGT augmentation in Ang II-dependent hypertension, which leads to the development of kidney injury.

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.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor‐alpha (TNF‐α) levels increase. However, TNF‐α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF‐α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild‐type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail‐cuff plethysmography, and 24‐h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

ENHANCED PRODUCTIONS OF INTRARENAL NLRP-3 INFLAMMASOME AND ANGIOTENSINOGEN IN ANGIOTENSIN II-DEPENDENT HYPERTENSION ARE PREVENTED BY IMMUNOSUPPRESSANT TREATMENT

Objective: Activated inflammasomes enhance maturation of pro-inflammatory cytokines, which facilitates the development of kidney injury. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), one of major subunits in the inflammasome complex, plays a crucial role in innate immunity and inflammation. NLRP3 inflammasome is activated by ATP-P2X7 axis and reactive oxygen species. Expression of pro-NLRP3 is promoted by activated NF-κB by cytokines or PAMPs/DAMPs. In angiotensin II (Ang II)-dependent hypertension, there is augmentation of renal immune cells leading to enhanced production of cytokines and consequent activation of NF-κB. Therefore, we hypothesized that intrarenal NLRP3 levels are increased in Ang II-dependent hypertension in association with the kidney injury, and treatment with mycophenolate mofetil (MMF), an immunosuppressant, prevents the augmentation of NLRP3 and attenuates the consequent progression of kidney injury. Design and method: Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to male rats for 2 weeks. mRNA levels of intrarenal NLRP3 and AIM2, which forms another type of inflammasome complex by viral or bacterial infections, were measured by droplet digital PCR. Furthermore, levels of intrarenal angiotensinogen (AGT), a critical contributor to activation of intrarenal renin-angiotensin system leading to the development of hypertension and associated kidney injury, were evaluated. Histological analyses of kidney injury were also performed. Results: MMF prevented increases in intrarenal macrophage/monocyte and IL-6 levels induced by Ang II infusion, confirming immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 levels (control group: 4.12 ± 1.1 copies/ng RNA vs. Ang II-infused group: 9.96 ± 1.8 copies). The elevated NLRP3 expression was attenuated by MMF (6.24 ± 1.4 copies). In contrast, intrarenal AIM2 levels were not altered by Ang II infusion or MMF. Urinary protein and AGT levels were elevated in Ang II-infused rats, which were prevented by MMF. Histological analyses showed mesangial expansion and tubulointerstitial fibrosis observed in Ang II-infused group, but these injury markers were in normal ranges in the group receiving Ang II + MMF. Conclusions: These results demonstrate activation of the NLRP3 inflammasome in Ang II dependent hypertension and indicate that immunosuppression by MMF mitigates the inflammasome activation, which contributes to attenuation of the kidney injury.

MicroRNA-133a-Dependent Inhibition of Proximal Tubule Angiotensinogen by Renal TNF (Tumor Necrosis Factor).

We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression.

Comparison of the effects of insulin and SGLT2 inhibitor on the Renal Renin-Angiotensin system in type 1 diabetes mice

AimsSGLT2 inhibitors have been proposed as an adjunct to insulin therapy for glycemic control in type 1 diabetes (T1D) patients. However, concern has been raised due to an increase in renin–angiotensin-system (RAS) activity reported in a clinical trial in which an SGLT2 inhibitor was added while insulin dose was reduced in T1D patients. We previously reported that insulin inhibits intrarenal angiotensinogen (Agt) gene transcription and RAS activation. We hypothesized that insulin, rather than SGLT2 inhibition might regulate the intrarenal RAS. MethodsWe compared RAS activity in non-diabetic wild type mice, Akita mice (T1D model) and Akita mice treated with insulin or the SGLT2 inhibitor canagliflozin. ResultsTreatment of Akita mice with insulin or canagliflozin produced similar reductions in blood glucose, whereas insulin, but not canagliflozin, reduced elevated systolic blood pressure. Akita mice exhibited increased renal Agt mRNA/protein expression, which was attenuated by insulin, but not by canagliflozin. Furthermore, insulin was more effective than canagliflozin in lowering kidney weight and albuminuria. ConclusionsInsulin, but not canagliflozin, lowers intrarenal RAS activity in Akita mice. Our findings can be of potential clinical importance, especially for T1D patients who are not on RAS inhibitors at the time of adding SGLT2 inhibitors.

Abstract P2048: Epigenetic Modification By Angiotensin II Mediates Augmentation Of Intrarenal Angiotensinogen Expression

Inappropriately activated intrarenal renin-angiotensin system, including proximal tubular angiotensinogen (AGT) augmentation, contributes to the progression of hypertension and associated kidney injury. It has been demonstrated that interleukin 6 (IL-6) is required for angiotensin II (Ang II)-induced AGT augmentation in renal proximal tubular cells (PTC). However, molecular mechanisms remain unelucidated. Recent studies suggest that Ang II downregulates cardiac class II histone deacetylases (HDAC), which exhibit protective effects against hypertension related tissue injury. Furthermore, HDAC9 (a class II HDAC) has been shown to act as an epigenetic repressor of proximal tubular AGT. Accordingly, the present study investigated the hypothesis that Ang II decreases HDAC9 levels, thereby enabling IL-6 to promote AGT expression in PTC. Ang II (400 ng/kg/min) was chronically perfused to male mice (N=7) for 14 days to investigate the regulation of intrarenal HDAC9. Moreover, cultured human PTC (hPTC) were used to create wild-type, HDAC9 overexpression, and HDAC9 knockdown by shRNA models and were treated with 100 nM Ang II, 10 ng/ml IL-6, or both for 24 hours. Chronic Ang II infusion elevated blood pressure and suppressed renal cortical HDAC9 levels. A decrease in HDAC9 was also observed in Ang II-treated hPTC. Treatment of hPTC with both Ang II and IL-6 increased AGT mRNA levels (1.50±0.10, ratio to the control, N=6), whereas individual treatments with Ang II or IL-6 did not alter AGT expression levels as previously demonstrated. This upregulation of AGT expression by Ang II and IL-6 co-stimulation was attenuated by the overexpression of HDAC9 in hPTC (1.10±0.08, ratio to the control). Importantly, AGT expression was enhanced by IL-6 without Ang II (2.31±0.08 by IL-6 and 2.27±0.15 by Ang II+IL-6, ratio to the control) in cells receiving the HDAC9 gene knockdown, suggesting that HDAC9 down-regulation mediates IL-6-induced AGT upregulation in hPTC. These results indicate that Ang II-induced epigenetic modification, such as the suppression of intrarenal HDAC9, results in increased accessibility of IL-6-derived transcription factors to the AGT promoter and consequent augmentation of intrarenal AGT expression in Ang II-dependent hypertension.

Evidence of Augmented Intrarenal Angiotensinogen Associated With Glomerular Swelling in Gestational Hypertension and Preeclampsia: Clinical Implications.

BackgroundAGT (angiotensinogen) synthesis occurs in renal proximal tubular epithelial cells, independent from systemic AGT, as a component of the intrarenal renin–angiotensin system. We investigated urinary AGT, as a biomarker for renin–angiotensin system activation, and electrolyte concentrations, in relation to glomerular volume, as a proxy for glomerular endotheliosis in renal biopsy tissue from pregnant normotensive control and hypertensive women.Methods and ResultsUrine samples were collected from normotensive control (n=10), gestational hypertensive (n=6), and pre‐eclamptic (n=16) women at the time a renal biopsy was obtained. Samples were collected from Lund University Hospital between November 1999 and June 2001. Urinary AGT, potassium, and sodium were measured, normalized to urinary creatinine. Mean glomerular volume was estimated from biopsy sections. AGT protein expression and localization were assessed in renal biopsies by immunohistochemistry. Urinary AGT concentrations were higher in hypertensive pregnancies (median, gestational hypertension: 11.3 ng/mmol [interquartile range: 2.8–13.6]; preeclampsia: 8.4 ng/mmol [interquartile range: 4.2–29.1]; normotensive control: 0.6 ng/mmol [interquartile range: 0.4–0.8]; P<0.0001) and showed a positive relationship with estimated mean glomerular volume. Urinary potassium strongly correlated with urinary AGT (P<0.0001). Although numbers were small, AGT protein was found in both glomeruli and proximal tubules in normotensive control but was present only in proximal tubules in women with hypertensive pregnancy.ConclusionsThis study shows that pregnant women with gestational hypertension or preeclampsia have increased urinary AGT and potassium excretion associated with signs of glomerular swelling. Our data suggest that the kidneys of women with hypertensive pregnancies and endotheliosis have inappropriate intrarenal renin–angiotensin system activation, which may contribute toward the pathogenesis of hypertension and renal injury.

The protein expression of TNF‐α receptor type 1 (TNFR1) is reduced by nitric oxide (NO) synthase inhibition in cultured renal proximal tubular (HK‐2) cells

Tumor necrosis factor‐alpha (TNF‐α) production, stimulated by chronic high salt (HS) intake, has been implicated in salt sensitive hypertension (SSH) though the mechanism is not yet clearly defined. TNF‐α exerts its biological responses via interaction with two cell surface receptors, TNF‐α receptor type 1 (TNFR1) and type 2 (TNFR2). To determine the roles of TNF‐α receptors during the development of SSH induced by NO deficiency, experiments in our laboratory demonstrated earlier that chronic HS intake alone in mice increases TNFR1 protein expression in renal cortical tissues. However, HS intake in mice chronically treated with a non‐specific pharmacological inhibitor of NOS or in knockout mice lacking endothelial NOS decreases TNFR1 protein expression in the renal cortical tissues. We have also demonstrated that chronic angiotensin induced intrarenal angiotensinogen (AGT) generation is augmented in TNFR1 knockout mice, indicating that TNFR1 activity suppressed AGT formation in the kidney. The present study examines the hypothesis that NO linearly regulates TNFR1 activity in the renal tubular cells in a way that NO deficiency decreases TNFR1 expression. The cultured HK‐2 cells (human kidney‐2 cells; HK‐2 (ATCC® CRL‐2190™) are used in these in‐vitro experiments. These cells were plated at a 125,000 cell per density and grown RPMI medium containing 10% FBS and 1% Penstrep, Non‐essential amino acids, Glutamine, Amphotericin B, Gentimicin at 37°c 5% CO2, 99% relative humidity for 24–48h until they reach confluency. Cells were then split into petry‐dishes and treated with drugs Nitro‐L‐arginine methyl ester(L‐NAME) at 0.5 and 1.0 mM concentrations as well as vehicle for 24 hours, harvested, and lysed. Protein concentration was calculated (BCA assay), and the predetermine volume was loaded in each well of the gel (mini‐Protean TGX gel 456–1096). Blot was treated with TNFα receptor 1 (19139 Abcam) antibody and GAPDH. It was observed that TNFR1 expression significantly decreases to 60 ± 25% (n=3) at 0.5 mM and 35 ± 15 % (n=5) at 1.0 mM conc. of L‐NAME treatment, compared to vehicle treatment in HK‐2 cells. The results show that L‐NAME reduces TNFR1 protein expression in HK‐2 cells after treatment for 24 hours in dose dependent manner. As proximal tubular cells are the prime site for intrarenal AGT generation, these data suggest that such decreases in TNFR1 activity induces enhanced AGT production which is involved in the exaggerated hypertensive responses to HS intake in NO deficient condition.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Canagliflozin Prevents Intrarenal Angiotensinogen Augmentation and Mitigates Kidney Injury and Hypertension in Mouse Model of Type 2 Diabetes Mellitus

Background: Hypertension and renal injury are common complications of type 2 diabetes mellitus (T2DM). Hyperglycemia stimulates renal proximal tubular angiotensinogen (AGT) expression via elevated oxidative stress contributing to the development of high blood pressure and diabetic nephropathy. The sodium glucose cotransporter 2 (SGLT2) in proximal tubules is responsible for the majority of glucose reabsorption by renal tubules. We tested the hypothesis that SGLT2 inhibition with canagliflozin (CANA) prevents intrarenal AGT augmentation and ameliorates kidney injury and hypertension in T2DM. Methods: We induced T2DM in New Zealand obese mice with a high fat diet (DM, 30% fat) with control mice receiving regular fat diet (ND, 4% fat). When DM mice exhibited > 350 mg/dL blood glucose levels, both DM- and ND-fed mice were treated with 10 mg/kg/day CANA or vehicle by oral gavage for 6 weeks. We evaluated intrarenal AGT, blood pressure, and the development of kidney injury. Results: Systolic blood pressure in DM mice (133.9 ± 2.0 mm Hg) was normalized by CANA (113.9 ± 4.0 mm Hg). CANA treatment ameliorated hyperglycemia-associated augmentation of renal AGT mRNA (148 ± 21 copies/ng RNA in DM, and 90 ± 16 copies/ng RNA in DM + CANA) and protein levels as well as elevation of urinary 8-isoprostane levels. Tubular fibrosis in DM mice (3.4 ± 0.9-fold, fibrotic score, ratio to ND) was suppressed by CANA (0.9 ± 0.3-fold). Furthermore, CANA attenuated DM associated increased macrophage infiltration and cell proliferation in kidneys of DM mice. Conclusions: CANA prevents intrarenal AGT upregulation and oxidative stress and which may mitigate high blood pressure, renal tubular fibrosis, and renal inflammation in T2DM.

Abstract P380: Inverse Circadian Pattern in the Renal Generation of Tumor Necrosis Factor-Alpha (TNFå) and Angiotensinogen (AGT) in Salt-Sensitive Hypertension (SSH) Induced by Angiotensin II (AngII) in Mice

Individuals with SSH show a non-dipping blood pressure (BP) during the inactive night period, a phenomenon which is significantly associated with enhanced cardiovascular morbidity and mortality. However, the pathophysiological mechanism for this phenomenon is not yet clearly understood. Chronic AngII administration in mice (reversed circadian rhythm) induced SSH with an increase in intra-renal AGT formation, a response that was exaggerated in TNFα receptor type 1 (TNFR1) knockout mice, indicating that renal AGT formation is suppressed by TNFR1 activation by TNFα. The present study examined the hypothesis that AngII-induced TNFα formation follows a circadian pattern with increases during the active period compared to the inactive period, that facilitates less AGT formation during the active period. Experiments were performed in mice chronically treated with or without AngII (25 ng/min; osmotic mini-pump) + high salt (4% NaCl) intake for 4 weeks that increased BP from 90±2 to 108±3 mmHg (tail-cuff method). Circadian rhythm in urinary parameters was assessed by 12 hour collections of urine using metabolic cages during the active night period (7PM to 7AM) and the inactive day period (7 AM to 7 PM). In normal mice (n=6), urinary excretion of TNFα (uTNFα) was higher (0.5±0.2 vs 0.13±0.08 pg/hour) but urinary excretion of AGT (uAGT) was lower (0.28±0.18 vs 0.46±0.14 ng/hour) during the active period compared to that during the inactive period. This inverse relationship between uTNFα and uAGT was exaggerated in AngII+HS treated mice (n=6). From the baseline (0 week) to the 2nd and 4 th week period, there were marked incremental changes in uTNFα during active periods (0.68±0.20 to 14.2±5.4 and 44.6±14.6 pg/hour) which were less during inactive periods (0.73±0.48 to 10.3±3.3 and 12.1±4.7 pg/hour). Interestingly, uAGT changed minimally during active periods (0.21±0.14 to 0.05±0.04 and 1.42±0.97 ng/hour) but increased significantly during inactive periods (0.32±0.14 to 1.57±0.71 and 3.98±1.67 ng/hour). These data suggest that an increase in TNFα generation suppresses intra-renal AGT formation during active periods but this phenomenon is inversed during inactive periods. The increase in AGT during the inactive period could lead to a non-dipping BP pattern in SSH.

The Relationship between the Intrarenal Dopamine System and Intrarenal Renin-angiotensin System Depending on the Renal Function

Objective The mechanisms underlying the intrarenal renin-angiotensin system (RAS) activation depend on the conditions of kidney diseases. In angiotensin II (AngII) infusion models, the circulating AngII is filtered into the renal tubular lumens, activating intrarenal RAS. However, in the chronic kidney disease (CKD) models, plasma angiotensinogen (AGT) is filtered into the tubular lumens because of glomerular injury, activating intrarenal RAS. The intrarenal dopamine system activation reduces intrarenal AGT expression and suppresses the intrarenal RAS activity in AngII infusion models. However, the relationship between the intrarenal dopamine system and intrarenal RAS has not been elucidated. Therefore, this study was conducted to determine that relationship in CKD patients. Methods We recruited 46 CKD patients (age: 51.1±20.0 years; 16 men; causes of CKD: chronic glomerulonephritis, 34; diabetic nephropathy, 2; nephrosclerosis, 4; and others, 6) not undergoing dialysis or taking RAS blockers. The urinary dopamine (U-DOPA) level, an indicator of intrarenal dopamine activity, and the urinary AGT (U-AGT) level, a surrogate marker of intrarenal RAS activity, were measured. Results As the CKD stages progressed, the U-DOPA levels decreased while the U-AGT levels increased. The U-DOPA levels were significantly and negatively correlated with the U-AGT levels but significantly and positively correlated with the estimated glomerular filtration rate (eGFR). A multiple regression analysis revealed that the U-DOPA levels were associated with the U-AGT levels after adjusting for age, sex, body mass index, and blood pressure (β=-0.38, p=0.045). However, no correlation was observed when eGFR was also adjusted (β=-0.17, p=0.29). Conclusion The negative correlation between the intrarenal dopamine system and intrarenal RAS in CKD patients may be affected by the renal function.

Melatonin ameliorates intrarenal renin-angiotensin system in a 5/6 nephrectomy rat model.

Activation of the intrarenal renin-angiotensin system (RAS) plays a critical role in the pathophysiology of chronic kidney disease (CKD) and hypertension. It has been reported that reactive oxygen species (ROS) are important components of intrarenal RAS activation. Melatonin is recognized as a powerful antioxidant, and we recently reported that impaired nighttime melatonin secretion correlates negatively with urinary angiotensinogen excretion, the surrogate marker of intrarenal RAS activity in patients with CKD. However, whether melatonin supplementation ameliorates the augmentation of intrarenal RAS in CKD has remained unknown. We aimed to clarify whether exogenous melatonin ameliorates intrarenal RAS activation via the reduction of ROS production. 5/6 Nephrectomized (Nx) rats were used as a chronic progressive CKD model and compared with sham-operated control rats. The Nx rats were divided into untreated Nx rats and melatonin-treated Nx rats. The levels of intrarenal RAS, ROS components, and renal injury were evaluated after 4weeks of treatment. Compared with the control rats, the untreated Nx rats exhibited significant increases in intrarenal angiotensinogen, angiotensin II (AngII) type 1 receptors, and AngII, accompanied by elevated blood pressure, higher oxidative stress (8-hydroxy-2'-deoxyguanosine), lower antioxidant (superoxide dismutase) activity, and increased markers of interstitial fibrosis (α-smooth muscle actin, Snail, and type I collagen) in the remnant kidneys. Treatment with melatonin significantly reversed these abnormalities. Antioxidant treatment with melatonin was shown to ameliorate intrarenal RAS activation and renal injury in a 5/6 Nx rat model.

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

BackgroundDuring high sodium intake, the renin‐angiotensin‐aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid‐electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin‐angiotensin‐aldosterone system–epithelial sodium channel axis.Methods and ResultsMale control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin‐converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin‐angiotensin‐aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls.ConclusionsThese data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin‐angiotensin‐aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis.

Abstract P464: Sodium Glucose Cotransporter 2 Inhibition by Canagliflozin Attenuates Intrarenal Angiotensinogen Augmentation in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a complex disease where hyperglycemia occurs as a result of the development of insulin resistance. T2DM leads to complications, including renal and cardiovascular injury. Renal proximal tubular angiotensinogen (AGT) is stimulated by hyperglycemia via elevated oxidative stress, and is associated with activation of the intrarenal renin-angiotensin system which contributes to development of high blood pressure and diabetic nephropathy in diabetes mellitus. Sodium glucose co-transporter 2 (SGLT2) is mainly expressed in early renal proximal tubules and is responsible for most of the glucose reabsorption by the tubules. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on intrarenal AGT regulation in T2DM mice. Male New Zealand Obese mice were fed a regular fat diet (RFD, 4% fat) or a high fat diet (HFD, 40% fat) to induce diabetes. When the mice fed with HFD exhibited &gt;350 mg/dl blood glucose levels (week 0), both RFD and HFD fed mice were treated with 10 mg/kg/day CANA or vehicle for 6 weeks by daily oral gavage. Systolic blood pressure (SBP) levels were measured by a tail cuff system and 24-hour urine samples were collected using metabolic cages. Intrarenal AGT mRNA and protein levels were determined by digital PCR and western blot analysis. CANA treatment decreased blood glucose levels in HFD mice, which remained suppressed for duration of the study. SBP in HFD groups were higher than in the RFD and RFD+CANA groups (134.7±3.6, 118.7±10.8 and 108.3±7.6 mmHg) at week 6. The elevated SBP was normalized by CANA (110.0±6.0 mmHg). The HFD group exhibited greater renal cortical AGT mRNA levels than the RFD group (3.7±0.4 vs. 7.4±1.0 copies/reaction). Likewise, intrarenal AGT mRNA expression was lower in CANA treated group (4.5±0.8 copies/reaction). Western blot analysis also showed lower AGT protein levels in CANA treated group (1.02±0.08-fold compared with RFD) than in HFD group (1.49±0.08-fold). In the HFD group, CANA treatment also suppressed elevated urinary 8-isoprostane levels, a marker of renal oxidative stress. These results demonstrate that CANA attenuates intrarenal AGT augmentation that occurs in T2DM, which may mitigate the development of diabetic nephropathy and progression of high blood pressure.

Abstract P414: Blockade of Sodium Glucose Cotransporter 2 by Canagliflozin Suppresses High Glucose-induced Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Intrarenal angiotensinogen (AGT) is mainly expressed in proximal tubular cells (PTC). AGT is increased by hyperglycemia (HG) in type 1 and 2 diabetes mellitus, which causes elevated intrarenal angiotensin formation contributing to the development of hypertension and kidney injury. Sodium glucose co-transporter 2 (SGLT2) is abundantly expressed in early PTC and may promote increased intrarenal AGT by increasing intracellular glucose levels. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTC. Mouse PTC were treated with 5, 10 or 25 mM glucose. 0-10 μM CANA was applied one hour before glucose treatment. AGT mRNA and protein levels were measured by digital PCR and western blot analysis. Levels of intracellular reactive oxygen species (ROS) were determined with H 2 DCF-DA. Tempol, an antioxidant, was used to test if elevated ROS is involved in HG-induced AGT upregulation. 10 mM glucose increased AGT protein levels at 12 hours (3.06±0.48-fold compared with 5 mM glucose) and treatment with 10 μM CANA attenuated the AGT augmentation (1.68±0.05-fold). AGT protein levels were also increased by 25 mM glucose; but CANA did not suppress the AGT levels caused by this glucose concentration. In PTC treated with 10 mM glucose for 12 hours, the suppressing effect on AGT upregulation was observed with 1 and 10 μM CANA. Lower concentrations of CANA (0.01 and 0.1 μM) did not lower AGT protein levels significantly. Elevated AGT mRNA expression by glucose was also attenuated by CANA. Treatment of PTC with 1 mM pyruvate also increased AGT expression levels, indicating that glycolysis is involved in HG-induced AGT upregulation. After incubation of PTC with 10 mM glucose for 12 hours, intracellular ROS levels were elevated compared to baseline (4.24±0.23-fold) and these were also inhibited by CANA (0.2±0.08-fold). Furthermore, tempol attenuated AGT upregulation in HG-treated PTC. These results indicate that enhanced glucose entry via SGLT2 into PTC elevates intracellular ROS generation by stimulation of glycolysis and consequent AGT augmentation. Thus, SGLT2 inhibition by CANA may limit HG-induced AGT stimulation which suppress intrarenal angiotensin formation and reduce kidney injury in diabetes mellitus.

HDAC9 is an epigenetic repressor of kidney angiotensinogen establishing a sex difference

BackgroundSexual difference has been shown in the pathogenesis of chronic kidney disease induced by hypertension. Females are protected from hypertension and related end-organ damage. Augmentation of renal proximal tubular angiotensinogen (AGT) expression can promote intrarenal angiotensin formation and the development of associated hypertension and kidney injury. Female rodents exhibit lower intrarenal AGT levels than males under normal conditions, suggesting that the suppressed intrarenal AGT production by programmed mechanisms in females may provide protection from these diseases. This study was performed to examine whether epigenetic mechanisms serve as repressors of AGT.MethodsMale and female Sprague Dawley rats were used to investigate sex differences of systemic, hepatic, and intrarenal AGT levels. All histone deacetylase (HDAC) mRNA levels in the kidneys were determined using a PCR array. HDAC9 protein expression in the kidneys and cultured renal proximal tubular cells (PTC) was analyzed by Western blot analysis and immunohistochemistry. The effects of HDAC9 on AGT expression were evaluated by using an inhibitor and siRNA. ChIP assay was performed to investigate the interaction between the AGT promoter and HDAC9.ResultsPlasma and liver AGT levels did not show differences between male and female Sprague-Dawley rats. In contrast, females exhibited lower AGT levels than males in the renal cortex and urine. In the absence of supplemented sex hormones, primary cultured renal cortical cells isolated from female rats sustained lower AGT levels than those from males, suggesting that the kidneys have a unique mechanism of AGT regulation controlled by epigenetic factors rather than sex hormones. HDAC9 mRNA and protein levels were higher in the renal cortex of female rats versus male rats (7.09 ± 0.88, ratio to male) while other HDACs did not exhibit a sex difference. HDAC9 expression was localized in PTC which are the primary source of intrarenal AGT. Importantly, HDAC9 knockdown augmented AGT mRNA (1.92 ± 0.35-fold) and protein (2.25 ± 0.50-fold) levels, similar to an HDAC9 inhibitor. Furthermore, an interaction between HDAC9 and a distal 5’ flanking region of AGT via a histone complex containing H3 and H4 was demonstrated.ConclusionsThese results indicate that HDAC9 is a novel suppressing factor involved in AGT regulation in PTC, leading to low levels of intrarenal AGT in females. These findings will help to delineate mechanisms underlying sex differences in the development of hypertension and renin-angiotensin system (RAS) associated kidney injury.

Abstract 015: Immunosuppression Attenuates Intrarenal Angiotensinogen Augmentation in Angiotensin II Dependent Hypertension

Augmented intrarenal angiotensinogen (AGT) is a critical contributor to activation of intrarenal renin-angiotensin system (RAS) leading to the development of hypertension and associated kidney injury. It has been shown that treatment with mycophenolate mofetil (MMF), an immunosuppressive drug, mitigates the increased intrarenal angiotensin (Ang) II levels and blood pressure in hypertensive animal models, suggesting that an activated immune system mediates intrarenal RAS activation and consequent hypertension. Associated macrophage (MΦ) infiltration augments pro-inflammatory cytokine levels including interleukin-6 (IL-6), which plays a crucial role in augmentation of AGT expression in cultured renal proximal tubular cells. Accordingly, this study was performed to establish pathophysiological relevance for the effects of stimulated MΦ and IL-6 on intrarenal AGT augmentation in Ang II-dependent hypertension. Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7±6.1 mmHg) than MAP in Ang II+MMF group (154.7±2.0 mmHg) which was not statistically different than in control group. The augmentation of urinary AGT and urinary protein by Ang II infusion was attenuated by MMF treatment (AGT, control: 89.3±25.2, Ang II: 1,194±305.1, and Ang II+MMF: 389±192.0 ng/day). Importantly, the augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Urinary 8-isoprostane levels were not altered by Ang II and/or MMF during the 2-week treatments. MMF treatment suppressed Ang II-induced renal MΦ infiltration and IL-6 elevation (IL-6 mRNA, Ang II: 32.4±7.5 and Ang II+MMF: 3.6±1.7, ratio to control). qRT-PCR, western blot and immunohistochemistry revealed elevated intrarenal AGT mRNA and protein levels in Ang II infused rats which were normalized by the MMF treatment (AGT mRNA, Ang II: 2.5±0.2 and Ang II+MMF: 1.5±0.1, ratio to control). These results indicate that stimulated IL-6 production in infiltrated MΦ contributes to intrarenal AGT augmentation in early stages of Ang II-dependent hypertension, which contributes to the development of kidney injury.