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The intrarenal renin–angiotensin system is critical for the regulation of tubule sodium reabsorption, renal haemodynamics and blood pressure. The excretion of renin in urine can result from its increased filtration, the inhibition of renin reabsorption by megalin in the proximal tubule, or its secretion by the principal cells of the collecting duct. Modest increases in circulating or intrarenal angiotensin II (ANGII) stimulate the synthesis and secretion of angiotensinogen in the proximal tubule, which provides sufficient substrate for collecting duct-derived renin to form angiotensin I (ANGI). In models of ANGII-dependent hypertension, ANGII suppresses plasma renin, suggesting that urinary renin is not likely to be the result of increased filtered load. In the collecting duct, ANGII stimulates the synthesis and secretion of prorenin and renin through the activation of ANGII type 1 receptor (AT1R) expressed primarily by principal cells. The stimulation of collecting duct-derived renin is enhanced by paracrine factors including vasopressin, prostaglandin E2 and bradykinin. Furthermore, binding of prorenin and renin to the prorenin receptor in the collecting duct evokes a number of responses, including the non-proteolytic enzymatic activation of prorenin to produce ANGI from proximal tubule-derived angiotensinogen, which is then converted into ANGII by luminal angiotensin-converting enzyme; stimulation of the epithelial sodium channel (ENaC) in principal cells; and activation of intracellular pathways linked to the upregulation of cyclooxygenase 2 and profibrotic genes. These findings suggest that dysregulation of the renin–angiotensin system in the collecting duct contributes to the development of hypertension by enhancing sodium reabsorption and the progression of kidney injury.

Renal proximal tubular angiotensinogen (AGT) is increased by hyperglycemia (HG) in diabetes mellitus, which augments intrarenal angiotensin II formation, contributing to the development of hypertension and kidney injury. Sodium-glucose cotransporter 2 (SGLT2) is abundantly expressed in proximal tubular cells (PTCs). The present study investigated the effects of canagliflozin (CANA), a SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTCs. Mouse PTCs were treated with 5–25 mM glucose. CANA (0–10 µM) was applied 1 h before glucose treatment. Glucose (10 mM) increased AGT mRNA and protein levels at 12 h (3.06 ± 0.48-fold in protein), and 1 and 10 µM CANA as well as SGLT2 shRNA attenuated the AGT augmentation. CANA did not suppress the elevated AGT levels induced by 25 mM glucose. Increased AGT expression induced by treatment with pyruvate, a glucose metabolite that does not require SGLT2 for uptake, was not attenuated by CANA. In HG-treated PTCs, intracellular reactive oxygen species levels were elevated compared with baseline (4.24 ± 0.23-fold), and these were also inhibited by CANA. Furthermore, tempol, an antioxidant, attenuated AGT upregulation in HG-treated PTCs. HG-induced AGT upregulation was not inhibited by an angiotensin II receptor antagonist, indicating that HG stimulates AGT expression in an angiotensin II-independent manner. These results indicate that enhanced glucose entry via SGLT2 into PTCs elevates intracellular reactive oxygen species generation by stimulation of glycolysis and consequent AGT augmentation. SGLT2 blockade limits HG-induced AGT stimulation, thus reducing the development of kidney injury in diabetes mellitus.

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, We examined the hypothesis that elevated AngII levels during HS intake reduce 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 and TNFR2 receptors. HS +AngII treatment for 4 weeks increased mean blood pressure (MBP) in all strains of mice. However, the increase in MBP in TNFR1KO was greater than that in WT or in TNFR2KO. The increase in urinary angiotensinogen excretion (UAGT) at the 4th week was also greater in TNFR1KO mice than that in WT or in TNFR2KO mice. The results indicate that TNFR1 activity exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

Glomerular arteriolar vasoconstriction and tubulointerstitial injury are observed before glomerular damage occurs in models of hypertension. High interstitial ATP concentrations, caused by the increase in arterial pressure, alter renal mechanisms involved in the long-term control of blood pressure, autoregulation of glomerular filtration rate and blood flow, tubuloglomerular feedback (TGF) responses, and sodium excretion. Elevated ATP concentrations and augmented expression of P2X receptors have been demonstrated under a genetic background or induction of hypertension with vasoconstrictor peptides. In addition to the alterations of the microcirculation in the hypertensive kidney, the vascular actions of elevated intrarenal angiotensin II levels may be mitigated by the administration of broad purinergic P2 antagonists or specific P2Y12, P2X1, and P2X7 receptor antagonists. Furthermore, the prevention of tubulointerstitial infiltration with immunosuppressor compounds reduces the development of salt-sensitive hypertension, indicating that tubulointerstitial inflammation is essential for the development and maintenance of hypertension. Inflammatory cells also express abundant purinergic receptors, and their activation by ATP induces cytokine and growth factor release that in turn contributes to augment tubulointerstitial inflammation. Collectively, the evidence suggests a pathophysiological activation of purinergic P2 receptors in angiotensin-dependent hypertension. Coexistent increases in intrarenal angiotensin II and activates Ang II AT1 receptors, which interacts with over-activated purinergic receptors in a complex manner, suggesting convergence of their post-receptor signaling processes.

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.

Background Elevated advanced glycation end products (AGE) in diabetes mellitus (DM) are implicated in the progression of DM-associated tissue injury, including diabetic nephropathy. The intrarenal renin-angiotensin system, in particular augmentation of angiotensinogen (AGT) in proximal tubular cells (PTC), plays a crucial role in the development of diabetic nephropathy. This study investigated hypothesis that AGE stimulates AGT production in PTC. Materials and Methods Urinary AGT and AGE levels in streptozotocin-induced DM mice were measured by enzyme-linked immunosorbent assays. AGT expression and secretion were evaluated in cultured rat PTC receiving 0-200 µg/ml AGE-BSA treatments for 24 hours. Furthermore, intracellular signaling pathways activated by AGE were elucidated. Results DM mice exhibited greater urinary AGT and AGE levels compared to control mice (AGT: 21.6 ± 5.5 ng/day vs. 190.1 ± 57.8 ng/day, AGE: 139.1 ± 21.6 μg/day vs. 332.8 ± 102.7 μg/day). In cultured PTC, treatment with AGE-BSA enhanced AGT mRNA expression (3.43 ± 0.11-fold compared to control), intracellular AGT protein levels (3.60 ± 0.38-fold), and secreted AGT levels (2.11 ± 0.18-fold). On the other hand, AGT levels were not altered in PTC receiving nonglycated BSA. Recombinant soluble AGE receptor, which competes with endogenous AGE receptor, diminished the AGE-induced AGT upregulation, suggesting that AGE-BSA stimulates AGT expression via activation of the AGE receptor. Enhanced phosphorylation of ERK1/2 and c-Jun, but not p38 MAP kinase, were observed in AGE-BSA-treated PTC. AGE-induced AGT augmentation was attenuated by an ERK inhibitor. Conclusions The findings indicate that AGE enhances proximal tubular AGT expression via ERK1/2, which can exacerbate the development of diabetic related kidney injury.

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.

Angiotensin II (Ang II)-induced hypertension increases afferent and efferent arteriole resistances via the actions of Ang II on the AT1 receptor. In addition to the increased interstitial levels of Ang II, the increased arterial pressure increases interstitial ATP concentrations which act on the purinergic receptors P2X1 and P2X7, to constrict the AA, preventing increases in plasma flow and single nephron GFR (SNGFR). Blockade of the P2 receptors also mitigates the effects of Ang II, thus increasing blood flow and SNGFR, but the resulting increases in mechanical stresses (shear stress and circumferential hoop stress) on the glomerular microvasculature have not been quantified. A mathematical microvascular hemodynamic glomerular model was developed to simulate blood flow and plasma filtration at each of 320 capillary segments in an anatomically-accurate rat glomerular capillary network topology. Afferent and efferent arteriole resistances and network hydraulic conductivity were adjusted to match glomerular hemodynamic data for control, Ang II-induced hypertension and P2X1-blocked conditions (Franco, Martha, et al. Amer. J. Physiology-Renal 313.1 (2017): F9-F19). Ang II infusion increased both afferent and efferent resistances, reducing blood flow while slightly raising glomerular pressure. Blockade of the purinergic receptors reduced both afferent and efferent resistances, maintaining glomerular pressure at hypertensive levels but increasing blood flow significantly, increasing shear stress from 24.9 dynes/cm 2 in hypertensive conditions to 71.3 dynes/cm 2 after purinergic blockade. Because glomerular pressure was maintained, hoop stress barely changed from 69.5 kPa in hypertensive conditions to 70.9 kPa after purinergic blockade. Purinergic blockade also increased hydraulic conductivity and filtering surface area, increasing SNGFR. In hypertension, purinergic stimulation does not prevent the transmission of increased arterial pressure to the glomerular capillaries to reduce capillary hoop stress. However, activation of the purinergic system protects the glomerular microvasculature from increases in shear stress caused by a marked increase in blood flow that would occur in the absence of purinergic stimulation.

In ANG II-dependent hypertension, ANG II activates ANG II type 1 receptors (AT1Rs), elevating blood pressure and increasing renal afferent arteriolar resistance (AAR). The increased arterial pressure augments interstitial ATP concentrations activating purinergic P2X receptors (P2XRs) also increasing AAR. Interestingly, P2X1R and P2X7R inhibition reduces AAR to the normal range, raising the conundrum regarding the apparent disappearance of AT1R influence. To evaluate the interactions between P2XRs and AT1Rs in mediating the increased AAR elicited by chronic ANG II infusions, experiments using the isolated blood perfused juxtamedullary nephron preparation allowed visualization of afferent arteriolar diameters (AAD). Normotensive and ANG II-infused hypertensive rats showed AAD responses to increases in renal perfusion pressure from 100 to 140 mmHg by decreasing AAD by 26 ± 10% and 19 ± 4%. Superfusion with the inhibitor P2X1Ri (NF4490; 1 μM) increased AAD. In normotensive kidneys, superfusion with ANG II (1 nM) decreased AAD by 16 ± 4% and decreased further by 19 ± 5% with an increase in renal perfusion pressure. Treatment with P2X1Ri increased AAD by 30 ± 6% to values higher than those at 100 mmHg plus ANG II. In hypertensive kidneys, the inhibitor AT1Ri (SML1394; 1 μM) increased AAD by 10 ± 7%. In contrast, treatment with P2X1Ri increased AAD by 21 ± 14%; combination with P2X1Ri plus P2X7Ri (A438079; 1 μM) increased AAD further by 25 ± 8%. The results indicate that P2X1R, P2X7R, and AT1R actions converge at receptor or postreceptor signaling pathways, but P2XR exerts a dominant influence abrogating the actions of AT1Rs on AAR in ANG II-dependent hypertension.

During the early phase of ANG II-dependent hypertension, tubular PGE2is increased. Renin synthesis and secretion in the collecting duct (CD) are upregulated by ANG II, contributing to further intratubular ANG II formation. However, what happens first and whether the triggering mechanism is independent of tubular ANG II remain unknown. PGE2stimulates renin synthesis in juxtaglomerular cells via E-prostanoid (EP) receptors through the cAMP/cAMP-responsive element-binding (CREB) pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2in CD cells. The M-1 CD cell line expressed EP1, EP3, and EP4 but not EP2. Dose-response experiments, in the presence of ANG II type 1 receptor blockade with candesartan, demonstrated that 10−6M PGE2maximally increases renin mRNA (approximately 4-fold) and prorenin/renin protein levels (approximately 2-fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10-fold increase). To evaluate further the signaling pathway involved, we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative. Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation, and augmentation of renin. Knockdown of the EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2increases CD renin expression through the EP1 receptor via the PKC/cAMP/CREB pathway. Therefore, we conclude that during the early stages of ANG II-dependent hypertension, there is augmentation of PGE2that stimulates renin in the CD, resulting in increased tubular ANG II formation and further stimulation of renin.

Deleterious effects of purinergic P2X1and P2X7receptors (P2XRs) in ANG II-dependent hypertension include increased renal vascular resistance, and impaired autoregulation and pressure natriuresis. However, their specific effects on the determinants of glomerular hemodynamics remain incompletely delineated. To investigate the P2XR contributions to altered glomerular hemodynamics in hypertension, the effects of acute blockade of P2X1R, P2X7R, and P2X4R with NF449, A438079, and PSB12054, respectively, were evaluated in ANG II-infused rats (435 ng·kg−1·min−1). P2X1R or P2X7R blockade reduced afferent (6.85 ± 1.05 vs. 2.37 ± 0.20 dyn·s−1·cm−5) and efferent (2.85 ± 0.38 vs. 0.99 ± 0.07 dyn·s−1·cm−5) arteriolar resistances, leading to increases in glomerular plasma flow (75.82 ± 5.58 vs. 206.7 ± 16.38 nl/min), ultrafiltration coefficient (0.0198 ± 0.0024 vs. 0.0512 ± 0.0046 nl·min−1·mmHg−1), and single-nephron glomerular filtration rate (22.73 ± 2.02 vs. 51.56 ± 3.87 nl/min) to near normal values. Blockade of P2X4R did not elicit effects in hypertensive rats. In normotensive sham-operated rats, only the P2X1R antagonist caused an increase plasma flow and single-nephron glomerular filtration rate, whereas the P2X4R antagonist induced glomerular vasoconstriction that was consistent with evidence that P2X4R stimulation increases release of nitric oxide from endothelial cells. Mean arterial pressure remained unchanged in both hypertensive and normotensive groups. Western blot analysis showed overexpression of P2X1R, P2X7R, and P2X4R proteins in hypertensive rats. Whereas it has been generally assumed that the altered glomerular vascular resistances in ANG II hypertension are due to AT1receptor-mediated vasoconstriction, these data indicate a predominant P2X1R and P2X7R control of glomerular hemodynamics in ANG II hypertension.

In Ang II dependent hypertension, the increased afferent arteriolar resistance (AAR) is assumed to be primarily due to the increased intrarenal levels of Ang II activating AT1 receptors. Because the renal interstitial ATP levels increase with increased renal perfusion pressure (RPP), purinergic P2X receptors (P2XR) also influence AAR. However, the interactions between the respective receptors has not been fully established. Accordingly, experiments were performed to test the hypothesis that P2XR and AT1R share post-receptor signaling pathways which allow dominance of P2XR to control AAR. Experiments using the in vitro juxtamedullary nephron preparation allowed direct visualization of afferent arteriolar diameters (AAD). Normotensive and Ang II infused hypertensive rats showed AAD responses to increases in RPP from 100 to 140 mmHg by decreasing AAD by 26% ± 10% and 19% ± 4% demonstrating autoregulation. In kidneys from normotensive rats, superfusion at 100 mmHg RPP with 5 μM ATP induced a rapid vasoconstriction from a control of 14.5 ± 0.9 μm to 11.2 ± 0.7 μm (n=8, P < 0.05). P2X1 receptor inhibition with NF449 (1 μM) increased AAD by 28% ± 7% returning it close to control values (14.0 ± 0.8 μm). P2X7 receptor inhibition with A438079 (1 μM) did not significantly alter the AAD (12.2 ± 0.6 μm), but combined treatment with P2X1R plus P2X7R inhibitors fully returned the AAD to control values (14.8 ± 0.7 μm), reflecting a dominant role of P2X1R in mediating ATP afferent vasoconstriction in normotensive kidneys. In Ang II infused hypertensive rats, at RPP 140 mmHg, treatment with AT1R inhibitor by SML1394 (1 μM) increased AAD by 10% ± 7% (13.44 ± 0.73 μm vs. 12.21 ± 0.63 μm, n=10, P

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.

Mechanisms facilitating progression of hypertension via cross stimulation of the renin-angiotensin system (RAS) and inflammation have been proposed. Accordingly, we review and update evidence for regulation of RAS components by pro-inflammatory factors. Angiotensin II (Ang II), which is produced by RAS, induces vasoconstriction and consequent blood pressure elevation. In addition to this direct action, chronically elevated Ang II stimulates several pathophysiological mechanisms including generation of oxidative stress, stimulation of the nervous system, alterations in renal hemodynamics, and activation of the immune system. In particular, an activated immune system has been shown to contribute to the development of hypertension. Recent studies have demonstrated that immune cell-derived pro-inflammatory cytokines regulate RAS components, further accelerating systemic and local Ang II formation. Specifically, regulation of angiotensinogen (AGT) production by pro-inflammatory cytokines in the liver and kidney is proposed as a key mechanism underlying the progression of Ang II-dependent hypertension.

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.

SGLT2 inhibitors lower blood glucose (BG) levels and have the potential to reduce cardiovascular and renal complications of type 2 diabetes mellitus (T2DM). Since BG levels can vary significantly during a treatment regime, hemoglobin A1c is often utilized as a stable indicator of BG status but dynamic measurements of BG may provide greater insights. A blood glucose telemetry systems was used to investigate BG dynamics in canagliflozin (CANA, an SGLT inhibitor)-treated T2DM mice. Male New Zealand Obese mice were fed a high fat diet to induce diabetes. When the mice exhibited a BG of ~350 mg/dl, the mice were treated with vehicle for 5 days followed by 10 mg/kg/day CANA for 5 days by daily oral gavage (single dosing/day). BG levels were monitored continuously via implanted telemetry devices. Chronic treatment (6 weeks) was also performed to investigate blood pressure monitored by a telemetry system and to assess kidney injury. During vehicle treatment for 5 days, BG levels in the mice averaged 336.7 mg/dl. Lower BG levels during early morning compared to night time (active time) were observed during vehicle treatment. BG levels demonstrated a large variation over 24 hours during the vehicle treatment (maximum minus minimum BG: 368.5 mg/dl on day 5). CANA rapidly reduced BG levels within 3 hours following treatment (214.8±25.4 mg/dl), and the lowered BG was sustained until the next dosing. The average 24-hour BG was gradually suppressed during the CANA treatment reaching 170.1 mg/dl on the last day of CANA treatment. Moreover, CANA reduced the variation of BG (maximum minus minimum BG: 214.8 mg/dl on day 5 of CANA treatment). In mice receiving chronic treatment, CANA started lowering systolic blood pressure on week 2 and significant suppression was observed on week 6 (vehicle: 157.9±2.2 vs. CANA: 124.7±7.6 mmHg). The development of kidney injury, especially renal tubular fibrosis and inflammation, was attenuated by CANA. These findings demonstrate that CANA treatment results in rapid and sustained reductions of both BG levels and BG variability which precede the reduction of blood pressure. The temporal dissociation between the lowering of BG and of arterial pressure levels by CANA suggests hyperglycemia-induced factors mediate the development of hypertension in T2DM.

Two important mechanisms regulating afferent arterioles (AA) in angiotensin II (ANG II) hypertension are the purinergic receptors (P2R) and the angiotensin AT1 receptors (AT1R); however, the nature of the interactions between the respective receptors has not been established. Accordingly, studies were performed in rats subjected to 2 weeks of ANG II infusion (80 ng/min via osmotic minipumps) which has been shown to increase interstitial ATP and ANG II concentrations. Experiments using the in vitro isolated juxtamedullary nephron preparation allowed direct visualization of the AA. To determine the interaction between P2XR and AT1R on AA at hypertensive renal perfusion pressure, afferent arteriolar inside diameters (AAD) were measured in response to increases in renal perfusion pressure (RPP) from 100 mmHg to 140 mmHg followed by superfusion with the inhibitors (i). P2X1Ri (NF-449) 1 μM, P2X7Ri (A-438079) 1 μM and P2X1Ri 1 μM plus P2X7Ri 1 μM followed by superfusion with AT1Ri (SML-1394) 1 μM. Increases in RPP to 140 mmHg decreased AAD from 14.75±0.19 μm to 11.69±0.23 μm (n=15, P

Experimental models of hypertension and patients with inappropriately increased renin formation due to a stenotic kidney, arteriosclerotic narrowing of the renal arterioles or a rare juxtaglomerular cell tumor have shown a progressive augmentation of the intrarenal/intratubular renin–angiotensin system (RAS). The increased intrarenal angiotensin II (Ang II) elicits renal vasoconstriction and enhanced tubular sodium reabsorption in proximal and distal nephron segments. The enhanced intrarenal Ang II levels are due to both increased Ang II type 1 (AT1) receptor mediated Ang II uptake and AT1 receptor dependent stimulation of renal angiotensinogen (AGT) mRNA and augmented AGT production. The increased AGT formation and secretion into the proximal tubular lumen leads to local formation of Ang II, which stimulates proximal transporters such as the sodium/hydrogen exchanger. Enhanced AGT production also leads to spillover of AGT into the distal nephron segments as reflected by AGT in the urine, which provides an index of intrarenal RAS activity. There is also increased Ang II concentration in distal nephron with stimulation of distal sodium transport. Increased urinary excretion of AGT has been demonstrated in patients with hypertension, type 1 and type 2 diabetes mellitus, and several types of chronic kidney diseases indicating an upregulation of intrarenal RAS activity.

BACKGROUND Indirect evidence suggests that angiotensin 1-7 (Ang1-7) may counterbalance prohypertensive actions of angiotensin II (AngII), via activation of vascular and/or renal tubular receptors to cause vasodilation and natriuresis/diuresis. We examined if Ang1-7 would attenuate the development of hypertension, renal vasoconstriction, and decreased natriuresis in AngII-infused rats and evaluated the mechanisms involved. METHODS AngII, alone or with Ang1-7, was infused to conscious Sprague-Dawley rats for 13 days and systolic blood pressure (SBP) and renal excretion were repeatedly determined. In anesthetized rats, acute actions of Ang1-7 and effects of blockade of angiotensin AT1 or Mas receptors (candesartan or A-779) were studied. RESULTS Chronic AngII infusion increased SBP from 143 ± 4 to 195 ± 6 mm Hg. With Ang1-7 co-infused, SBP increased from 133 ± 5 to 161 ± 5 mm Hg (increase reduced, P < 0.002); concurrent increases in urine flow (V) and sodium excretion (UNaV) were greater. In anesthetized normotensive or AngII-induced hypertensive rats, Ang1-7 infusion transiently increased mean arterial pressure (MABP), transiently decreased renal blood flow (RBF), and caused increases in UNaV and V. In normotensive rats, candesartan prevented the Ang1-7-induced increases in MABP and UNaV and the decrease in RBF. In anesthetized normotensive, rats intravenous A-779 increased MABP (114 ± 5 to 120 ± 5 mm Hg, P < 0.03) and urine flow. Surprisingly, these changes were not observed with A-779 applied during background Ang1-7 infusion. CONCLUSIONS The results suggest that in AngII-dependent hypertension, Ang1-7 deficit contributes to sodium and fluid retention and thereby to BP elevation; a correction by Ang1-7 infusion seems mediated by AT1 and not Mas receptors.

Serelaxin is a novel recombinant human relaxin-2 that has been investigated for the treatment of acute heart failure. However, its effects on renal function, especially on the renal microcirculation, remain incompletely characterized. Our immunoexpression studies localized RXFP1 receptors on vascular smooth muscle cells and endothelial cells of afferent arterioles and on principal cells of collecting ducts. Clearance experiments were performed in male and female normotensive rats and Ang II-infused male rats. Serelaxin increased mean arterial pressure slightly and significantly increased renal blood flow, urine flow, and sodium excretion rate. Group analysis of all serelaxin infusion experiments showed significant increases in GFR. During infusion with subthreshold levels of Ang II, serelaxin did not alter mean arterial pressure, renal blood flow, GFR, urine flow, or sodium excretion rate. Heart rates were elevated during serelaxin infusion alone (37 ± 5%) and in Ang II-infused rats (14 ± 2%). In studies using the in vitro isolated juxtamedullary nephron preparation, superfusion with serelaxin alone (40 ng/ml) significantly dilated afferent arterioles (10.8 ± 1.2 vs. 13.5 ± 1.1 µm) and efferent arterioles (9.9 ± 0.9 vs. 11.9 ± 1.0 µm). During Ang II superfusion, serelaxin did not alter afferent or efferent arteriolar diameters. During NO synthase inhibition (l-NNA), afferent arterioles also did not show any vasodilation during serelaxin infusion. In conclusion, serelaxin increased overall renal blood flow, urine flow, GFR, and sodium excretion and dilated the afferent and efferent arterioles in control conditions, but these effects were attenuated or prevented in the presence of exogenous Ang II and NO synthase inhibitors.

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.

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 >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.

During the early phase of ANG II-dependent hypertension, tubular PGE2 is increased. Renin synthesis and secretion in the collecting duct (CD) are upregulated by ANG II, contributing to further intratubular ANG II formation. However, what happens first and whether the triggering mechanism is independent of tubular ANG II remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular cells via E-prostanoid (EP) receptors through the cAMP/cAMP-responsive element-binding (CREB) pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. The M-1 CD cell line expressed EP1, EP3, and EP4 but not EP2. Dose-response experiments, in the presence of ANG II type 1 receptor blockade with candesartan, demonstrated that 10−6 M PGE2 maximally increases renin mRNA (approximately 4-fold) and prorenin/renin protein levels (approximately 2-fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10-fold increase). To evaluate further the signaling pathway involved, we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative. Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation, and augmentation of renin. Knockdown of the EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through the EP1 receptor via the PKC/cAMP/CREB pathway. Therefore, we conclude that during the early stages of ANG II-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANG II formation and further stimulation of renin.

Cytochrome P450 1B1 protects against angiotensin II (Ang II)–induced hypertension and associated cardiovascular changes in female mice, most likely via production of 2-methoxyestradiol. This study was conducted to determine whether 2-methoxyestradiol ameliorates Ang II–induced hypertension, renal dysfunction, and end-organ damage in intact Cyp1b1 −/− , ovariectomized female, and Cyp1b1 +/+ male mice. Ang II or vehicle was infused for 2 weeks and administered concurrently with 2-methoxyestradiol. Mice were placed in metabolic cages on day 12 of Ang II infusion for urine collection for 24 hours. 2-Methoxyestradiol reduced Ang II–induced increases in systolic blood pressure, water consumption, urine output, and proteinuria in intact female Cyp1b1 −/− and ovariectomized mice. 2-Methoxyestradiol also reduced Ang II–induced increase in blood pressure, water intake, urine output, and proteinuria in Cyp1b1 +/+ male mice. Treatment with 2-methoxyestradiol attenuated Ang II–induced end-organ damage in intact Cyp1b1 −/− and ovariectomized Cyp1b1 +/+ and Cyp1b1 −/− female mice and Cyp1b1 +/+ male mice. 2-Methoxyestradiol mitigated Ang II–induced increase in urinary excretion of angiotensinogen in intact Cyp1b1 −/− and ovariectomized Cyp1b1 +/+ and Cyp1b1 −/− female mice but not in Cyp1b1 +/+ male mice. The G protein–coupled estrogen receptor 1 antagonist G-15 failed to alter Ang II–induced increases in blood pressure and renal function in Cyp1b1 +/+ female mice. These data suggest that 2-methoxyestradiol reduces Ang II–induced hypertension and associated end-organ damage in intact Cyp1b1 −/− , ovariectomized Cyp1b1 +/+ and Cyp1b1 −/− female mice, and Cyp1b1 +/+ male mice independent of G protein–coupled estrogen receptor 1. Therefore, 2-methoxyestradiol could serve as a therapeutic agent for treating hypertension and associated pathogenesis in postmenopausal females, and in males.

The (pro)renin receptor [(P)RR] upregulates cyclooxygenase-2 (COX-2) in inner medullary collecting duct (IMCD) cells through ERK1/2. Intrarenal COX-2 and (P)RR are upregulated during chronic ANG II infusion. However, the duration of COX-2 and (P)RR upregulation has not been determined. We hypothesized that during the early phase of ANG II-dependent hypertension, membrane-bound (P)RR and COX-2 are augmented in the renal medulla, serving to buffer the hypertensinogenic and vasoconstricting effects of ANG II. In Sprague-Dawley rats infused with ANG II (0.4 μg·min−1·kg−1), systolic blood pressure (BP) increased by day 7 (162 ± 5 vs. 114 ± 10 mmHg) and continued to increase by day 14 (198 ± 15 vs. 115 ± 13 mmHg). Membrane-bound (P)RR was augmented at day 3 coincident with phospho-ERK1/2 levels, COX-2 expression, and PGE2in the renal medulla. In contrast, membrane-bound (P)RR was reduced and COX-2 protein levels were not different from controls by day 14. In cultured IMCD cells, ANG II increased secretion of the soluble (P)RR. In anesthetized rats, COX-2 inhibition decreased the glomerular filtration rate (GFR) and renal blood flow (RBF) during the early phase of ANG II infusion without altering BP. However, at 14 days of ANG II infusions, COX-2 inhibition decreased mean arterial BP (MABP), RBF, and GFR. Thus, during the early phase of ANG II-dependent hypertension, the increased (P)RR and COX-2 expression in the renal medulla may contribute to attenuate the vasoconstrictor effects of ANG II on renal hemodynamics. In contrast, at 14 days the reductions in RBF and GFR caused by COX-2 inhibition paralleled the reduced MABP, suggesting that vasoconstrictor COX-2 metabolites contribute to ANG II hypertension.

While it is clearly recognized that increased intrarenal nitric oxide (NO) levels elicit natriuresis, confounding data showing that systemic nitric oxide synthase inhibition (NOSi) also increases sodium excretion (UNaV) poses a conundrum. This response has been attributed to the associated increases in arterial pressure (AP); however, the increases in AP and in UNaV are temporally dissociated. The changes in regional renal haemodynamics induced by NOSi could also contribute to the alterations of UNaV. To evaluate the roles of AP and non-AP mechanisms mediating the natriuresis, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) was infused i.v. at doses ranging from 5 to 50 μg/kg/min in anaesthetized rats. UNaV, perfusion of the cortex (cortical blood flow, CBF) and medulla (medullary blood flow, MBF) with laser-Doppler flowmetry and glomerular filtration rate (GFR) were measured. UNaV increased from 0.6 ± 0.2 to 1.6 ± 0.1 μmol/kg/min (P < 0.05) with the lower nonpressor doses. With the higher doses, AP increased from 116 ± 4 to 122 ± 4 mmHg and UNaV increased from 1.1 ± 0.3 to 3.3 ± 0.7 μmol/min/g (P < 0.002). UNaV increased similarly in a group where renal AP was maintained at baseline levels. The associated reductions in CBF (17 ± 5 and 38 ± 5 %) and MBF (27 ± 6 and 52 ± 6 %) would be expected to attenuate rather than contribute to the natriuresis. Plasma atrial natriuretic peptide (ANP) concentrations increased significantly following NOSi. Anantin, a natriuretic peptide receptor-A blocker, prevented or reversed the L-NAME-induced natriuresis without altering the L-NAME-induced changes in AP or CBF. The results indicate that increased ANP and related natriuretic peptides mediate the AP-independent natriuresis, at least partly, elicited by systemic L-NAME infusion and help resolve the conundrum of natriuresis during systemic NOSi.

In angiotensin II (ANG II)-dependent hypertension, the augmented intrarenal ANG II constricts the renal microvasculature and stimulates Rho kinase (ROCK), which modulates vascular contractile responses. Rho may also stimulate angiotensinogen (AGT) expression in preglomerular vascular smooth muscle cells (VSMCs), but this has not been established. Therefore, the aims of this study were to determine the direct interactions between Rho and ANG II in regulating AGT and other renin-angiotensin system (RAS) components and to elucidate the roles of the ROCK/NF-κB axis in the ANG II-induced AGT augmentation in primary cultures of preglomerular VSMCs. We first demonstrated that these preglomerular VSMCs express renin, AGT, angiotensin-converting enzyme, and ANG II type 1 (AT1) receptors. Furthermore, incubation with ANG II (100 pmol/l for 24 h) increased AGT mRNA (1.42 ± 0.03, ratio to control) and protein (1.68 ± 0.05, ratio to control) expression levels, intracellular ANG II levels, and NF-κB activity. In contrast, the ANG II treatment did not alter AT1a and AT1b mRNA levels in the cells. Treatment with H-1152 (ROCK inhibitor, 10 nmol/l) and ROCK1 small interfering (si) RNA suppressed the ANG II-induced AGT augmentation and the upregulation and translocalization of p65 into nuclei. Functional studies showed that ROCK exerted a greater influence on afferent arteriole responses to ANG II in rats subjected to chronic ANG II infusions. These results indicate that ROCK is involved in NF-κB activation and the ROCK/NF-κB axis contributes to ANG II-induced AGT upregulation, leading to intracellular ANG II augmentation.

Deleterious effects of purinergic P2X

The inability of mineralocorticoid receptor (MR) blockade to reduce hypertension associated with high angiotensin (Ang) II suggests direct actions of Ang II to regulate tubular sodium reabsorption via the epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron. We used freshly isolated aldosterone-sensitive distal nephron from mice to delineate the synergism and primacy between aldosterone and Ang II in controlling functional ENaC activity. Inhibition of MR specifically prevented the increased number of functionally active ENaC, but not ENaC open probability elicited by a low sodium diet. In contrast, we found no functional role of glucocorticoid receptors in the regulation of ENaC activity by dietary salt intake. Simultaneous inhibition of MR and Ang II type 1 receptors ameliorated the enhanced ENaC activity caused by low dietary salt intake and produced significantly greater natriuresis than either inhibitor alone. Chronic systemic Ang II infusion induced more than 2 times greater increase in ENaC activity than observed during dietary sodium restriction. Importantly, ENaC activity remained greatly above control levels during maximal MR inhibition. We conclude that during variations in dietary salt intake both aldosterone and Ang II contribute complementarily to the regulation of ENaC activity in the aldosterone-sensitive distal nephron. In contrast, in the setting of Ang II–dependent hypertension, ENaC activity is upregulated well above the physiological range and is not effectively suppressed by inhibition of the aldosterone–MR axis. This provides a mechanistic explanation for the resistance to MR inhibition that occurs in hypertensive subjects having elevated intrarenal Ang II levels.

Various models of experimental hypertension and clinical examples of increased renin formation from a stenotic kidney or a juxtaglomerular cell tumor have shown that increased circulating angiotensin II (Ang II) stimulates the intrarenal/intratubular renin–angiotensin system (RAS) that elicits renal vasoconstriction, enhanced tubular sodium reabsorption, and progressive development of hypertension and renal injury. The enhanced intrarenal Ang II activity is due to both receptor-mediated Ang II uptake and Ang II type 1 (AT1) receptor–mediated stimulation of renal angiotensinogen (AGT) mRNA and protein by proximal tubule cells. The increased AGT secretion leads to local formation of Ang II and spillover of AGT into the distal nephron segments as reflected by increased AGT excretion in the urine, which provides an index of intrarenal RAS activity. In clinical studies, increased urinary excretion of AGT has been demonstrated in hypertension, type 1 and type 2 diabetes mellitus, and several types of chronic kidney diseases. In addition, renin secretion from principal cells of the collecting ducts is increased by AT1 receptor activation and acts on AGT from the proximal tubule to form more Ang I. Renin and/or (pro)renin activity is enhanced by binding to the (pro)renin receptor (PRR) on intercalated cells or secreted as soluble PRR contributing further to AGT cleavage, thus making more substrate available for Ang II conversion by local angiotensin-converting enzyme. The augmented intratubular Ang II concentrations together with elevated renal interstitial Ang II concentrations contribute to sustained stimulation of sodium reabsorption, vasoconstriction, development of hypertension, and progressive renal injury and fibrosis.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension.

Diabetes mellitus is one of the most prevalent diseases and is associated with increased incidence of structural and functional derangements in the kidneys, eventually leading to end-stage renal disease in a significant fraction of afflicted individuals. The renoprotective effects of renin-angiotensin system (RAS) blockade have been established; however, the mechanistic pathways have not been fully elucidated. In this review article, the cardinal role of an activated RAS in the pathogenesis of diabetic nephropathy is discussed with a focus on 4 themes: 1) Introduction to RAS cascade, 2) Intrarenal RAS in diabetes, 3) Clinical outcomes of RAS blockade in diabetic nephropathy, and 4) Potential of urinary angiotensinogen (AGT) as an early biomarker of intrarenal RAS status in diabetic nephropathy. This review article provides a mechanistic rational supporting the hypothesis that an activated intrarenal RAS contributes to the pathogenesis of diabetic nephropathy, and that urinary AGT levels provide an index of intrarenal RAS activity.