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application/pdf, Owing to aging populations, the prevalence of hypertension and associated cardiovascular events has been increasing worldwide. The morbidity and mortality due to cancer have also been increasing with aging populations. Several small-molecule inhibitors have been used in cancer therapy, which have a positive impact on the prognosis and survival of patients with cancer. Consequently, the number of cancer survivors with hypertension has been rapidly increasing. Anticancer therapy, including vascular endothelial growth factor inhibitors, increases blood pressure. However, both clinical and laboratory evidence are lacking regarding optimal blood pressure control in patients with hypertension with cancer. Here, we propose the concept of onco-hypertension, which is an evolving subspecialty focused on the complex pathophysiology of hypertension and cancer. In this review, we highlight blood pressure changes in cancer, hypertension induced by anticancer therapy, and optimal blood pressure management in patients with hypertension with cancer. In addition, we discuss needed studies to further establish this new onco-hypertension concept.

Background: The number of cancer survivors coincides with cardiovascular diseases is increasing, therefore, we are promoting the concept of “Onco-Hypertension” to clarify the mechanism linking cancer and blood pressure. In this study, we evaluated body osmolyte and water imbalance in hepatocellular cancer (HCC) model rats. Methods: Wistar rats were administered diethylnitrosamine (DEN) (1.5 μg/day, p.o.), a carcinogenic drug, for 8 weeks to establish liver cancer. Three weeks after the completion of DEN administration, we investigated blood pressure, tissue osmolyte and water content, and its association with aldosterone secretion. Results: HCC rats significantly reduced blood pressure, skin sodium, potassium, and water content. In the carcass (muscle + bone), dry weight, sodium, potassium, and water content were dramatically reduced without changing bone mass in HCC rats, suggesting that HCC causes muscle wasting to supply osmolyte and water for the dehydrated organs. These osmolytes and water loss were significantly associated with increased urinary aldosterone excretion. Supplementation of 0.25% salt water to drink improved body sodium and water loss and muscle wasting in HCC rats, which were completely suppressed by treatment with spironolactone (75 mg/kg/day, p.o.), a mineralocorticoid receptor (MR) blocker. Conclusion: These findings suggest that HCC causes body osmolyte and water loss, which leads to aldosterone hypersecretion and muscle catabolism to compensate for dehydration. A relatively small amount of salt supplement ameliorates the HCC-induced dehydration and muscle wasting via aldosterone/MR-mediated sodium and water restoration.

We have previously shown that podocyte injury increases the glomerular filtration of liver-derived angiotensinogen (Agt) and the generation of intrarenal angiotensin (Ang) II and that the filtered Agt is reabsorbed by proximal tubules in a manner dependent on megalin. In the present study, we aimed to study the role of megalin in the generation of renal Ang II and sodium handling during nephrotic syndrome. We generated proximal tubule-specific megalin knockout (KO) mice and crossed these animals with NEP25 mice, in which podocyte-specific injury can be induced by injection of the immunotoxin LMB2. Without podocyte injury, renal Agt staining was markedly diminished and urinary Agt increased in KO mice. However, renal Ang II was similar between KO and control mice on average: 117 (95% CI 101–134) vs. 101 (68–133) fmol/g tissue. We next tested the effect of megalin KO on intrarenal Ang II generation with podocyte injury. Control NEP25 mice showed markedly increased renal Agt staining and renal Ang II levels: 450 (336–565) fmol/g tissue. Megalin KO/NEP25 mice showed markedly diminished Agt reabsorption and attenuated renal Ang II: 199 (156–242) fmol/g tissue (p < 0.001). Compared with control NEP25 mice, megalin KO/NEP25 mice excreted 5-fold more sodium in the urine. Western blot analysis showed that megalin KO decreased NHE3 and the cleaved α and γ forms of ENaC. These data indicate that Agt reabsorbed by proximal tubules via megalin in nephrotic syndrome is converted to Ang II, which may contribute to sodium retention and edema formation by activating NHE3 and ENaC. SUMMARY: This study shows that in nephrotic syndrome, plasma angiotensinogen leaked into the renal tubular lumen is reabsorbed by proximal tubules via megalin and converted to angiotensin II, which may contribute to sodium retention and edema formation by activating NHE3 and ENaC.

Renal expression of klotho is reduced in hypertension. Experiments were performed to examine whether exogenous klotho protein supplementation ameliorates pressure natriuresis in early phase of hypertension, using stroke-prone spontaneously hypertensive rats (sp-SHR). The interactions between klotho protein and renal renin-Ang (angiotensin) system were examined with immunoprecipitation and cell culture methods. Uninephrectomy was performed in sp-SHRs to induce nephrosclerosis, and they were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to sp-SHR decreased blood pressure, renal Ang II levels, AGT (angiotensinogen) expression, HIF (hypoxia-inducible factor)-1α abundance, and medullary fibronectin levels, with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt, and mTOR (mammalian target of rapamycin) abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and enhanced pressure-induced natriuresis in sp-SHR. Klotho protein bound to AT1R (Ang II type-1 receptor) and decreased the presence of AT1R on HK-2 (human proximal tubular) cells, attenuating inositol triphosphate generation. Klotho protein suppressed Ang II–induced increments of AGT expression in HK-2 cells. Collectively, the present data demonstrate that klotho binds with the AT1R to suppress Ang signal transduction, participating in inactivating renal renin-Ang system. Our results also suggest that exogenous klotho supplementation represses Akt-mTOR signaling to reduce renal hypertrophy and restore the autoregulatory ability of glomerular filtration rate in uninephrectomized sp-SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway and medullary fibrosis, contributing to enhancements of pressure natriuresis and reduction in blood pressure.

Introduction: We recently reported that kidneys accumulate urea as an alternative osmolyte in the renal medulla to concentrate sodium into the urine during high salt intake. This urea-driven water conservation process is coupled with the energy-intensive nature of hepatic urea production, which leads to a catabolic state in mice fed high salt. In this study, we examined the effects of renal denervation on the salt-driven catabolic state since previous studies found that the renal sympathetic nervous system regulates urinary sodium excretion and energy metabolism of some organs such as liver and muscle. Methods: Sham-operated (sham) or renal denervated (RDX) male C57/B6J mice were fed on 0.3% NaCl diet with tap water (NS) or 4% NaCl high salt diet + 0.9% NaCl water to drink (HS) for 4 consecutive weeks (ad libitum), followed by 2 weeks of pair-feeding to match energy intake in all groups. We measured daily food intake and body weight, 24 hours urinary sodium excretion, body sodium content, and activity of liver arginase, a urea producing enzyme. Results: NS + sham and HS + sham mice showed similar body weight gain (NS + sham: +3.6 ± 0.2, HS + sham: +3.4 ± 0.2 g) during ad libitum feeding although HS significantly increased food intake (NS + sham: 3.1 ± 0.04, HS + sham: 3.3 ± 0.04 g/day). After the pair-feeding, HS mice significantly decreased their body weight (NS + sham: +0.9 ± 0.07, HS + sham: -0.6 ± 0.05 g) and increased liver arginase activity (NS + sham: 3792 ± 322, HS + sham: 5412 ± 499 units/L/tissue), confirming the high salt-induced catabolic hepatic urea production. On the other hand, HS did not increase food intake (NS + RDX: 3.2 ± 0.1, HS + RDX: 3.1 ± 0.01 g/day) and liver arginase activity (NS + RDX: 3599 ± 316, HS + RDX: 3363 ± 369 units/L/tissue) in RDX group. In addition, HS + RDX mice did not show pair feeding-induced body weight loss (NS + RDX: +0.6 ± 0.3, HS + RDX: +0.3 ± 0.3 g). HS + RDX mice did not alter 24 hours urinary sodium excretion (HS + sham: 135 ± 6.3, HS + RDX: 142 ± 13 mmol/d/kg) and body sodium content (HS + sham: 0.15 ± 0.003, HS + RDX: 0.15 ± 0.006 mmol/d/kg) compared with HS + sham mice. Conclusion: These findings suggest that renal denervation attenuates the HS-induced body weight loss and catabolic urea production independently of urinary sodium excretion.

A 5-10% reduction of body mass by caloric restriction (CR) has been effective in normalizing SBP in hypertensive patients. Moreover, SGLT2 inhibitors lowered SBP in spontaneous hypertensive rats and are considered a therapeutic alternative to patients with T2DM concomitant with hypertension. However, these potential benefits have not been fully addressed in a model of metabolic syndrome, which is characterized by multiple factors. Lean LETO and obese, insulin resistant OLETF rats from 2 independent studies were divided in the following groups (n=7): 1) ad lib LETO, 2) ad lib OLETF, 3) LETO 50% CR (10 d at 15 wks of age), 4) OLETF 50% CR, 5) LETO 30% CR (14 d at 12 wks of age), 6) OLETF 30% CR, 7) OLETF SGLT2i (Luseoglifozin 10 mg/kg/day x 4 wks at 10 wks of age), and 8) OLETF SGLT2i + 30% CR. SBP was measured weekly by tail-cuff plethysmography.SBP increased 19% (23 mmHg, P =0.002) consistently in OLETF compared to LETO (124 mmHg), but neither the CR-induced reduction in body mass nor the supplementation of SGLT2i modified the strain-associated increase in SBP (Fig. 1). Mean body mass in OLETF decreased by 18% with 50% CR (409 to 334 g) but decreased only 7% (34 g) with 30% CR and 8% (38 g) with SGLT2i and CR (454 g control). Serum Na + remained unaltered by 50% CR. Urinary Na+ excretion also was unchanged in 30% CR. While the CR treatments induced profound changes in body mass (and adiposity, not shown), they were not sufficient to alter Na+ balance and SBP. Furthermore, the addition of SGLT2i did not reduce SBP suggesting that in the OLETF model of metabolic syndrome factors beyond adiposity and impaired glucose regulation contribute to the maintenance of elevated arterial pressure.

We previously reportedthat silencing of the PRR gene, which encodes the(pro)renin receptor ((P)RR) significantly reduced Wnt/β-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking monoclonal antibodies (mAbs) directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro andin vivo. We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0/G1 phase of the cell cycle and significantly reduced proliferation in vitroconcomitant with a significant reduction in the expression of active β-catenin and cyclin D1. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active β-catenin and the proliferation marker Ki-67 and reduced tumor growth. In contreffects ast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro orin vivo, indicating that the effects of the anti-(P)RR mAbs was independent of the renin-angiotensin system. These data indicatethat mAbs against human (P)RR can suppress PDAC cells proliferation by hindering activation of the Wnt/β-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.

Introduction: We have recently reported that high salt intake induces the energy-intensive nature of hepatic urea osmolyte production that accompanied the decrease in heart rate (HR). These findings suggest that high salt intake decreases sympathetic nerve activity to reduce cardiovascular energy expenditure for the hepatic urea production. Therefore, we examined the effects of high salt intake on renal sympathetic nerve activity (RSNA) by using a radiotelemetry system in rats. Methods: We inserted the radiotelemetry system into male Sprague Dawley (SD) rats for recording RSNA and HR. After 2 weeks recovery, we fed the rats 0.3% NaCl diet (NS) with tap water to drink (NS + tap), 4% NaCl diet with tap water (HS + tap), and 4% NaCl diet with saline (HS + saline) for 4 days in this order, and kept monitoring their HR and RSNA. In the separate experiment, we also fed male SD rats NS + tap or HS + saline for 6 consecutive weeks to confirm a catabolic state in HS + saline rats. Results: Rats exhibited marked reductions in RSNA and HR during HS + saline phase compared with other phases. HS + saline rats significantly decreased body weight (369±35 g) compared with NS + tap rats (405±48 g) despite of the similar daily food intake (18.5±1.3 vs. 19.2±1.6 g/day). And HS + saline rats increased liver arginase, a urea producing enzyme, activity (1561±121 units/L/mg) compared with NS + tap rats (1337±131 units/L/mg) at 6 weeks after the feeding. Conclusion: High salt intake decreased RSNA accompanied by the catabolic urea production in rats. These findings suggest that the sympathetic nervous system is one of the key regulators to reduce cardiovascular energy expenditure, which could support the energy-intensive nature of hepatic urea production in high salt-fed rats.

Recent data indicate that klotho interacts with the renin-angiotensin system (RAS). However, the effects of klotho protein supplementation on hypertensive renal injury have not been examined yet. First, the in vivo experiments were performed using spontaneously hypertensive rats (SHR). To elucidate in vivo observations, the in vitro studies were carried out to examine the interactions between klotho protein and angiotensin type-1 (AT1) receptor with immunoprecipitation and cell culture (HK-2 cell) methods. Uninephrectomized SHRs were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to uninephrectomized SHRs decreased blood pressure, renal angiotensin II levels, angiotensinogen expression, HIF-1α abundance, and medullary fibronectin with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt and mTOR abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and renal plasma flow, and improved pressure-induced natriuresis in SHR. Klotho protein bound to AT1 receptors without affecting the bindings of angiotensin II to AT1 receptor. Klotho decreased the presence of AT1 receptors on HK-2 cells, attenuating inositol triphosphate generation. Angiotensin II elevated angiotensinogen expression in HK-2 cells, and klotho protein suppressed angiotensin II-induced increases in angiotensinogen expression. Of note, although angiotensin II improves glomerular autoregulatory ability especially at lower pressure range, angiotensin II impairs pressure-induced natriuresis. Collectively, the present data demonstrate that klotho binds with the AT1 receptors to suppress angiotensin signal transduction and inactivate the renal RAS. In addition, our results suggest that exogenous klotho supplementation suppresses Akt-mTOR signaling to decline renal hypertrophy and restores renal autoregulatory ability in uninephrectomized SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway to suppress medullary fibrosis, presumably involved in improvements of pressure-natriuresis and reduction in blood pressure.

Inappropriate activation of the intrarenal renin–angiotensin system induces generation of reactive oxygen species and tubulointerstitial inflammation, which contribute to salt-sensitive hypertension (SSHT). Liver-type fatty acid–binding protein is expressed in proximal tubules in humans, but not in rodents, and may play an endogenous antioxidative role. The objective of the present study was to examine the antioxidative effect of liver-type fatty acid–binding protein on post–angiotensin II SSHT model in transgenic mice with selective overexpression of human liver-type fatty acid–binding protein in the proximal tubules. The transgenic mice showed marked protection against angiotensin II–induced SSHT. Overexpression of tubular liver-type fatty acid–binding protein prevented intrarenal T-cell infiltration and also reduced reactive oxygen species generation, intrarenal renin–angiotensin system activation, and monocyte chemotactic protein-1 expression. We also performed an in vitro study using the murine proximal tubular cell lines with or without recombinant liver-type fatty acid–binding protein and murine proximal tubular cell lines transfected with human liver-type fatty acid–binding protein, and found that gene transfection of liver-type fatty acid–binding protein and, in part, recombinant liver-type fatty acid–binding protein administration had significantly attenuated angiotensin II–induced reactive oxygen species generation and the expression of angiotensinogen and monocyte chemotactic protein-1 in murine proximal tubular cell lines. These findings indicated that liver-type fatty acid–binding protein in the proximal tubules may protect against angiotensin II–induced SSHT by attenuating activation of the intrarenal renin–angiotensin system and reducing oxidative stress and tubulointerstitial inflammation. Present data suggest that liver-type fatty acid–binding protein in the proximal tubules may be a novel therapeutic target for SSHT.

We demonstrated previously that the blood pressure of patients with IgA nephropathy becomes salt sensitive as renal damage progresses. We also showed that increased urinary angiotensinogen levels in such patients closely correlate with augmented renal tissue angiotensinogen gene expression and angiotensin II levels. Here, we investigated the relationship between urinary angiotensinogen and salt sensitivity of blood pressure in patients with IgA nephropathy. Forty-one patients with IgA nephropathy consumed an ordinary salt diet (12 g/d of NaCl) for 1 week and a low-salt diet (5 g/d of NaCl) for 1 week in random order. The salt-sensitivity index was calculated as the reciprocal of the slope of the pressure-natriuresis curve drawn by linking 2 data points obtained during consumption of each diet. The urinary angiotensinogen:creatinine ratio was significantly higher in patients who consumed the ordinary salt diet compared with the low-salt diet (17.5 μg/g [range: 7.3 to 35.6 μg/g] versus 7.9 μg/g [range: 3.1 to 14.2 μg/g] of creatinine, respectively; P r =0.43; P =0.008) and changes in logarithmic urinary angiotensinogen:creatinine ratio ( r =0.37; P =0.017) but not with changes in urinary protein excretion ( r =0.18; P =0.49). In contrast, changes in sodium intake did not alter the urinary angiotensinogen:creatinine ratio in patients with Ménière disease and normal renal function (n=9). These data suggest that the inappropriate augmentation of intrarenal angiotensinogen induced by salt and associated renal damage contribute to the development of salt-sensitive hypertension in patients with IgA nephropathy.

(Pro)renin receptor ((P)RR) is a component of the Wnt receptor complex (Science, 2010). We have recently demonstrated that (P)RR plays an important role in the tumorigenesis of pancreatic ductal adenocarcinoma via the activation of Wnt/β-catenin signaling pathway (Shibayama et al. Sci Rep. 2015). Since the patients with colon cancer often show aberrantly activated Wnt/β-catenin-dependent signaling pathway by the mutations of its components, we investigated the possible role of (P)RR and Wnt/β-catenin signaling pathway in carcinogenesis of colon cancer. Real-time PCR was used for measuring mRNA levels of (P)RR. Protein levels of (P)RR was determined by Western blotting and immunohistochemistry. Activated β-catenin levels were determined by Western blotting. Cell proliferative ability was evaluated by counting the cell number in cultured colon cancer cell lines, HCT116 and DLD-1 cells. As compared to normal colon tissues (n=6), mRNA and protein levels of (P)RR were increased by 2.6- and 2.2-fold, respectively, in colon cancer tissues (n=9), which were associated with increased activated β-catenin levels (by 2.8-fold, P

In contrast to proteolytic activation of inactive prorenin by cleavage of the N-terminal 43 residue peptide, we found that prorenin is activated without proteolysis by binding of the prorenin receptor to the pentameric “handle region” I 11P LLKK 15P . We hypothesized that such activation occurs in hypertensive rats and causes cardiac renin–angiotensin system (RAS) activation and end-organ damage. To test this hypothesis, we devised methods of specifically inhibiting nonproteolytic activation by decapeptide spanning the pentameric handle region peptide as a decoy. In stroke-prone spontaneously hypertensive rats (SHRsp) fed a high-salt diet, arterial pressure started to rise significantly with a marked increase in the cardiac prorenin receptor mRNA level at 8 weeks of age, and cardiac fibrosis had developed by 12 weeks of age. By immunohistochemistry using antibodies to the active site of the renin molecule, we demonstrated increased proteolytic or nonproteolytic activation of prorenin in the heart but not in plasma of SHRsp. Continuous subcutaneous administration of the handle region peptide completely inhibited the increased staining by antibodies to the active site of the renin molecule, indicating the increased nonproteolytic but not proteolytic activation of prorenin in the heart of SHRsp. Administration of the handle region peptide also inactivated tissue RAS without affecting circulating RAS or arterial pressure and significantly attenuated the development and progression of cardiac fibrosis. These results clearly demonstrate the significant role of nonproteolytically activated tissue prorenin in tissue RAS activation leading to cardiac fibrosis and significant inhibition of the cardiac damage produced by chronic infusion of the handle region peptide.

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti–phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [ 3 H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [ 3 H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 μmol/L), a selective MR antagonist, or PD98059 (10 μmol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [ 3 H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [ 3 H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho–ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 μmol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [ 3 H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 μmol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.

The relations among hypertensive response, oxidative stress, and mitogen-activated protein kinase (MAPK) in cardiovascular tissues have not been fully established. We investigated the involvement of reactive oxygen species on changes in the hemodynamics and cardiovascular MAPKs activities induced by acutely administered angiotensin II (Ang II) in conscious normotensive rats with or without treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol). Intravenous infusion of a pressor dose of Ang II rapidly increased mean arterial blood pressure (MBP) by 53±5 mm Hg. After a 30-minute treatment with Ang II, phosphorylated MAPKs (ERK1/2, JNK, p38) as well as thiobarbital reactive substances (T-BARS) were increased in the aorta and cardiac left ventricle. Tempol had no significant effect on the elevation of MBP elicited by Ang II; however, it dose-dependently suppressed the augmented phosphorylation of cardiovascular MAPKs and increased T-BARS levels in plasma and tissues induced by Ang II. An acutely administered pressor dose of phenylephrine, an α-adrenoceptor agonist, also showed tempol-sensitive cardiovascular MAPK activation and tempol-insensitive blood pressure elevation. These in vivo data indicate that acute administration of Ang II or phenylephrine provoked an increase in oxidative stress in the cardiovascular tissues leading to the activation of MAPKs, whether it was mediated by pressure overload or the direct action of these vasoconstrictors, and that oxidative stress might not have a major contribution to the acute hypertensive responses elicited by the vasoconstrictors.

This study was performed to examine whether there is an inappropriate regulation of intrarenal angiotensinogen in Dahl-salt sensitive rats (DS) fed a high salt diet (HS). Dahl salt-resistant rats (DR) and DS were maintained on HS (8% NaCl) or low salt diet (LS, 0.3% NaCl) for 4 weeks. Systolic blood pressure (SBP), measured by tail-cuff plethysmography, was unaltered in DR (DR+HS, 127+/-3 mm Hg, n=5; DR+LS, 126+/-3, n=5); however, SBP was significantly increased in DS+HS (208+/-7, n=9) compared with DS+LS (134+/-2, n=5). HS suppressed plasma renin activity in both strains (0.7+/-0.2 ng of angiotensin I/mL per hour in DS+HS, 3.1+/-0.5 in DS+LS, 0.8+/-0.2 in DR+HS, 5.1+/-0.7 in DR+LS). Plasma angiotensinogen levels, measured by Western blot analysis, were also suppressed by HS in both strains (36 919+/-2170 integrated densitometric unit in DS+HS, 53 028+/-2752 in DS+LS, 44 722+/-1721 in DR+HS, 55782+/-3785 in DR+LS). However, kidney angiotensinogen levels were significantly increased in DS+HS (75 850+/-4171, integrated densitometric unit) compared with DS+LS (47 232+/-3470), DR+HS (44 748+/-8236), and DR+LS (42 504+/-4052). Urinary excretion of angiotensinogen, measured by radioimmunoassay of angiotensin I after incubation with excess renin, had a similar profile. Urinary excretion of angiotensinogen was significantly increased in DS+HS (2958+/-531 pmol/d) compared with DS+LS (56+/-4), DR+HS (31+/-12), and DR+LS (21+/-7). These data indicate that intrarenal angiotensinogen is enhanced in DS+HS, which is reflected by the increased urinary excretion of angiotensinogen. The results suggest that DS on HS have an inappropriate augmentation of intrarenal angiotensinogen, which may contribute to impaired sodium excretion during a high salt diet and the development of hypertension in this strain.

Recent studies have implicated a contribution of oxidative stress to the development of hypertension. Studies were performed to determine the effects of the superoxide dismutase (SOD) mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine- N -oxyl (Tempol) on vascular superoxide production and renal sympathetic nerve activity (RSNA) in anesthetized Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Compared with WKY rats (n=6), SHR showed a doubled vascular superoxide production, which was normalized by treatment with Tempol (3 mmol/L, n=7). In WKY rats (n=6), Tempol (30 mg/kg IV) significantly decreased mean arterial pressure (MAP) from 108±5 to 88±6 mm Hg and HR from 304±9 to 282±6 beats/min. In SHR (n=6), Tempol significantly decreased MAP from 166±4 to 123±9 mm Hg and HR from 380±7 to 329±12 beats/min. Furthermore, Tempol significantly decreased RSNA in both WKY rats and SHR. On the basis of group comparisons, the percentage decreases in MAP (−28±4%), HR (−16±3%) and integrated RSNA (−63±6%) in SHR were significantly greater than in WKY rats (−17±3%, −9±2%, and −30±4%, respectively). In SHR, changes in integrated RSNA were highly correlated with changes in MAP (r=0.85, P

To examine the potential role of superoxide anion (O 2 − ) and its interaction with NO in the regulation of renal hemodynamics and excretory function, we have evaluated the renal responses to enhancement in O 2 − activity before and during NO synthase inhibition in anesthetized dogs (n=6). Intraarterial infusion of a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DETC; 0.1 and 0.5 mg/kg per min) was made to enhance O 2 − activity in the kidney. Cortical (CBF), medullary (MBF), and total renal blood flow (RBF) responses were assessed using laser-Doppler needle flow probes and an electromagnetic flow probe. DETC caused dose-dependent changes in renal parameters, which were recovered within 30 minutes after the termination of DETC infusion. The high-dose infusion of DETC for 25 minutes resulted in an increase of 29±10% in renal vascular resistance (control, 35.4±4.4 mm Hg/mL per min per g) and decreases of 21±5% in RBF (control, 3.5±0.5 mL/min per g), 20±5% in CBF, 21±7% in MBF, 62±11% in urine flow (control, 10.5±2.2 μL/min per g), and 47±11% in sodium excretion (control, 2.1±0.2 μmol/min per g), without a significant change (−10±6%) in glomerular filtration rate (control, 0.74±0.09 mL/min per g). During NO synthase inhibition with intraarterial administration of nitro- l -arginine (50 μg/kg per min), the same dose of DETC showed a greater increase in renal vascular resistance (73±15%) and reductions in RBF (39±4%), CBF (32±5%), MBF (34±6%), urine flow (78±5%), and sodium excretion (67±10%), with a marked reduction in glomerular filtration rate (59±7%). These data indicate that O 2 − exerts renal vasoconstriction as well as antidiuretic and antinatriuretic effects. These responses are enhanced during NO synthase blockade, suggesting that NO serves a renoprotective effect against these action of O 2 − .

Previous studies have indicated that angiotensin II (Ang II) concentrations in renal interstitial fluid are much higher than plasma levels. In the present study, we performed experiments to explore renal interstitial fluid concentrations of Ang I and Ang II further and to determine whether these levels are altered by acute arterial infusion of an ACE inhibitor (enalaprilat) or by volume expansion. Microdialysis probes (molecular weight cutoff point: 30 000 Da) were implanted in the renal cortex of anesthetized rats and were perfused at a rate of 2 microL/min. Using relative equilibrium rates, the basal renal interstitial fluid Ang II concentration averaged 3.07+/-0.43 nmol/L, a value much higher than the plasma Ang II concentration of 107+/-8 pmol/L (n=7). Interstitial fluid Ang I concentrations (0.84+/-0.04 nmol/L) were consistently lower than the Ang II concentrations but higher than the plasma Ang I concentrations (112+/-14 pmol/L). Intra-arterial infusion of enalaprilat (7.5 micromol/kg/min, n=5) for 120 minutes resulted in a significant decrease in mean arterial pressure (from 114+/-4 to 68+/-4 mm Hg) along with reductions in plasma and renal ACE activity (by -99% and -52%, respectively). Enalaprilat resulted in a significant increase in plasma Ang I from 133+/-21 to 1167+/-328 pmol/L and a decrease in plasma Ang II from 110+/-12 to 67+/-9 pmol/L. During enalaprilat infusion, interstitial fluid concentration of Ang I was significantly increased from 0.78+/-0.06 to 0.97+/-0.08 nmol/L; however, Ang II concentrations were not altered significantly (3.67+/-0.28 versus 3.67+/-0.25 nmol/L). Acute volume loading with Ringer's solution containing 1% bovine serum albumin at a rate of 150 microL/min for 2 hours (6% to 7% of body weight) lowered plasma concentrations of Ang I from 110+/-23 to 16+/-2 pmol/L and Ang II from 100+/-23 to 36+/-6 pmol/L; however, renal interstitial fluid concentrations of Ang I and Ang II were not altered significantly during volume expansion (Ang I, from 0.77+/-0.05 to 0.69+/-0.03 nmol/L; Ang II, from 3.76+/-0.43 to 3.59+/-0.39 nmol/L, n=5). These data indicate that renal interstitial fluid concentrations of Ang I and Ang II are substantially higher than the corresponding plasma concentrations. Furthermore, the fact that the high interstitial fluid concentrations of Ang II are not responsive to acute ACE inhibition or volume expansion suggests the compartmentalization and independent regulation of renal interstitial fluid Ang II.

Proximal tubular sodium-glucose co-transporter 2 (SGLT2) transports glucose and sodium with a 1:1 stoichiometry. However, studies have indicated that treatment with SGLT2 inhibitors does not substantially increase the urinary excretion rate of sodium, while that of glucose is markedly increased. These data suggest that urinary sodium is reabsorbed by other mechanisms distal to the nephron during SGLT2 inhibition. Here, we aimed to investigate whether diuretics affect the SGLT2 inhibitor-induced changes in the urinary excretion rate of sodium in obese metabolic syndrome SHR/NDmcr-cp(+/+) (SHRcp) rats. Male 13-week-old SHRcp rats were treated with: 1) vehicle (0.5% carboxymethylcellulose), 2) a SGLT2 inhibitor, luseogliflozin (10 mg/kg/day, p.o.), 3) luseogliflozin + hydrochlorothiazide (10 mg/kg/day, p.o.) or 4) luseogliflozin + hydrochlorothiazide + furosemide (5 mg/kg/day, p.o.) for 5 weeks (n = 6-8 per group). Blood pressure and glucose metabolism were evaluated by telemetry and oral glucose tolerance test respectively. Vehicle-treated SHRcp rats developed non-dipper type hypertension (night and day time systolic blood pressure; 186 ± 2 and 185 ± 2 mmHg, respectively) and insulin resistance. As compared with vehicle-treated animals, luseogliflozin-treated rats showed an approximately 4,000-fold increase in urinary glucose excretion and improved glucose metabolism. Luseogliflozin also slightly decreased blood pressure, which was associated with an approximately 30% increase in urinary excretion of sodium. The addition of hydrochlorothiazide or hydrochlorothiazide + furosemide further decreased blood pressure and improved blood pressure circadian rhythm to a dipper profile in luseogliflozin-treated animals. In these animals, urinary sodium excretion tended to be increased by diuretics, although these changes were not statistically significant. These data indicate that SGLT2 inhibitor-induced natriuresis is not enhanced by diuretics. Thus, SGLT2 inhibitors may elicit their beneficial effects on glucose metabolism and hypertension in patients who are treated with diuretics.

Several studies have indicated that treatment with dipeptidyl peptidase-4 (DPP-4) inhibitors decreases blood pressure. We aimed to examine the effects of vildagliptin, a specific DPP-4 inhibitor, on blood pressure and its dipping pattern in Dahl salt-sensitive (DSS) rats. Male DSS rats were treated with a high salt (8% NaCl) diet and vehicle (0.5% carboxymethylcellulose) or vildagliptin (3 or 10 mg/kg, twice daily, p.o.) for 7 days (n = 7 per group). Mean arterial pressure (MAP) was measured by telemetry. High salt diet for 7 days significantly increased MAP with an extreme dipping pattern of blood pressure in DSS rats. Vildagliptin dose-dependently inhibited DPP-4 enzymatic activity and increased plasma GLP-1 levels. Furthermore, vildagliptin dose-dependently attenuated the development of salt-induced hypertension. Interestingly, vildagliptin significantly increased urine sodium excretion and normalized the dipping pattern. In anesthetized high salt-diet DSS rats, acute intra-cerebroventricular infusion of vildagliptin (50, 500 or 2500 μg in 10 μl solution) did not alter MAP or heart rate (n = 4). These data suggest that treatment with the DPP-4 inhibitor, vildagliptin, attenuates the extreme dipping pattern of blood pressure and development of salt sensitive hypertension by increasing urinary sodium excretion. These effects of vildagliptin may not be mediated through the central nervous system.

Abstract —Recent studies have indicated that angiotensin II (Ang II) can stimulate oxidative stress. The present study was conducted to assess the contribution of oxygen radicals to hypertension and regional circulation during Ang II–induced hypertension. With radioactive microspheres, the responses of systemic and regional hemodynamics to the membrane-permeable, metal-independent superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) were assessed in conscious Ang II–infused hypertensive rats. Ang II–infused rats (80 ng/min SC for 12 days: n=25) showed higher mean arterial pressure (MAP: 161±4 mm Hg) and total peripheral resistance (TPR: 1.59±0.08 mm Hg · min −1 · mL −1 ) than vehicle-infused normotensive rats (116±3 mm Hg and 0.95±0.04 mm Hg · min −1 · mL −1 , respectively; n=23). The blood flow rates in the brain, spleen, large intestine, and skin were significantly reduced in Ang II–infused rats compared with vehicle-infused rats, whereas rates in the lung, heart, liver, kidney, stomach, small intestine, mesenterium, skeletal muscle, and testis were similar. Vascular resistance was significantly increased in every organ studied except the lung, in which the resistance was similar. Tempol (216 μmol/kg IV) significantly reduced MAP by 30±4% from 158±7 to 114±5 mm Hg and TPR by 35±6% from 1.57±0.17 to 0.95±0.04 mm Hg · min −1 · g −1 in Ang II–infused rats (n=9) but had no effect on these parameters in vehicle-infused rats (n=8). In Ang II–infused rats, tempol did not affect regional blood flow but significantly decreased vascular resistance in the brain (29±6%), heart (31±6%), liver (37±7%), kidney (30±7%), small intestine (38±6%), and large intestine (47±7%). Ang II–infused hypertensive rats showed doubled vascular superoxide production (assessed with lucigenin chemiluminescence), which was normalized by treatment with tempol (3 mmol/L, n=7). Further studies showed that the NO synthase inhibitor, N ω -nitro- l -arginine methyl ester (11 μmol · kg −1 · min −1 IV, n=11) markedly attenuated the systemic and regional hemodynamic responses of tempol in Ang II–infused rats. These results suggest that in this model of hypertension, oxidative stress may have contributed to the alterations in systemic blood pressure and regional vascular resistance through inactivation of NO.

We previously reported that streptozotocin (STZ)-induced type 1 diabetes caused renal tubular cell senescence through blood glucose- and p21-dependent pathway at week 4 of STZ administration in mice. We investigated the involvement of the cell senescence on accumulation of iron in the renal tubular cells in the present study. STZ mice (n=5) did not show the significant iron accumulation in the kidney at week 4. STZ mice (n=10) showed increases in renal tubular iron accumulation (Perl’s Prussian blue: 278.1±71.9 fold vs. non-diabetic control, p

Insulin resistance (IR) is associated with elevated renin-angiotensin system (RAS), impaired urinary Na + excretion (U Na V), and high blood pressure. RAS blockade lowers blood pressure, decreases IR and fasting plasma glucose (FPG). Glucagon-like peptide 1 (GLP-1), has been reported to have natriuretic effects as well as blood glucose lowering effects, however, these findings are still controversial. To assess the contributions of GLP-1 receptor and angiotensin receptor type 1(AT1) activation on renal Na + handling, systolic blood pressure (SBP) and FPG, metabolic measurements were taken at days 0, 2, 7, and every week thereafter for six weeks, SBP was measured daily using radio telemetry in five groups of rats: 1) untreated, lean LETO (n=7), 2) untreated, obese OLETF (n=5), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan /kg/d; n=4), 4) OLETF + GLP-1 mimetic (Ex; 10 ug exenatide/kg/d; n=7), and 5) OLETF + ARB + Exenatide (combo; n=6). FPG increased (101 ± 4 vs 131 ± 3 mg/dL) in OLETF compared to LETO, and ARB treatment decreased it (120 ± 2 mg/dL); however, the changes in Ex and combo groups were not significant. OLETF rats exhibited elevated SBP at the onset of the study compared to LETO (119 ± 1 vs. 130 ± 1 mmHg). U Na V decreased (1.07 ± 0.07 vs 0.43 ± 0.14 & 0.31 ± 0.11 mmol/day) in Ex and combo groups on day 2 compared to OLETF, and the decrease in U Na V in Ex was associated with increased (132 ± 1 vs 142 ± 1 mmHg) SBP. However, reduced U Na V in combo group was associated with reduced (114 ± 3 mmHg) SBP suggesting that activation of AT1 has a greater contribution to the insulin resistance-associated increase in SBP than the impaired U Na V. After this initial increase in SBP in Ex, it decreased to a nadir (124 ± 2 mmHg) at day 11 of treatment before increasing to 130 for the duration of the study. After six weeks of treatment U Na V increased (1.02 ± 0.05 vs 1.35 ± 0.09 mmol/day) only in combo group compared to OLETF. The results suggest that chronic GLP-1 receptor activation acutely increases SBP by decreasing U Na V, but co-treatment with an ARB abolishes this effect. These results also suggest that despite the lack of improved FBG, chronic GLP-1 receptor activation may protect against the development of high blood pressure associated with insulin resistance independent of increased U Na V.

Vitamin D diminishes renin expression. In addition, we previously reported that vitamin D increased renal expression of klotho in rats with normal kidney function. In the present study, effects of vitamin D on renal injury was assessed in four groups of rats (n=6-8 for each group); uni-nephrectomized stroke-prone spontaneously hypertensive rats fed high salt (6%) diet as a control (C), those treated with irbesartan (100 mg/kg/day) (I), rats treated with calcitriol (30 ng/kg/day) (V), and rats treated with both irbesartan and calcitriol (I+V). Six weeks later, rats were killed with over-anesthesia, and harvested right kidney for analysis. As shown in table, systolic blood pressure (SBP) in C (p

Introduction: Parietal epithelial cells (PECs) contribute to the glomerular crescent formation of crescentic glomerulonephritis. Co-cultured model consisting of mesangial cells (MCs), PECs, and macrophages which play a pivotal role in the development of crescentic glomerulonephritis showed an increase in Angiotensin (Ang) II-induced cell proliferation and collagen secretion in PECs. Moreover, Ang II type 1 receptor blocker (ARB) treatment completely prevented an increase in cell proliferation and collagen secretion in PECs under the co-cultured model. However, it is not clear whether Ang II directly promotes PEC fibrosis and proliferation under the monoculture in PECs. Aim: The aim of present study is to demonstrate that Ang II stimulation directly affects PEC proliferation and fibrosis. Methods: Primary fluorescein isothiocyanate (FITC)-labeled PEC population was collected from glomeruli in rats using high-speed cell sorter. ERK1/2 phosphorylation was evaluated by Western blotting. Cell proliferation ability was measured by water-soluble tetrazolium salts (WST)-1 and the soluble collagen levels in culture supernatants was determined by the Sircol collagen assay. Results: FITC-labeled PECs could be identified as a distinct cell population by high-speed cell sorter. The ERK 1/2 phosphorylation in PECs was stimulated by Ang II (100 nmol/l) by about 1.7-fold. After the treatment with 100 nmol/l Ang II for 24 hrs, cell proliferation ability significantly increased in PECs (mean ± SEM: 1.60 ± 0.03 vs. 1.00 ± 0.04, relative ratio). However, the proliferation ability in Ang II-stimulated PECs was suppressed by Ang II plus 10 nmol/l olmesartan (an ARB) or Ang II plus 100 nmol/l PD98059 (a MEK inhibitor). PECs also promoted a collagen secretion after the stimulation by 100 nmol/l Ang II for 24 hrs (52.1 ± 8.1 vs. 16.8 ± 2.4 μg/ml). Furthermore, the significant decrease in soluble collagen secretion was observed by the treatments with Ang II plus 10 nmol/l olmesartan or Ang II plus 10 μg/ml pan-specific neutralizing TGF-β antibody. Conclusion: These data indicate that Ang II-stimulated PECs promote proliferation and fibrosis and may suggest contributing to the development of crescentic glomerulonephritis by PECs independently from MCs or macrophages.

The development of dipeptidyl peptidase-IV (DPP-4) inhibitors, such as alogliptin, has led to the improvement in glycemic control in type 2 diabetes (T2D) by preventing the degradation of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide. However, the renoprotective effects of alogliptin have not been addressed yet. This 12-week study in Japanese patients with T2D was performed to address the renoprotective effects of alogliptin. In addition, urinary angiotensinogen (AGT), a marker of intrarenal renin-angiotensin system (RAS) activity, was examined to demonstrate the clinical usage as a prognostic marker. Forty-three patients with T2D (18 women, age: 66.1+/-11.2) were recruited in Miyazaki University and its affiliated hospitals, and alogliptin (25 mg/day) was added on the top of the traditional hypoglycemic agents. The urinary concentrations of albumin (Alb) and AGT were measured using commercially available ELISA kits before and after the alogliptin treatment, and normalized by the urinary concentration of creatinine (Cr) (UAlbCR and UAGTCR, respectively). The alogliptin treatment tended to decrease UAlbCR (99.6 +/- 26.8 vs. 114.6 +/- 36.0, mg/g Cr). However, this change was not statistically significant (p = 0.1976). Then, we defined good responders to the alogliptin treatment in terms of %change in UAlbCR less than -25% after the 12-week treatment, and a logistic analysis of UAGTCR before the treatment showed the area under the curve (AUC) as 0.644. When we set the cutoff value of UAGTCR as 20.8 μg/g Cr, the maximum specificity (17/27 = 63.0%) and sensitivity (10/16 = 62.5%) were obtained (Youden index = 0.255). Based on this cutoff value of UAGTCR before the treatment, we divided all patients into 2 groups as higher (group H, N = 20) and lower (group L) values of UAGTCR at the baseline. %Change in UAlbCR was significantly lower in the group H compared with the group L (-14.6% +/- 8.6% vs. +22.8% +/- 16.8%, p = 0.0327). These data indicate that the T2D patients with the higher UAGTCR before the treatment would show more decrease in UAlbCR by the alogliptin treatment. In conclusion, urinary AGT could be a prognostic marker of renoprotective effects of alogliptin in T2D patients.

Activation of the renin-angiotensin system (RAS) leads to an increase in blood pressure and onset of insulin resistance (IR); however, the contributions of increased blood pressure and AT1 activation independently on the manifestation of IR are not well defined. The goal of this study was to determine the contribution of elevated blood pressure, independent of RAS, to the onset of IR in a model of metabolic syndrome. To address the hypothesis that AT1 activation, and not elevated blood pressure independently, is the principal contributor of obesity-associated IR, we measured changes in systolic blood pressure (SBP), adiposity, plasma triglycerides (TG), glucose tolerance, and insulin resistance index (IRI) in four groups of rats: 1) lean strain-control Long Evans Tokushima Otsuka (LETO; n=5), 2) obese Otsuka Long-Evans Tokushima Fatty (OLETF; n=7), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg; n=8), and 4) OLETF + calcium channel blocker (CCB; 5 mg amlodipine/kg; n=7). ARB treatment alleviated the obesity-related increase in mean SBP, while the decrease with CCB remained 10.2 mmHg greater than LETO. ARB (0.51 g/100g body mass) and CCB (0.64 g/100g body mass) reduced mean relative retroperitoneal fat mass and mean plasma triglycerides (28.4 and 27.4 mg/dL respectively) compared to OLETF, but both remained greater (1.7, 1.57 g/100g body mass and 28.94, 29.94 mg/dL respectively) than LETO. ARB improved glucose tolerance by 4605.94 of 34965.94 and mean calculated IRI by 5581429.5 of 17398363, but CCB had no detectable effect on either. Despite relatively similar reductions in SBP, adiposity and plasma TG (principal components of metabolic syndrome), CCB did not improve glucose tolerance and IRI (additional metrics of metabolic syndrome), while ARB did, demonstrating that AT1 activation is the primary factor contributing to the development of impaired glucose metabolism and regulation during metabolic syndrome, independent of the hypertension, adiposity and dyslipidemia. Thus, targeting RAS to improve the consequences of metabolic syndrome appears to be prudent and effective.

Recent basic and clinical data demonstrated that the intrarenal renin-angiotensin system (RAS) plays an important role in the progression of chronic kidney disease (CKD). The urinary angiotensinogen (AGT) excretion rate could be a novel biomarker for the activity of the RAS in the kidney. We previously reported that the healthy volunteers do not have a circadian rhythm of AGT level in urine (Hypertension. 2011;57:e78) or in plasma (Hypertension. 2012;60:A399). However, the circadian rhythm of AGT level in urine and in plasma in patients with CKD has not been reported yet. Therefore, this study was performed to investigate the circadian rhythm of AGT level in urine and in plasma in patients with CKD. We recruited 6 CKD patients with continuous proteinuria admitted to the Kagawa University Hospital from 06/2011 to 10/2011 for the purpose of diagnostic renal biopsy. Plasma samples were collected at 06:00, 12:00, and 18:00. Urine samples were collected at 06:00, 09:00, 12:00, and 18:00. Plasma renin activities (PRAs), plasma and urinary AGT concentrations, and urinary albumin (Alb) concentration were measured using commercially available kits. The urinary concentrations of AGT and Alb were normalized by the urinary concentration of creatinine (Cr) (UAGT/Cr and UAlb/Cr, respectively). PRA (2.07 +/- 0.77 ng of angiotensin I/mL/hr at 06:00, 2.45 +/- 0.77 at 12:00, and 2.58 +/- 0.76 at 18:00, P = 0.8853) or plasma AGT (19.9 +/- 2.4 μg/mL at 06:00, 24.1 +/- 3.1 at 12:00, and 23.0 +/- 3.9 at 18:00, P = 0.6300) did not show a circadian rhythm. Moreover, UAlb/Cr (511 +/- 319 mg/g Cr at 06:00, 827 +/- 459 at 09:00, 1479 +/- 862 at 12:00, and 1370 +/- 818 at 18:00, P = 0.6964) or UAGT/Cr (54 +/- 22 μg/g Cr at 06:00, 119 +/- 41 at 09:00, 350 +/- 194 at 12:00, and 198 +/- 104 at 18:00, P = 0.3035) did not show a circadian rhythm. In conclusion, in addition to healthy volunteers, patients with CKD do not have a circadian rhythm of AGT level in urine or in plasma.

Others and we showed the increased levels of intrarenal angiotensinogen (AGT) in diabetic humans, mice, and rats. However, the precise localization of the augmented AGT mRNA and protein in proximal tubule segments of kidneys in diabetes remains to be established. To investigate the detailed localization of AGT in 3 proximal tubule segments of the kidneys in diabetes, the Otsuka Long-Evans Tokushima fatty (OLETF) rat (15 weeks) and the age-matched genetic control, the Long-Evans Tokushima Otsuka (LETO) rat were used. Kidney tissues were also prepared from OLETF rats that were administered with angiotensin II type 1 receptor blocker, olmesartan (0.02% in chow) or a combination of vasodilator agents (0.03% hydralazine, 0.006% reserpine, and 0.012% hydrochlorothiazide in chow; HRH). Moreover, biopsied samples of human kidney cortex were obtained by fine-needle aspiration to confirm the below-mentioned results of the rat localization study. Using 3-μm sections of zinc-saturated formalin-fixed paraffin-embedded slides, we examined the co-localization of AGT mRNA or protein with segment-specific protein markers (Segment (S)1-specific anti-sodium glucose cotransporter 2 antibody, S2-specific anti-carbonic anhydrase IV antibody, and S3-specific anti-ecto-adenosine triphosphatase antibody) by double staining using fluorescence in situ hybridization and/or immunofluorescence. AGT mRNA expression was not recognized much in S1 segments of either OLETF or LETO rat kidneys. In S3 segments, the AGT mRNA expression area was augmented in the OLETF rats than in the LETO rats (2.31 ± 0.02-fold; P = 0.0029). AGT protein expression areas in S1 and S3 segments were also increased (8.50 ± 2.78-fold; P = 0.0476, and 3.71 ± 0.83-fold; P = 0.0317, respectively). Chronic treatment with olmesartan, but not with HRH, ameliorated the augmented AGT mRNA and protein expression areas. AGT mRNA and protein expression areas in S2 segments were unchanged in OLETF rats compared with LETO rats. Biopsied samples of human kidney cortex showed the same results as for rat AGT mRNA and protein expression in the S1 and S2 segments. These data suggest that the augmented AGT mRNA in S3 and AGT protein in S1 and S3 play an important role in the development and the progression of diabetic nephropathy.

Background: We have previously reported that urinary angiotensinogen (AGT) excretion did not have a circadian rhythm and could be a novel biomarker for the activity of the renin-angiotensin system (RAS) in kidney. However, there have been few reports investigating the circadian rhythm of plasma AGT in human body. Thus, this study was performed to examine the circadian rhythm in plasma AGT in human. METHODS: Evaluating RAS in clinical practice is generally performed in a recumbent position after a 30-minute stabilization period. However, to determine the necessity of recumbent position, we first compared plasma AGT concentrations measured right after waking up and after a 5-minute sitting rest. Next, we examined the circadian rhythm of plasma AGT in 43 healthy volunteers who had shown no abnormalities in the medical examinations in 2011. Plasma AGT was measured at three time points (9 a.m., 1 p.m., and 4 p.m.) in the above volunteers. Blood was collected by a micro hematocrit capillary tube with heparin, frozen for storage after centrifugation, and thawed for the measurement of plasma AGT using an ELISA kit. Results: There was no significant difference between the plasma AGT values of the two measuring methods (P = 0.1202, n = 5). Based on the result, we performed blood sampling after a 5-minute sitting rest in the volunteers consisting of 17 men and 26 women. Average blood pressure was 116.3/75.1 mmHg at 9 a.m., 116.3/71.9 mmHg at 1 p.m., and 115.5/70.1 mmHg at 4 p.m.; average pulse rate was 78.7/min at 9 a.m., 77.1/min at 1 p.m., and 73.3/min at 4 p.m. Blood pressure and pulse rate did not change throughout the day. Average plasma AGT was 20.4 ± 6.0 ng/ml at 9 a.m., 20.7 ± 5.0 ng/ml at 1 p.m., and 19.8 ± 6.4 ng/ml at 4 p.m. Plasma AGT did not show a circadian rhythm (P = 0.3803). Conclusion: We found in this study that plasma AGT did not have a circadian rhythm. We also found that plasma AGT was not affected by daily life actions. Thus, future patients may not be required to rest nor wait for certain time points before measuring plasma AGT. We also have to unveil the normal AGT levels and the influence on the levels by diseases. As we think that plasma AGT and ratio of urinary AGT to plasma AGT can be a new surrogate marker of hypertension and kidney diseases, we further need to go into this research area.

We previously reported that hyperglycemia induces renal tubular cell senescence in type 1 diabetic animals. In the present study, we evaluated the tubular cell senescence in type 2 diabetic animals and investigated whether the renal cell senescence could regress in response to treatment with a reno-protective dosage of an angiotensin receptor blocker, telmisartan (10 mg/kg/day). Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneously developed type 2 diabetes, showed an age-dependent increase in renal cell senescence analyzed by p21 expression (6 weeks old: 1.3±0.3 fold, 22 weeks old: 2.6±0.3 fold, vs. age-matched lean control rats, n=8, respectively) and senescence-associated β galactosidase (SABG) staining. OLETF rats were separated into the following 3 groups at 22 weeks of age and were kept under treatment until 34 weeks of age (n=10 per group): 1) un-treated, 2) telmisartan, and 3) tempol (an anti-oxidant, 3 mmol/L in drinking water). Neither treatment affected the blood glucose levels. Telmisartan, but not tempol, markedly reduced SABG staining by 34 weeks of age compared with that at 22 weeks of age or with that of age-matched un-treated rats. Increase in the expression of p21 was significantly smaller in telmisartan-treated rats (1.3±0.2 fold vs. lean rats) than in un-treated rats (2.9±0.2 fold, p

Background: We previously reported that various pathological conditions including high blood pressure increase p21 expression in the kidney; however, the functional importance of renal p21 up-regulation has not been clarified yet. In the present study, we evaluated the role of p21 in acute kidney injury, a life-threatening disease that can occur independently of the pathological background of patients (whether renal p21 is up-regulated or not). Methods and Results: The mice lacking functional p21 (p21-KO, n=9) and its wild-type control (WT, n=7) underwent a 45-min renal ischemia followed by a 24-h reperfusion (I/R). I/R significantly increased both mRNA expression and nuclear immunoreactivity of p21 in the kidney of WT compared with sham surgery (p21/β-actin, 1.28±0.23 vs. 0.57±0.15, respectively, P Conclusion: Renal p21 plays a protective role against I/R injury and is necessary for the beneficial effect of renal IPC.

We previously reported that aldosterone/mineralocorticoid receptor activation caused cell senescence via p21, a cell cycle regulator, -dependent pathway in proximal tubules of rat kidney and human proximal tubular cells. However, functional influences of cell senescent on the kidney are still unclear. In the present study, we hypothesized that aldosterone-induced cell senescence caused cell apoptosis by increasing the secretion of apoptotic factors (n=6 in all groups). Aldosterone increased senescent cells at day 3 and day 5 in human proximal tubular cells, and the changes were accompanied by the increase in p21 protein expression (day 3: 2.9±0.7 fold, day 5: 6.5±0.9 fold, p