Ryousuke Satou

Urinary Angiotensinogen as a Novel Biomarker of the Intrarenal Renin-Angiotensin System Status in Hypertensive Patients

We reported previously that urinary angiotensinogen (UAGT) levels provide a specific index of the intrarenal renin-angiotensin system (RAS) status in angiotensin II–dependent hypertensive rats. To study this system in humans, we recently developed a human angiotensinogen ELISA. To test the hypothesis that UAGT is increased in hypertensive patients, we recruited 110 adults. Four subjects with estimated glomerular filtration levels <30 mL/min per 1.73 m2 were excluded because previous studies have already shown that UAGT is highly correlated with estimated glomerular filtration in this stage of chronic kidney disease. Consequently, 106 paired samples of urine and plasma were analyzed from 70 hypertensive patients (39 treated with RAS blockers [angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers; systolic blood pressure: 139±3 mm Hg] and 31 not treated with RAS blockers [systolic blood pressure: 151±4 mm Hg]) and 36 normotensive subjects (systolic blood pressure: 122±2 mm Hg). UAGT, normalized by urinary concentrations of creatinine, were not correlated with race, gender, age, height, body weight, body mass index, fractional excretion of sodium, plasma angiotensinogen levels, or estimated glomerular filtration. However, UAGT/urinary concentration of creatinine was significantly positively correlated with systolic blood pressure, diastolic blood pressure, urinary albumin:creatinine ratio (r=0.5994), and urinary protein:creatinine ratio (r=0.4597). UAGT/urinary concentration of creatinine was significantly greater in hypertensive patients not treated with RAS blockers (25.00±4.96 &mgr;g/g) compared with normotensive subjects (13.70±2.33 &mgr;g/g). Importantly, patients treated with RAS blockers exhibited a marked attenuation of this augmentation (13.26±2.60 &mgr;g/g). These data indicate that UAGT is increased in hypertensive patients, and treatment with RAS blockers suppresses UAGT, suggesting that the efficacy of RAS blockade to reduce the intrarenal RAS activity can be assessed by measurements of UAGT.

Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases

We previously reported that urinary excretion rates of angiotensinogen (AGT) provide a specific index of the activity of the intrarenal renin-angiotensin system in angiotensin II-dependent hypertensive rats. Meanwhile, we have recently developed direct enzyme-linked immunosorbent assays (ELISAs) to measure plasma and urinary AGT in humans. This study was performed to test a hypothesis that urinary AGT levels are enhanced in chronic kidney disease (CKD) patients and correlated with some clinical parameters. Eighty patients with CKD (37 women and 43 men, from 18 to 94 years old) and seven healthy volunteers (two women and five men, from 27 to 43 years old) were included. Plasma AGT levels showed a normal distribution; however, urinary AGT-creatinine ratios (UAGT/UCre) deviated from the normal distribution. When a logarithmic transformation was executed, Log(UAGT/UCre) levels showed a normal distribution. Therefore, Log(UAGT/UCre) levels were used for further analyses. Log(UAGT/UCre) levels were not correlated with age, gender, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, serum sodium levels, serum potassium levels, urinary sodium-creatinine ratios, plasma renin activity, or plasma AGT levels. However, Log(UAGT/UCre) levels were significantly correlated positively with urinary albumin-creatinine ratios, fractional excretion of sodium, urinary protein-creatinine ratios, and serum creatinine, and correlated negatively with estimated glomerular filtration rate. Log(UAGT/UCre) levels were significantly increased in CKD patients compared with control subjects (1.8801 +/- 0.0885 vs. 0.9417 +/- 0.1048; P = .0024). These data confirmed our earlier report and showed that a new ELISA assay is a valid approach for measuring urinary AGT.

Intrarenal angiotensin II and its contribution to the genesis of chronic hypertension

The increased activity of intrarenal renin-angiotensin system (RAS) in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis and renal injury. Increases in intrarenal and interstitial angiotensin (Ang) II levels are due to increased AT(1) receptor mediated Ang II uptake and stimulation of renal angiotensinogen (AGT) mRNA and protein expression. Augmented proximal tubule AGT production increases tubular AGT secretion and spillover of AGT into the distal nephron and urine. Increased renin formation by principal cells of the collecting ducts forms Ang I from AGT thus increasing Ang II. The catalytic actions of renin and prorenin are enhanced by prorenin receptors (PRRs) on the intercalated cells. The resultant increased intrarenal Ang II levels contribute to the genesis of chronic hypertension.

Intrarenal angiotensin II and angiotensinogen augmentation in chronic angiotensin II-infused mice

The objectives of this study were to determine the effects of chronic angiotensin II (ANG II) infusions on ANG II content and angiotensinogen expression in the mouse kidney and the role of the angiotensin II type 1 receptor (AT(1)R) in mediating these changes. C57BL/6J male mice were subjected to ANG II infusions at doses of 400 or 1,000 ng.kg(-1).min(-1) either alone or with an AT(1)R blocker (olmesartan; 3 mg.kg(-1).day(-1)) for 12 days. Systolic and mean arterial pressures were determined by tail-cuff plethysmography and radiotelemetry. On day 13, blood and kidneys were collected for ANG II determinations by radioimmunoanalysis and intrarenal angiotensinogen expression studies by quantitative RT-PCR, Western blotting, and immunohistochemistry. ANG II infusions at the low dose elicited progressive increases in systolic blood pressure (135 +/- 2.5 mmHg). In contrast, the high dose induced a rapid increase (152 +/- 2.5, P < 0.05 vs. controls, 109 +/- 2.8). Renal ANG II content was increased by ANG II infusions at the low dose (1,203 +/- 253 fmol/g) and the high dose (1,258 +/- 173) vs. controls (499 +/- 40, P < 0.05). Kidney angiotensinogen mRNA and protein were increased only by the low dose to 1.13 +/- 0.02 and 1.26 +/- 0.10, respectively, over controls (1.00, P < 0.05). These effects were not observed in mice infused at the high dose and those receiving olmesartan. The results indicate that chronic ANG II infusions augment mouse intrarenal ANG II content with AT(1)R-dependent uptake occurring at both doses, but only the low dose of infusion, which elicited a slow progressive response, causes an AT(1)R-dependent increase in intrarenal angiotensinogen expression.

Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.

Costimulation with angiotensin II and interleukin 6 augments angiotensinogen expression in cultured human renal proximal tubular cells

Augmented intrarenal ANG II stimulates IL-6, which contributes to renal injury. The expression of intrarenal angiotensinogen (AGT) is enhanced by increased intrarenal ANG II in human renin/human AGT double transgenic mice. ANG II also augments AGT expression in hepatocytes and cardiac myocytes. However, the mechanisms underlying AGT augmentation by ANG II and the contribution of IL-6 to this system are poorly understood. This study was performed in human renal proximal tubular epithelial cells (HRPTECs) to test the hypothesis that IL-6 contributes to the upregulation of AGT expression by ANG II. Human kidney-2 (HK-2) cells, immortalized HRPTECs, were incubated with 10(-7) M ANG II and/or 10 ng/ml IL-6 for up to 24 h. AGT mRNA and protein expressions were measured by real-time RT-PCR and ELISA, respectively. The activities of NF-kappaB and STAT3 were evaluated by Western blotting and EMSA. Stimulation with either ANG II or IL-6 did not significantly alter AGT mRNA or protein expression. In contrast, costimulation with ANG II and IL-6 significantly increased AGT mRNA and protein expressions (1.26 +/- 0.10 and 1.16 +/- 0.13 over control, respectively). Olmesartan, an ANG II type 1 receptor blocker, and an IL-6 receptor antibody individually inhibited this synergistic effect. NF-kappaB was also activated by costimulation with ANG II and IL-6. Phosphorylation and activity of STAT3 were increased by stimulation with IL-6 alone and by costimulation. The present study indicates that IL-6 plays an important role in ANG II-mediated augmentation of AGT expression in human renal proximal tubular cells.

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1–7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.

Intrarenal mouse renin-angiotensin system during ANG II-induced hypertension and ACE inhibition

Angiotensin-converting enzyme (ACE) inhibition (ACEi) ameliorates the development of hypertension and the intrarenal ANG II augmentation in ANG II-infused mice. To determine if these effects are associated with changes in the mouse intrarenal renin-angiotensin system, the expression of angiotensinogen (AGT), renin, ACE, angiotensin type 1 receptor (AT(1)R) mRNA (by quanitative RT-PCR) and protein [by Western blot (WB) and/or immunohistochemistry (IHC)] were analyzed. C57BL/6J male mice (9-12 wk old) were distributed as controls (n = 10), ANG II infused (ANG II = 8, 400 ng x kg(-1) x min(-1) for 12 days), ACEi only (ACEi = 10, lisinopril, 100 mg/l), and ANG II infused + ACEi (ANG II + ACEi = 11). When compared with controls (1.00), AGT protein (by WB) was increased by ANG II (1.29 +/- 0.13, P < 0.05), and this was not prevented by ACEi (ACEi + ANG II, 1.31 +/- 0.14, P < 0.05). ACE protein (by WB) was increased by ANG II (1.21 +/- 0.08, P < 0.05), and it was reduced by ACEi alone (0.88 +/- 0.07, P < 0.05) or in combination with ANG II (0.80 +/- 0.07, P < 0.05). AT(1)R protein (by WB) was increased by ANG II (1.27 +/- 0.06, P < 0.05) and ACEi (1.17 +/- 0.06, P < 0.05) but not ANG II + ACEi [1.15 +/- 0.06, not significant (NS)]. Tubular renin protein (semiquantified by IHC) was increased by ANG II (1.49 +/- 0.23, P < 0.05) and ACEi (1.57 +/- 0.15, P < 0.05), but not ANG II + ACEi (1.10 +/- 0.15, NS). No significant changes were observed in AGT, ACE, or AT(1)R mRNA. In summary, reduced responses of intrarenal tubular renin, ACE, and the AT(1)R protein to the stimulatory effects of chronic ANG II infusions, in the presence of ACEi, are associated with the effects of this treatment to ameliorate augmentations in blood pressure and intrarenal ANG II content during ANG II-induced hypertension.

Angiotensin-Converting Enzyme–Derived Angiotensin II Formation During Angiotensin II–Induced Hypertension

The extent to which endogenous angiotensin (Ang) II formation is responsible for increasing kidney Ang II content and blood pressure during Ang II–induced hypertension is unknown. To address this, mice were treated with an Ang-converting enzyme (ACE) inhibitor (ACEi) to block endogenous Ang II formation during chronic Ang II infusions. C57BL/6J male mice (8 to 12 weeks) were subjected to Ang II infusions (400 ng/kg per minute) with or without an ACEi (lisinopril, 100 mg/L in the drinking water) for 12 days. Blood pressure was monitored by tail-cuff method and telemetry. Ang II content was determined by radioimmunoanalysis. Ang II infusions increased 24-hour mean arterial pressure significantly (141.0±3.7 mm Hg) versus controls (110.0±1.0 mm Hg). ACEi prevented the increase in concentration in Ang II–infused mice (Ang II+ACEi; 114.0±7.4 mm Hg; P value not significant). Plasma Ang II content was significantly increased by Ang II (367±60 fmol/mL) versus controls (128±22 fmol/mL; P<0.05); plasma Ang II was not altered by ACEi alone (90±31) or in combination with Ang II infusions (76±27). Intrarenal Ang II content was significantly increased by Ang II (998±143 fmol/g) versus controls (524±60 fmol/g; P<0.05), and this was prevented by ACEi (Ang II+ACEi; 484±102 fmol/g; P value not significant). Thus, ACEi ameliorates the increases in blood pressure and intrarenal Ang II content caused by Ang II infusions, indicating that endogenous ACE-mediated Ang II formation plays a significant role in the increases of blood pressure and intrarenal Ang II during Ang II–induced hypertension.

Intrarenal Angiotensin-Converting Enzyme Induces Hypertension in Response to Angiotensin I Infusion

The contribution of the intrarenal renin-angiotensin system to the development of hypertension is incompletely understood. Here, we used targeted homologous recombination to generate mice that express angiotensin-converting enzyme (ACE) in the kidney tubules but not in other tissues. Mice homozygous for this genetic modification (ACE 9/9 mice) had low BP levels, impaired ability to concentrate urine, and variable medullary thinning. In accord with the ACE distribution, these mice also had reduced circulating angiotensin II and high plasma renin concentration but maintained normal kidney angiotensin II levels. In response to chronic angiotensin I infusions, ACE 9/9 mice displayed increased kidney angiotensin II, enhanced rate of urinary angiotensin II excretion, and development of hypertension. These findings suggest that intrarenal ACE-derived angiotensin II formation, even in the absence of systemic ACE, increases kidney angiotensin II levels and promotes the development of hypertension.