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The monocyte chemoattractant protein-1 (MCP-1)/CC-chemokine receptor 2 (CCR2) pathway plays a critical role in the development of antiglomerular basement membrane (anti-GBM) nephritis. We recently showed angiotensin II (Ang II) infusion in rats activated MCP-1 and transforming growth factor-β1 (TGF-β1), which in turn induced macrophage infiltration of renal tissues. This study was performed to demonstrate that combination therapy with a CCR2 antagonist (CA) and an Ang II type 1 receptor blocker (ARB) ameliorated renal injury in the anti-GBM nephritis model. An anti-GBM nephritis rat model developed progressive proteinuria and glomerular crescent formation, accompanied by increased macrophage infiltration and glomerular expression of MCP-1, angiotensinogen, Ang II, and TGF-β1. Treatment with CA alone or ARB alone moderately ameliorated kidney injury; however, the combination treatment with CA and ARB dramatically prevented proteinuria and markedly reduced glomerular crescent formation. The combination treatment also suppressed the induction of macrophage infiltration, MCP-1, angiotensinogen, Ang II, and TGF-β1 and reversed the fibrotic change in the glomeruli. Next, primary cultured glomerular mesangial cells (MCs) stimulated by Ang II showed significant increases in MCP-1 and TGF-β1 expression. Furthermore, cocultured model consisting of MCs, parietal epithelial cells, and macrophages showed an increase in Ang II-induced cell proliferation and collagen secretion. ARB treatment attenuated these augmentations. These data suggest that Ang II enhances glomerular crescent formation of anti-GBM nephritis. Moreover, our results demonstrate that inhibition of the MCP-1/CCR2 pathway with a combination of ARB effectively reduces renal injury in anti-GBM nephritis. [ABSTRACT FROM AUTHOR]

Intrarenal angiotensinogen (AGT) is expressed highly in renal proximal tubular cells (RPTCs) and contributes to the regulation of intrarenal angiotensin II levels. Inhibition of nuclear factor (NF)-κB suppressed human (h)AGT expression in human RPTCs. However, the presence and localization of an NF-κB binding site in the hAGT promoter region have not been determined. Therefore, this study was performed to demonstrate that an NF-κB binding site in the hAGT promoter region contributes to hAGT promoter activity in human RPTCs. The hAGT promoter region was cloned from -4358 to +122 and deletion analysis was performed. A possible NF-κB binding site was removed from the hAGT promoter region (M1) and mutated (M2). Human RPTCs were transfected, and hAGT promoter activity was determined by luciferase assay. The identity of DNA binding proteins from binding assays were determined by Western blot. Progressive 5'-end deletions demonstrated removal of a distal promoter element in hAGT_-2414/+122 reduced promoter activity (0.61 ± 0.12, ratio to hAGT_-4358/+122). Inhibition of NF-κB suppressed promoter activity in hAGT_-4358/+122 (0.51 ± 0.14, ratio to control) and hAGT_-3681/+122 (0.48 ± 0.06, ratio to control) but not in the construct without the NF-κB binding site. Promoter activity was reduced in the domain mutants M1 (0.57 ± 0.08, ratio to hAGT_-4358/+122) and M2 (0.61 ± 0.16, ratio to hAGT_-4358/+122). DNA binding levels of NF-κB protein were reduced in M1. These data demonstrate the functional importance of an NF-κB binding site in the hAGT promoter region, which contributes to hAGT promoter activity in human RPTCs. [ABSTRACT FROM AUTHOR]

This article discusses research into the role of endogenous angiotensin (Ang) II formation as a cause of the physiological effects of Ang II-induced hypertension on the kidneys. The experimenters used Ang-converting enzyme inhibitor (ACEi) as a means of regulating Ang II induced hypertension. The role of Ang II regulation in the protection of the renal system from abnormalities in renin-angiotensin system regulation is explored.

This article discusses research indicating that angiotensins in urine (UAGT) can serve as a biomarker for the renin-angiotensin system (RAS) status of hypertensive patients. In this experiment urinary creatinine levels and UAGT levels were tested as indicators of renin-angiotensin system health, as indicated by blood pressure, urinary albumin:creatinine ratios, and glomerular filtration rates.

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. [ABSTRACT FROM AUTHOR]

In 2-kidney 1-clip (2K1C) hypertension, intrarenal angiotensin II (Ang II) levels are increased in both kidneys but the mechanisms for augmentation of Ang II may be different. We recently reported that urinary angiotensinogen (AGT) is increased only in the non-clipped kidneys. However, it has not been determined if these changes are accompanied by augmentation of intrarenal AGT mRNA synthesis in the non-clipped kidneys of 2K1C hypertensive rats. Experiments were performed on male Sprague-Dawley rats (n=11) subjected to left renal arterial clipping (.25 mm gap) and followed for 18-21 days prior to anesthesia and separate measurements of renal function. Systolic arterial pressure increased to 180±3 mmHg compared to 126±4 mmHg in sham operated rats. There were no significant differences in water intake, body weights, and 24 hour urine volume and sodium excretion in awake rats. Separate measurements of renal function showed that renal plasma flow and glomerular filtration rate were similar in clipped and non-clipped kidneys and not different from those in sham rats. However, urine flow, sodium excretion and urinary AGT (uAGT) excretion were significantly greater in non-clipped kidneys compared to clipped and sham kidneys. While kidney AGT protein levels were not increased significantly, AGT transcript measured by real time RT-PCR revealed that the AGT mRNA levels in the cortex were 2.15 fold significantly greater in the non-clipped kidneys than in sham (1.0±.1) or clipped kidneys (.98±.15). The results support the hypothesis that in the non-clipped kidneys of 2K1C rats there is an augmentation of intrarenal AGT mRNA synthesis which explains increased uAGT excretion rates and intrarenal Ang II levels. [ABSTRACT FROM AUTHOR]

In type 1 diabetes mellitus (T1DM) there is increased prorenin secretion by the principal cells of the collecting duct. Binding of prorenin to prorenin receptor (PRR) on intercalated cells increases its catalytic activity, increases local angiotensin (Ang) II formation, and stimulates intracellular MAPK signaling responsible for inflammation and tissue fibrosis. Thus, changes in the amount of membrane bound PRR may be a key factor in stimulating these pathways. However, it has not been established that activation of PRR in the collecting duct contributes to increased intrarenal Ang II and tubulointerstitial inflammation via stimulation of inflammatory pathways including transforming growth factor-beta (TGF-β). This study tested the hypothesis that hyperglycemia increases the PRR abundance at the plasma membrane (PM) in the collecting duct cells, thus allowing greater capability to be activated by locally produced prorenin. Streptozotocin (STZ; 60 mg/kg; ip single dose) was used to induce T1DM in Sprague-Dawley rats (N=10) and compared to control rats (N=8). After 7-days induction, STZ-rats showed plasma glucose levels of 428±13 vs. 138±9 mg/dL and insulin of 0.05±0.02 vs. 2.4±0.6 ng/mL, compared to control. Although PRR transcript in the renal medulla were not different between groups; PRR localized predominantly on the apical aspects of collecting duct cells in STZ-induced rats; while in controls it was primarily found intracellularlly. These changes were accompanied by greater levels of active renin and Ang II in the urine and increased TGF-β mRNA levels in the renal medulla of STZ-rats (Renin: 186± 34 vs. 6± 3 ng Ang I/mL/h; P<0.01; Ang II: 884± 147 vs. 42± 14 fmol/h; P<0.05; TGF-β: 1.22 ± 0.06 vs. 0.97 ± 0.03 mRNA ratio; P<0.01). To further assess if hyperglycemia induced in vitro PRR trafficking alterations, collecting duct M-1 cells were treated with normal glucose (NG; 1mM glucose + 1 mM mannitol) and high glucose (HG; 4mM) for 5, 60, and 360 min. PRR protein levels were higher in the PM fractions in cells treated with HG, compared to cells treated with NG. Thus, hyperglycemia increases PRR abundance in the PM of the collecting duct and stimulates TGF-β synthesis in the renal medulla which may underlie the development of tubulointerstitial inflammation. [ABSTRACT FROM AUTHOR]

In angiotensin II (AngII) dependent hypertension, an inappropriate elevation of intrarenal Ang II leads to the progression of hypertension and kidney injury. Intrarenal angiotensinogen (AGT), produced mainly by renal proximal tubular cells (PTC), is a crucial factor in regulation of intratubular Ang II. Increases in intrarenal AGT are associated with increases in intrarenal immune cells and interleukin 6 (IL-6) levels which contribute to Ang II-dependent AGT augmentation. These results suggest that Ang II and IL-6 synergize to increase AGT levels during progression of Ang II-dependent hypertension. However, the interactions among Ang II, Ang II type 1 receptors (AT1R) and IL-6 on AGT expression and the mechanisms have not been delineated. In this study, mouse PTC (WT-PTC) and AT1aR knockout strains (AT1aRKO-PTC) were used to test the existence of this synergism between Ang II and IL-6. Basal AGT mRNA expression levels in PTC were determined and compared with that in mouse mesangial cells which is another established AGT-expressing kidney cells. AGT mRNA expression levels were measured by real-time RT-PCR and normalized based on GAPDH levels. WT-PTC had 21.1-fold (± 2.0) higher basal AGT mRNA levels than mesangial cells, indicating markedly high AGT production by the PTC. Further, WT-PTC and AT1aRKO-PTC were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Stimulation with either Ang II or IL-6 alone did not significantly alter AGT mRNA expression in both WT-PTC and AT1RaKO-PTC. Co-stimulation with Ang II and IL-6 increased AGT mRNA levels (1.59 ± 0.04, ratio to control) in WT-, but not AT1aRKO-PTC. To test for involvement of the NF-κB signaling pathway in the synergism between Ang II and IL-6, activation of NF-κB as an AT1R signaling pathway was evaluated by western blot analysis. Furthermore, WT-PCT were treated with an NF-κB inhibitor during the co-stimulation. In WT-PTC, IκB levels were decreased by the co-stimulation (0.61 ± 0.04, ratio to control) indicating NF-κB activation. Ten μM Parthenolide, an NF-κB inhibitor, attenuated the AGT mRNA increase induced by the co-stimulation. These results suggest that simultaneous AT1R activation acts synergistically with IL-6 to increase AGT expression in mouse PTC and that the signaling pathway involves NF-κB. [ABSTRACT FROM AUTHOR]

The development of glomerulonephritis causes glomerular injury and reduced renal function and is associated with increased renin release. The aims of this study were to demonstrate activation of intrarenal RAS in glomerulonephritis and determine the effects of DRI with aliskiren on progression of glomerulonephritis associated renal injury induced by administration of an anti-Thy1.1 antibody. We divided rats into 3 groups; control group, anti-Thy1.1 glomerulonephritis group and anti-Thy1.1 glomerulonephritis treated with aliskiren (30 mg/kg/day) group. In the anti-Thy1.1 rats, protein excretion and micro albumin excretion levels increased markedly after anti-Thy1.1 antibody injection. The increases in protein (94.7 ± 13.0 mg/day) and micro albumin (7.5 ± 2.6 mg/day) excretions were attenuated by aliskiren treatment (43.6 ± 4.5 mg/day of protein and 2.6 ± 0.7 mg/day of micro albumin). The glomerular expansion score was significantly higher in the anti-Thy1.1 group compared to the control group. The anti-Thy1.1 group also showed elevation of intrarenal TGF-β and PAI-1 mRNA levels compared with control rats. The progression of glomerular expansion and elevation of these mRNA levels was prevented by aliskiren. Importantly, the anti-Thy1.1 group had markedly increased urinary angiotensinogen (AGT) excretion at days 3 (3102.9 ± 683.8 ng/day), but these changes were markedly suppressed by aliskiren treatment (441.3 ± 104.8 ng/day). AGT protein expression in the kidney was significantly lower in the aliskiren treatment group compared with anti-Thy1.1 group (western blot, 0.8 ± 0.04 and immunohistochemistry, 0.7 ± 0.11 relative to anti-Thy1.1 group). Furthermore, plasma renin activity (PRA) and plasma angiotensin (Ang) II levels were significantly increased in the anti-Thy1.1 group and aliskiren treatment prevented the elevation of PRA and plasma Ang II. Anti-Thy1.1 antibody also elicited left ventricular hypertrophy and fibrosis, which were not ameliorated by aliskiren. These results demonstrate that DRI prevents or reduces intrarenal RAS activation and glomerular injury in progressive glomerulonephritis. Accordingly, DRI may be an effective approach to treat glomerulonephritis as well as other intrarenal RAS associated kidney diseases. [ABSTRACT FROM AUTHOR]

High prorenin levels in the circulation are associated with the progression of diabetic nephropathy and microvascular changes in diabetes. In spite of high plasma prorenin, the urine of diabetes mellitus patients exhibits very low pH and contains high levels of “active” renin, but low levels of prorenin. Prorenin binding to the prorenin receptor (PRR) results in non-proteolytic activation of prorenin and stimulation of intracellular synthesis of transforming growth factor-beta (TGF-β). Furthermore, the PRR cytosolic domain is associated with the vacuolar H+-ATPase, which is involved in acid-base homeostasis by renal intercalated cells. In diabetes, the collecting duct is the major source of prorenin and PRR is upregulated in the kidneys of diabetic rats. We examined the relationships among urine levels of renin (uRen), prorenin (uPro), soluble PRR (sPRR) and TGF-β (uTGF-β) in 93 subjects, including non-diabetes mellitus (Non-DM: n=64); and type-2 diabetes mellitus (DM; n=29) patients. Urine albumin (uAlb), creatinine (uCr) and pH were also assessed. DM patients showed higher uRen levels (DM: 81±21 vs Non-DM: 29±4.5 ng AngI/mL/hr; p<0.001), which were positively associated with uAlb/uCr (p<0.05, r=0.21) and inversely correlated with urine pH (p<0.01, r=-0.38). These values contrasted with uPro levels, which were no different between groups. DM patients showed increased uTGF-β compared to Non-DM (DM: 22±5.3 vs Non-DM: 5.6±1.5 pg/mg uCr; p<0.0001). Urine sPRR levels were positively correlated with urine pH (p<0.0001, r=0.56). No differences were observed in sPRR plasma and urine levels between non-DM and DM patients. However, the plasma levels of sPRR were 12.5 and 18.5-fold higher than in urine of Non-DM and DM, respectively. Treatment with RAS blockers increased sPRR in plasma (DM with RAS blockade: 24,455±2,151 vs DM without RAS blockade: 17,726±1,255 pg/mL, p<0.001) but not in urine. These data suggest that the cell membrane-bound PRR, not the soluble form, might be responsible for the increases in renin activity and TGF-β, as well as in the low pH in the urine of type-2 DM patients. It is likely that prorenin-mediated activation of membrane bound PRR promotes diabetic nephropathy by increasing urinary formation of TGF-β. [ABSTRACT FROM AUTHOR]

Young female humans and animals are protected from the development of hypertension and related end-organ damage when compared to age-matched males. We recently showed that high salt (HS) worsened preexisting hypertension and renal injury to a greater extent in Sprague-Dawley (SD) male than female rats. It has been shown that epigenetic modifications can contribute to hypertension and that HS increases the correlation between histone and DNA modifications and gene expression. However, it remains unclear if HS diet is the leading cause of these epigenetic modifications, thereby increasing the predisposition to hypertension. We hypothesize that HS consumption exacerbates sex disparity in the expression of chromatin modification genes, explaining the gender disparity in the susceptibility to hypertension. To test this hypothesis we used SD rats (7±1 week of age), a model of non-sensitive salt hypertension to minimize the influence of genetic susceptibility. We determined the expression levels of chromatin modification genes in male and female rats and the effects of HS diet (8% NaCl for 2-weeks) on the epigenetic changes. We used cDNAs that were prepared from RNA samples extracted from kidney cortexes and PCR array to screen 84 rat epigenetic chromatin modification enzyme genes (RT2 Profiler PCR Array, QIAGEN, Inc, Valencia, CA). Analysis of 9 genes (Aof1, AurkA, AurkB, AurkC, Hdac9, Pak1, Setd6, Smyd3 and Suv420h2) showed different expression levels between sexes, and these were further altered by HS diet. Importantly, the histone deacetylase 9 (Hdac9) gene was expressed significantly higher in the female controls compared to males (p<0.05); however, this difference decreased in response to HS diet. Hdac9 has recently been reported to be associated with a high risk for hypertension. We concluded that in the rat kidney the expression of genes involved in histone modification is sexually dimorphic and these genes are differentially altered by HS diet. Thus, the epigenetic effects of HS consumption may help to explain the mechanisms underlying the sexual disparities in genetic susceptibility to diseases with complex environment-gene interaction, such as hypertension. [ABSTRACT FROM AUTHOR]

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC. [ABSTRACT FROM AUTHOR]