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1.  Angiotensin II Infusion Induces Nephrin Expression Changes and Podocyte Apoptosis 
American Journal of Nephrology  2008;28(3):500-507.
Background/Aim
In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion.
Methods
Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng·kg–1·min–1) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.
Results
The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = −0.63, p < 0.05).
Conclusion
These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.
doi:10.1159/000113538
PMCID: PMC2630486  PMID: 18204248
Angiotensin II; Proteinuria; Nephrin expression; Podocyte; Apoptosis
2.  Angiotensin II induces nephrin dephosphorylation and podocyte injury: Role of caveolin-1 
Cellular signalling  2011;24(2):443-450.
Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10−6 M) pretreated with or without losartan (10−5 M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.
Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.
doi:10.1016/j.cellsig.2011.09.022
PMCID: PMC3237911  PMID: 21982880
Caveolin-1; Podocyte; Angiotensin II; Nephrin
3.  Disparate effects of eplerenone, amlodipine and telmisartan on podocyte injury in aldosterone-infused rats 
Background. Several studies in patients with primary aldosteronism (PA) have suggested that aldosterone (ALD) is directly contributing to albuminuria. However, there are limited data pertaining to the direct role of ALD in in vivo models in regard to the induction of renal injury and the involved mechanisms. In the present study, we established a high-dose ALD-infused rat model to evaluate urinary albumin excretion rate (UAER) and podocyte damage. Moreover, we studied the effect of eplerenone (EPL), telmisartan (TEL) and amlodipine (AML) on ALD-induced renal structural and functional changes.
Methods. Immunohistochemical and real-time PCR analyses, and TUNEL assays were performed to evaluate nephrin expression and podocyte injury.
Results. ALD-receiving rats (ARR) showed a progressive increase in BP, UAER and proteinuria when compared with control rats (CR). Conversely, BP was significantly reduced in ALD + EPL (A/ERR)-, ALD + AML (A/ARR)- and ALD + TEL (A/TRR)-treated rats. However, UAER and proteinuria were decreased only in A/ERR and A/TRR, but not in A/ARR. Only EPL administration provided protection against ALD-induced podocyte apoptosis. Renal tissue of ARR revealed enhanced expression of nephrin protein and mRNA. This effect of ALD was inhibited by EPL, but not by TEL or AML.
Conclusions. ALD induces direct glomerular injury independent of its haemodynamic effects; this effect of ALD is, at least in part, mediated through activation of the mineralocorticoid receptor.
doi:10.1093/ndt/gfq514
PMCID: PMC3108348  PMID: 20729265
aldosterone; amlodipine; eplerenone; podocyte; telmisartan
4.  Angiotensin II regulation of renal vascular ENaC proteins 
American journal of hypertension  2009;22(6):593-597.
Background
The importance of β and γ Epithelial Na+ Channel (ENaC) proteins in vascular smooth muscle cell mediated pressure-induced constriction in renal interlobar arteries has been demonstrated recently. In renal epithelial tissue, ENaC expression is regulated by angiotensin II (Ang II). However, whether or not Ang II regulates vascular ENaC expression has never been determined. Therefore, the goal of the current investigation was to determine if Ang II affects vascular ENaC expression and its contribution to pressure-induced constriction.
Methods
To address this goal, Sprague-Dawley rats were infused with Ang II (50 ng/kg/min) via osmotic mini-pump for 1 week. Mean arterial pressure was measured using radiotelemetry. Interlobar arteries were isolated from these animals to assess VSMC ENaC protein expression, pressure-induced constriction and agonist induced vascular reactivity.
Results
MAP was not different in control (113 ± 2) and Ang II (114 ± 2 mm Hg) infused mice. We found that Ang II infusion decreased renal VSMC β- and γENaC immunolabeling by 18%. Consistent with this finding, we also found that ENaC-dependant peak pressure-induced constriction was inhibited from 38 ± 3% to 25 ± 1% at 125 mm Hg. Vasoreactivity to KCl, phenylephrine and acetylcholine were unchanged.
Conclusions
Ang II suppression of pressure-induced constrictor responses in renal interlobar arteries may be mediated, at least in part, by inhibition of β and γENaC protein expression.
doi:10.1038/ajh.2009.59
PMCID: PMC2787100  PMID: 19325536
5.  CHBPR: Decreased cGMP level contributes to increased contraction in arteries from hypertensive rats: role of PDE1 
Hypertension  2011;57(3):655-663.
Recent evidence suggests that angiotensin II (Ang II) upregulates phosphodiesterase (PDE)-1A expression. We hypothesized that Ang II augmented PDE1 activation, decreasing the bioavailability of cyclic cyclic guanosine 3', 5'-monophosphate (cGMP), contributing to increased vascular contractility. Male Sprague-Dawley rats received mini-osmotic pumps with Ang II (60 ng.min−1) or saline for 14 days. PE-induced contractions were increased in aorta (Emax168±8 vs. 136±4%) and small-mesenteric arteries [(SMA), Emax170±6 vs. 143±3%] from Ang II infused rats compared to control. PDE1 inhibition with vinpocetine (10µM) reduced PE-induced contraction in aortas from Ang II rats (Emax94±12%) but not in control (154±7%). Vinpocetine decreased the sensitivity to PE in SMA from Ang II rats compared to vehicle (pD2 5.1±0.1 vs. 5.9±0.06), but not in control (6.0±0.03 vs. 6.1±0.04). Sildenafil (10µM), a PDE5 inhibitor reduced PE-induced maximal contraction similarly in Ang II and control rats. Arteries were contracted with PE (1µM) and concentration-dependent relaxation to vinpocetine and sildenafil was evaluated. Aortas from Ang II rats displayed increased relaxation to vinpocetine compared to control (Emax82±12 vs. 44±5%). SMA from Ang II rats showed greater sensitivity during vinpocetine-induced relaxation, compared to control (pD2 6.1±0.3 vs. 5.3±0.1). No differences in sildenafil-induced relaxation were observed. PDE1A and PDE1C expressions in aorta and PDE1A expression in SMA were increased in Ang II rats. cGMP production, which is decreased in arteries from Ang II rats, was restored after PDE1 blockade. We conclude that PDE1 activation reduces cGMP bioavailability in arteries from ANG II, contributing to increased contractile responsiveness.
doi:10.1161/HYPERTENSIONAHA.110.164327
PMCID: PMC3074298  PMID: 21282562
PDE1; angiotensin II; cGMP; hypertension; vinpocetine
6.  Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production 
Kidney international  2002;61(2):579-585.
Background
In rats maintained on a high salt diet (H/S) to suppress basal renal angiotensinogen levels, angiotensin II (Ang II) infusion for 13 days increased renal angiotensinogen mRNA and protein, thus providing a mechanism for further augmentation of intrarenal Ang II levels. The present study tested the hypothesis that enhanced intrarenal angiotensinogen formation during Ang II infusion is reflected by secretion into the tubular fluid leading to increased urinary excretion of angiotensinogen (UAGT).
Methods
The effects of chronic Ang II infusion were examined on kidney and plasma Ang II levels and UAGT in male Sprague-Dawley rats maintained on an 8% salt diet for three weeks (N = 10). Following one week on the H/S diet, Ang II (40 ng/min) was administered for two weeks via an osmotic minipump to one group (H/S + Ang II, N = 5), while the remaining rats were sham-operated (H/S + Sham, N = 5). Additionally, a control group was prepared with normal salt diet and sham-operation (N/S + Sham, N = 5).
Results
H/S alone did not alter systolic blood pressure (BP) (103 ± 2 vs. 104 ± 2 mm Hg), while Ang II infusion to H/S rats significantly increased systolic BP from 103 ± 2 to 154 ± 2 after two weeks. Intrarenal Ang II content in H/S + Ang II was significantly greater than H/S + Sham (435 ± 153 vs. 65 ± 14 fmol/g). Ang II infusion significantly increased UAGT (4.0 ± 0.5 vs. 1.0 ± 0.2 nmol Ang I/day by radioimmunoassay of generated Ang I; 57 ± 15 vs. 14 ± 2 densitometric units by Western blotting analysis) compared to Sham. UAGT by radioimmunoassay was highly correlated with kidney Ang II content (r = 0.79); but not with plasma Ang II concentration (r = 0.20).
Conclusions
These data demonstrate that chronic Ang II infusion increases urinary excretion rate of angiotensinogen, and suggest that UAGT provides a specific index of intrarenal angiotensinogen production in Ang II-dependent hypertension.
doi:10.1046/j.1523-1755.2002.00155.x
PMCID: PMC2575665  PMID: 11849399
Ang II; kidney; urine; high salt diet; hypertension; reninangiotensin system; blood pressure
7.  Puerarin Attenuated Early Diabetic Kidney Injury through Down-Regulation of Matrix Metalloproteinase 9 in Streptozotocin-Induced Diabetic Rats 
PLoS ONE  2014;9(1):e85690.
Radix puerariae, a traditional Chinese herbal medication, has been used successfully to treat patients with early stage of diabetic nephropathy. However, the underlined mechanism of this renal protective effect has not been determined. In the current study, we investigated the effects and the mechanism of puerarin in Streptozotocin (STZ)-induced diabetic rats. We treated STZ-rats with either puerarin or losartan, an angiotensin II receptor blocker, as compared to those treated with vehicle. We found that both puerarin and losartan attenuated kidney hypertrophy, mesangial expansion, proteinuria, and podocyte foot process effacement in STZ rats. In addition, both puerarin and losartan increased expression of podocyte slit diaphragm proteins such as nephrin and podocin. Interestingly, we found that puerarin treatment induced a more pronounced suppression of oxidative stress production and S-nitrosylation of proteins in the diabetic kidneys as compared to losartan treatment. Furthermore, we found that matrix metalloproteinase-9 (MMP-9), which is known to be activated by oxidative stress and S-nitrosylation of proteins, was also suppressed more extensively by puerarin than losartan. In conclusion, these data provide for the first time the potential mechanism to support the use of puerarin in the treatment of early diabetic nephropathy.
doi:10.1371/journal.pone.0085690
PMCID: PMC3893265  PMID: 24454919
8.  CENTRAL ANG-(1–7) ENHANCES BAROREFLEX GAIN IN CONSCIOUS RABBITS WITH HEART FAILURE 
Hypertension  2011;58(4):627-634.
In chronic heart failure (CHF), arterial baroreflex function is impaired, in part, by activation of the central renin-angiotensin system. A metabolite of Angiotensin II (Ang II), Ang-(1–7), has been shown to exhibit cardiovascular effects that are in opposition to that of Ang II. However, the action of Ang-(1–7) on sympathetic outflow and baroreflex function is not well understood, especially in CHF. The aim of this study was to determine the effect of intracerebroventricular infusion of Ang-(1–7) on baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) in conscious rabbits with CHF. We hypothesized that central Ang-(1–7) would improve baroreflex function in CHF. Ang-(1–7) (2 nmol/1 μl/hour) or artificial cerebrospinal fluid (1 μl/hour) was infused by an osmotic mini-pump for 4 days in sham and pacing-induced CHF rabbits (n=3–6/group). Ang-(1–7) treatment had no effects in sham rabbits but reduced HR and increased baroreflex gain (7.4±1.5 bpm/mm Hg vs. 2.5±0.4 bpm/mm Hg, P<0.05) in CHF rabbits. The Ang-(1–7) antagonist A779 (8 nmol/1 μl/hr) blocked the improvement in baroreflex gain in CHF. Baroreflex gain increased in CHF+Ang-(1–7) animals when only the vagus was allowed to modulate baroreflex control by acute treatment with the β-1 antagonist metoprolol, indicating increased vagal tone. Baseline RSNA was significantly lower and baroreflex control of RSNA was enhanced in CHF rabbits receiving Ang-(1–7). These data suggest that augmentation of central Ang-(1–7) inhibits sympathetic outflow and increases vagal outflow in CHF thus contributing to enhanced baroreflex gain in this disease state.
doi:10.1161/HYPERTENSIONAHA.111.177600
PMCID: PMC3187859  PMID: 21844487
angiotensin-(1–7); heart failure; sympathetic nervous system; baroreflex; vagus nerve; blood pressure; heart rate
9.  Expression of Angiotensinogen mRNA and Protein in Angiotensin II-Dependent Hypertension 
Chronic elevations in circulating angiotensin II (AngII) levels produce sustained hypertension and increased intrarenal AngII contents through multiple mechanisms, which may include sustained or increased local production of AngII. This study was designed to test the hypothesis that chronic AngII infusion increases renal angiotensinogen mRNA and protein levels, thus contributing to the increase in intrarenal AngII levels. AngII (80 ng/min) was infused subcutaneously for 13 d into Sprague-Dawley rats, using osmotic minipumps. Control rats underwent sham operations. By day 12, systolic arterial BP increased to 184 ± 3 mmHg in AngII-treated rats, whereas values for sham-treated rats remained at control levels (125 ± 1 mmHg). Plasma renin activity was markedly suppressed (0.2 ± 0.1 versus 5.3 ± 1.2 ng AngI/ml per h); however, renal AngII contents were significantly increased in AngII-treated rats (273 ± 29 versus 99 ± 18 fmol/g). Western blot analyses of plasma and liver protein using a polyclonal anti-angiotensinogen antibody demonstrated two specific immunoreactive bands, at 52 and 64 kD, whereas kidney tissue exhibited one band, at 52 kD. Densitometric analyses demonstrated that AngII infusion did not alter plasma (52- or 64-kD), renal (52-kD), or hepatic (52-kD) angiotensinogen protein levels; however, there was a significant increase in hepatic expression of the highly glycosylated 64-kD angiotensinogen protein, of almost fourfold (densitometric value/control value ratios of 3.79 ± 1.16 versus 1.00 ± 0.35). Renal and hepatic expression of angiotensinogen mRNA, which was examined by semiquantitative reverse transcription-PCR, was significantly increased in AngII-treated rats, compared with sham-treated rats (kidney, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 0.82 ± 0.11 versus 0.58 ± 0.04; liver, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 2.34 ± 0.07 versus 1.32 ± 0.15). These results indicate that increases in circulating AngII levels increase intrarenal angiotensinogen mRNA levels, which may contribute to the sustained renal AngII-generating capacity that paradoxically occurs in AngII-treated hypertensive rats.
PMCID: PMC2573050  PMID: 11181790
10.  Time-Course and Mechanisms of Restored Vascular Relaxation by Reduced Salt Intake and Angiotensin II Infusion in Rats Fed a High-Salt Diet 
Objective:
This study determined the mechanisms and time-course of recovery of vascular relaxation in middle cerebral arteries (MCA) of salt-fed Sprague-Dawley rats returned to low salt (LS) diet (0.4% NaCl) or infused with low-dose angiotensin II (ANG II).
Methods:
Rats were fed high salt (HS) diet (4% NaCl) for 3 days or 4 weeks before return to LS diet for various periods. Other rats fed HS diet (HS + ANG II) received a chronic (3 days) i.v. infusion of a low dose of ANG II (5 ng·kg−1·min−1) to prevent salt-induced ANG II suppression.
Results:
HS diet eliminated the increase in cerebral blood flow in response to acetylcholine (ACh) infusion and the relaxation of MCA in response to ACh, iloprost, cholera toxin, and reduced PO2. Recovery of vascular relaxation was slow, requiring at least 2 weeks of LS diet, regardless of the duration of exposure to HS diet. Hypoxic dilation was mediated by cyclooxygenase metabolites and ACh-induced dilation was mediated via NO in LS rats and in HS rats returned to LS diet or receiving ANG II infusion.
Conclusions:
Return to LS diet for 2 weeks or chronic 3-day ANG II infusion restore the mechanisms that normally mediate cerebral vascular relaxation.
doi:10.1080/10739680802544177
PMCID: PMC2652508  PMID: 19235625
angiotensin II; salt; vascular relaxation; vasodilation; endothelium; vascular smooth muscle
11.  AT1 Receptor Mediated Augmentation of Intrarenal Angiotensinogen in Angiotensin II-Dependent Hypertension 
Hypertension  2004;43(5):1126-1132.
Angiotensin (Ang) II-infused hypertensive rats exhibit increases in renal angiotensinogen mRNA and protein, as well as urinary angiotensinogen excretion in association with increased intrarenal Ang II content. The present study was performed to determine if the augmentation of intrarenal angiotensinogen requires activation of Ang II type 1 (AT1) receptors. Male Sprague-Dawley rats (200 to 220 g) were divided into 3 groups: sham surgery (n=10), subcutaneous infusion of Ang II (80 ng/min, n=11), and Ang II infusion plus AT1 blocker (ARB), olmesartan (5 mg/d, n=12). Ang II infusion progressively increased systolic blood pressure (SBP) compared with sham (178±8 mm Hg versus119±4 at day 11). ARB treatment prevented hypertension (113±6 at day 11). Twenty-four-hour urine collections were taken at day 12, and plasma and tissue samples were harvested at day 13. The Ang II+ARB group had a significant increase in plasma Ang II compared with Ang II and sham groups (365±46 fmol/mL versus 76±9 and 45±14, respectively). Nevertheless, ARB treatment markedly limited the enhancement of kidney Ang II by Ang II infusion (65±17 fmol/g in sham, 606±147 in Ang II group, and 288±28 in Ang II+ARB group). Ang II infusion significantly increased kidney angiotensinogen compared with sham (1.69±0.21 densitometric units versus 1.00±0.17). This change was reflected by increased angiotensinogen immunostaining in proximal tubules. ARB treatment prevented this increase (1.14±0.12). Urinary angiotensinogen excretion rates were enhanced 4.7× in Ang II group (4.67±0.41 densitometric units versus 1.00±0.21) but ARB treatment prevented the augmentation of urinary angiotensinogen (0.96±0.23). These data demonstrate that augmentation of intrarenal angiotensinogen in Ang II-infused rats is AT1-dependent and provide further evidence that urinary angiotensinogen is closely linked to intrarenal Ang II in Ang II-dependent hypertension.
doi:10.1161/01.HYP.0000122875.91100.28
PMCID: PMC2637079  PMID: 15037565
angiotensin II; angiotensinogen; receptors; angiotensin II; rats; kidney
12.  Sustained renal interstitial macrophage infiltration following chronic angiotensin II infusions 
Chronic angiotensin (ANG) II infusions into rats lead to augmented intrarenal levels of ANG II and inflammatory factors, impaired renal function, and progressive hypertension. Residual effects persist after cessation of ANG II infusions, as manifested by a hypertensive response to high-salt intake. This study was performed to determine the residual cytokines and chemokines following the cessation of ANG II infusion. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG II-infused rats were further subdivided into three subgroups. Minipumps were removed on day 12 with subsequent harvesting of kidneys at 0, 3, and 6 days after cessation of ANG II infusion. After 12 days of ANG II infusion, systolic blood pressure, interstitial fibrosis, preglomerular hypertrophy, and interstitial macrophage infiltration were significantly enhanced compared with the shams. By 3 days following the cessation of ANG II infusion, systolic blood pressure was normalized; however, interstitial fibrosis and preglomerular hypertrophy were still present. Furthermore, increased interstitial macrophage infiltration was still present 6 days after cessation of ANG II infusion. Importantly, augmented mRNA levels of monocyte chemotactic protein (MCP)-1 (1.55 ± 0.15 vs. 1.00 ± 0.13, relative ratio) and transforming growth factor (TGF)-β1 (1.52 ± 0.16 vs. 1.00 ± 0.08) persisted 6 days after the withdrawal of ANG II infusion (1.60 ± 0.20 for MCP-1 and 1.43 ± 0.17 for TGF-β1). Thus, the ANG II-induced activation of MCP-1 and TGF-β1 is sustained and may account for the persistent effect of chronic ANG II infusions on interstitial macrophage infiltration, suggesting a possible mechanism for the development of salt sensitivity in ANG II-dependent hypertension.
doi:10.1152/ajprenal.00059.2006
PMCID: PMC2001287  PMID: 16804106
kidney; tissue injury; cytokines; chemokines; residual effects
13.  Enhancement of Angiotensinogen Expression in Angiotensin II–Dependent Hypertension 
Hypertension  2001;37(5):1329-1335.
Chronic infusion of angiotensin (Ang) II leads to the development of hypertension and enhances intrarenal Ang II content to levels greater than can be explained from the circulating concentrations of the peptide. We previously reported that renal angiotensinogen (Ao) mRNA is enhanced in Ang II–dependent hypertension and may contribute to augmented intrarenal Ang II levels, but the Ao protein levels were not significantly increased. Because a high-salt diet (H/S) has been shown to suppress renal expression of Ao mRNA, we examined the effects of chronic Ang II infusion on kidney and liver Ao mRNA and protein levels in male Sprague-Dawley rats (n=12) maintained on an 8% salt diet. Ang II was administered via osmotic minipumps (40 ng/min) to 1 group (n=6) while the remaining rats were sham-operated. A H/S diet alone did not alter systolic blood pressure in sham animals (109±6 mm Hg at day 12); however, Ang II infusions to the H/S rats significantly increased systolic blood pressure (167±7 at day 12) and intrarenal Ang II content (459±107 fmol/g versus 270±42) despite a marked suppression of plasma renin activity (0.9±0.2 ng Ang I·mL−1·h−1 versus 2.8±1.3). Ang II infusions significantly increased kidney Ao mRNA compared with the H/S diet alone by 1.9±0.1-fold. Western blot analysis of kidney protein extracts showed that the Ang II–infused rats had increased kidney Ao protein levels compared with the H/S diet alone (1.9±0.1-fold). Liver Ao mRNA and protein and plasma Ao protein were also significantly increased by Ang II infusions. These data demonstrate the effects of Ang II infusion to stimulate Ao mRNA and protein. Thus, the augmented intrarenal Ang II in Ang II–dependent hypertension may result, in part, by a positive amplification mechanism to activate renal expression of Ao.
PMCID: PMC2575649  PMID: 11358949
angiotensin II; angiotensinogen; kidney; sodium, dietary; Western blot; reverse transcriptase–polymerase chain reaction
14.  Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE−/− mice 
Clinical Science (London, England : 1979)  2010;118(Pt 11):681-689.
AngII (angiotensin II) induces atherosclerosis and AAAs (abdominal aortic aneurysms) through multiple proposed mechanisms, including chemotaxis. Therefore, we determined the effects of whole-body deficiency of the chemokine receptor CCR2 (CC chemokine receptor 2) on these diseases. To meet this objective, apoE (apolipoprotein E)−/− mice that were either CCR2+/+ or CCR2−/−, were infused with either saline or AngII (1000 ng·kg−1 of body weight·min−1) for 28 days via mini-osmotic pumps. Deficiency of CCR2 markedly attenuated both atherosclerosis and AAAs, unrelated to systolic blood pressure or plasma cholesterol concentrations. During the course of the present study, we also observed that AngII infusion led to large dilatations that were restricted to the ascending aortic region of apoE−/− mice. The aortic media in most of the dilated area was thickened. In regions of medial thickening, distinct elastin layers were discernable. There was an expansion of the distance between elastin layers in a gradient from the intimal to the adventitial aspect of the media. This pathology differed in a circumscribed area of the anterior region of ascending aortas in which elastin breaks were focal and almost transmural. All regions of the ascending aorta of AngII-infused mice had diffuse medial macrophage accumulation. Deficiency of CCR2 greatly attenuated the AngII-induced lumen dilatation in the ascending aorta. This new model of ascending aortic aneurysms has pathology that differs markedly from AngII-induced atherosclerosis or AAAs, but all vascular pathologies were attenuated by CCR2 deficiency.
doi:10.1042/CS20090372
PMCID: PMC2841499  PMID: 20088827
angiotensin; aneurysm; aorta; chemokine; elastin; macrophage; AngII, angiotensin II; AAA, abdominal aortic aneurysm; apoE, apolipoprotein E; AT1 receptor, AngII type 1 receptor; CCR2, CC chemokine receptor 2; MCP, monocyte chemoattractant protein; TGF-β, transforming growth factor-β
15.  Inhibiting Amadori-modified albumin formation improves biomarkers of podocyte damage in diabetic rats 
Physiological Reports  2013;1(4):e00083.
Recent studies have shown that urinary excretion of podocyte proteins is an indicator of podocyte injury, and that podocyte abnormalities and elevated concentrations of Amadori-modified glycated albumin (AGA) are linked to the development of diabetic nephropathy and to each other. We evaluated relationships between urinary markers of podocyte damage, increased AGA and filtration function in rats made diabetic by streptozotocin injection and treated for 8 weeks with a compound that inhibits the formation of AGA, with age-matched nondiabetic and diabetic rats serving as controls. Blood and urine were collected for measurement of glycated albumin, creatinine, albumin, nephrin, podocalyxin, and βig-h3 protein. The elevated circulating concentrations of glycated albumin and higher urinary levels of these podocyte markers as well as of albumin that were observed in diabetic rats compared with nondiabetic controls were significantly reduced in animals receiving test compound, and decrease in urinary biomarkers correlated with reduction in AGA. The results provide evidence that lowering the concentration of AGA, independent of filtration status and hyperglycemia, reduces urinary nephrin, podocalyxin, and βig-h3 protein, linking the increased glycated albumin associated with diabetes to podocyte abnormalities and shedding of podocyte proteins into the urine.
doi:10.1002/phy2.83
PMCID: PMC3831899  PMID: 24303153
Amadori-modified albumin; nephrin; podocalyxin; podocyte damage; βig-h3 protein
16.  Protective effects of PPARγ agonist in acute nephrotic syndrome 
Background.
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have beneficial effects on renal structure and function in models of diabetes and chronic kidney diseases. However, the increased incidence of weight gain and edema potentially limits their usefulness. We studied an acute minimal-change disease-like nephrotic syndrome model to assess effects of PPARγ agonist on acute podocyte injury and effects on fluid homeostasis.
Methods.
Acute podocyte injury and nephrotic syndrome were induced by puromycin aminonucleoside (PAN) injection in rats.
Results.
PPARγ agonist, given at the time or after, but not before PAN, reduced proteinuria, restored synaptopodin, decreased desmin and trended to improve foot process effacement. There was no significant difference in glomerular filtration, effective circulating volume, blood pressure or fractional sodium excretion. PAN-injured podocytes had decreased PPARγ, less nephrin and α-actinin-4, more apoptosis and reduced phosphorylated Akt. In PAN-injured cultured podocytes, PPARγ agonist also reversed abnormalities only when given simultaneously or after injury.
Conclusions.
These results show that PPARγ agonist has protective effects on podocytes in acute nephrotic syndrome without deleterious effects on fluid homeostasis. PPARγ agonist-induced decrease in proteinuria in acute nephrotic syndrome is dependent at least partially on regulation of peroxisome proliferator-response element-sensitive gene expression such as α-actinin-4 and nephrin and the restoration of podocyte structure.
doi:10.1093/ndt/gfr240
PMCID: PMC3276311  PMID: 21565943
nephrotic syndrome; podocytes; PPARγ; puromycin aminonucleoside
17.  Early Glomerular Filtration Defect and Severe Renal Disease in Podocin-Deficient Mice 
Molecular and Cellular Biology  2004;24(2):550-560.
Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2−/−) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2−/− mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2−/− kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.
doi:10.1128/MCB.24.2.550-560.2004
PMCID: PMC343810  PMID: 14701729
18.  Receptor-Induced Dilatation in the Systemic and Intrarenal Adaptation to Pregnancy in Rats 
PLoS ONE  2009;4(3):e4845.
Normal pregnancy is associated with systemic and intrarenal vasodilatation resulting in an increased glomerular filtration rate. This adaptive response occurs in spite of elevated circulating levels of angiotensin II (Ang II). In the present study, we evaluated the potential mechanisms responsible for this adaptation. The reactivity of the mesangial cells (MCs) cultured from 14-day-pregnant rats to Ang II was measured through changes in the intracellular calcium concentration ([Cai]). The expression levels of inducible nitric oxide synthase (iNOS), the Ang II-induced vasodilatation receptor AT2, and the relaxin (LGR7) receptor were evaluated in cultured MCs and in the aorta, renal artery and kidney cortex by real time-PCR. The intrarenal distribution of LGR7 was further analyzed by immunohistochemistry. The MCs displayed a relative insensitivity to Ang II, which was paralleled by an impressive increase in the expression level of iNOS, AT2 and LGR7. These results suggest that the MCs also adapt to the pregnancy, thereby contributing to the maintenance of the glomerular surface area even in the presence of high levels of Ang II. The mRNA expression levels of AT2 and LGR7 also increased in the aorta, renal artery and kidney of the pregnant animals, whereas the expression of the AT1 did not significantly change. This further suggests a role of these vasodilatation-induced receptors in the systemic and intrarenal adaptation during pregnancy. LGR7 was localized in the glomeruli and on the apical membrane of the tubular cells, with stronger labeling in the kidneys of pregnant rats. These results suggest a role of iNOS, AT2, and LGR7 in the systemic vasodilatation and intrarenal adaptation to pregnancy and also suggest a pivotal role for relaxin in the tubular function during gestation.
doi:10.1371/journal.pone.0004845
PMCID: PMC2653634  PMID: 19287481
19.  The Directed Differentiation of Human iPS Cells into Kidney Podocytes 
PLoS ONE  2012;7(9):e46453.
The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm’s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.
doi:10.1371/journal.pone.0046453
PMCID: PMC3460883  PMID: 23029522
20.  Increased activity and expression of Ca2+-dependent NOS in renal cortex of ANG II-infused hypertensive rats 
The American journal of physiology  1999;277(5 Pt 2):F797-F804.
We have previously demonstrated that nitric oxide (NO) exerts a greater modulatory influence on renal cortical blood flow in ANG II-infused hypertensive rats compared with normotensive rats. In the present study, we determined nitric oxide synthase (NOS) activities and protein levels in the renal cortex and medulla of normotensive and ANG II-infused hypertensive rats. Enzyme activity was determined by measuring the rate of formation of l-[14C]citrulline from l-[14C]arginine. Western blot analysis was performed to determine the regional expression of endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms in the renal cortex and medulla of control and ANG II-infused rats. Male Sprague-Dawley rats were prepared by the infusion of ANG II at a rate of 65 ng/min via osmotic minipumps implanted subcutaneously for 13 days and compared with sham-operated rats. Systolic arterial pressures were 127 ± 2 and 182 ± 3 mmHg in control (n = 13) and ANG II-infused rats (n = 13), respectively. The Ca2+-dependent NOS activity, expressed as picomoles of citrulline formed per minute per gram wet weight, was higher in the renal cortex of ANG II-infused rats (91 ± 11) than in control rats (42 ± 12). Likewise, both eNOS and nNOS were markedly elevated in the renal cortex of the ANG II-treated rats. In both groups of rats, Ca2+-dependent NOS activity was higher in the renal medulla than in the cortex; however, no differences in medullary NOS activity were observed between the groups. Also, no differences in medullary eNOS levels were observed between the groups; however, medullary nNOS was decreased by 45% in the ANG II-infused rats. For the Ca2+-independent NOS activities, the renal cortex exhibited a greater activity in the control rats (174 ± 23) than in ANG II-infused rats (101 ± 10). Similarly, cortical iNOS was greater by 47% in the control rats than in ANG II-treated rats. No differences in the activity were found for the renal medulla between the groups. There was no detectable signal for iNOS in the renal medulla for both groups. These data indicate that there is a differential distribution of NOS activity, with the Ca2+-dependent activity and protein expression higher in the renal cortex of ANG II-infused rats compared with control rats, and support the hypothesis that increased constitutive NOS activity exerts a protective effect in ANG II-induced hypertension to maintain adequate renal cortical blood flow.
PMCID: PMC2574501  PMID: 10564245
endothelial nitric oxide synthase; neuronal nitric oxide synthase; inducible nitric oxide synthase; osmotic minipump; angiotensin II-induced hypertension; Western blot
21.  Silencing of hypoxia inducible factor-1α gene attenuated angiotensin II-induced renal injury in Sprague-Dawley rats 
Hypertension  2011;58(4):657-664.
Although it has been shown that up-regulation of hypoxia-inducible factor (HIF)-1α is protective in acute ischemic renal injury, long-term over-activation of HIF-1α is implicated to be injurious in chronic kidney diseases. Angiotensin II (ANG II) is a well-known pathogenic factor producing chronic renal injury and has also been shown to increase HIF-1α. However, the contribution of HIF-1α to ANG II-induced renal injury has not been evidenced. The present study tested the hypothesis that HIF-1α mediates ANG II-induced renal injury in Sprague-Dawley rats. Chronic renal injury was induced by ANG II infusion (200ng/kg/min) for 2 weeks in uninephrectomized rats. Transfection of vectors expressing HIF-1α shRNA into the kidneys knocked down HIF-1α gene expression by 70%, blocked ANG II-induced HIF-1α activation and significantly attenuated ANG II-induced albuminuria, which was accompanied by inhibition of ANG II-induced vascular endothelial growth factor, a known glomerular permeability factor, in glomeruli. HIF-1α shRNA also significantly improved the glomerular morphological damage induced by ANG II. Furthermore, HIF-1α shRNA blocked ANG II-induced upregulation of collagen and α-smooth muscle actin in tubulointerstitial region. There was no difference in creatinine clearance and ANG II-induced increase in blood pressure. HIF-1α shRNA had no effect on ANG II-induced reduction in renal blood flow and hypoxia in the kidneys. These data suggested that over-activation of HIF-1α-mediated gene regulation in the kidney is a pathogenic pathway mediating ANG II-induced chronic renal injuries and normalization of over-activated HIF-1α may be used as a treatment strategy for chronic kidney damages associated with excessive ANG II.
doi:10.1161/HYPERTENSIONAHA.111.177626
PMCID: PMC3174356  PMID: 21896938
glomerular sclerosis; tubulointerstitial; fibrosis; albuminuria; renal blood flow
22.  Decidualized Pseudopregnant Rat Uterus Shows Marked Reduction in Ang II and Ang-(1-7) Levels 
Placenta  2011;33(1):17-23.
Previous studies showed that angiotensin (Ang) II and Ang-(1-7) concentrations were reduced in the implantation site at day 7 of pregnancy in Sprague-Dawley rats as compared to the site immediately adjacent to it, which does not have the embryo attached, clearly showing the importance of the blastocyst in the regulation of renin-angiotensin system (RAS).
Objective
The objective of this study was to evaluate the regulation of the RAS in the decidualized uterus in the pseudopregnant rat, a model without the presence of a conceptus.
Methods
Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments; decidualization was induced by oil-injection of the right horn; the left horn served as a control. The uterine content of Ang I, Ang II, and Ang-(1-7) was examined in the decidualized and non-decidualized uteri.
Results
Both Ang-(1-7) and Ang II and ACE and ACE2 mRNA were significantly reduced in the decidualized horn as compared to the non-decidualized horn. Immunocytochemical characterization of Ang II, Ang-(1-7), ACE and ACE2 demonstrated that Ang-(1-7), Ang II, and ACE2 polarize to the anti-mesometrial pole with decidualization.
Conclusion
The decidualization process elicits marked reduction in uterine Ang II and Ang-(1-7) content as compared to the non-decidualized horn. The differential immunocytochemical expression of Ang II and Ang-(1-7) with ACE2, but not ACE in the anti-mesometrial pole of the decidualized horn may favor the formation and action of Ang-(1-7) in the anti-mesometrial pole, an area which plays a role in triggering the decidualization process.
doi:10.1016/j.placenta.2011.10.016
PMCID: PMC3249021  PMID: 22101031
angiotensin peptides; uterus; decidualization; ACE; ACE2; pseudopregnancy
23.  Sexual Dimorphism in Urinary Angiotensinogen Excretion During Chronic Angiotensin II–Salt Hypertension 
Gender medicine  2012;9(4):207-218.
Background
The intrarenal renin–angiotensin system contributes to hypertension by regulating sodium and water reabsorption throughout the nephron. Sex differences in the intrarenal components of the renin–angiotensin system have been involved in the greater incidence of high blood pressure and progression to kidney damage in males than females.
Objective
This study investigated whether there is a sex difference in the intrarenal gene expression and urinary excretion of angiotensinogen (AGT) during angiotensin II (Ang II)–dependent hypertension and high-salt (HS) diet.
Methods
Male and female Sprague-Dawley rats were divided into 5 groups for each sex: Normal-salt control, HS diet (8% NaCl), Ang II–infused (80 ng/min), Ang II–infused plus HS diet, and Ang II–infused plus HS diet and treatment with the Ang II receptor blocker, candesartan (25 mg/L in the drinking water). Rats were evaluated for systolic blood pressure (SBP), kidney AGT mRNA expression, urinary AGT excretion, and proteinuria at different time points during a 14-day protocol.
Results
Both male and female rats exhibited similar increases in urinary AGT, with increases in SBP during chronic Ang II infusion. HS diet greatly exacerbated the urinary AGT excretion in Ang II–infused rats; males had a 9-fold increase over Ang II alone and females had a 2.5-fold increase. Male rats displayed salt-sensitive SBP increases during Ang II infusion and HS diet, and female rats did not. In the kidney cortex, males displayed greater AGT gene expression than females during all treatments. During Ang II infusion, both sexes exhibited increases in AGT gene message compared with same-sex controls. In addition, HS diet combined with Ang II infusion exacerbated the proteinuria in both sexes. Concomitant Ang II receptor blocker treatment during Ang II infusion and HS diet decreased SBP and urinary AGT similarly in both sexes; however, the decrease in proteinuria was greater in the females.
Conclusion
During Ang II–dependent hypertension and HS diet, higher intrarenal renin-angiotensin system activation in males, as reflected by higher AGT gene expression and urinary excretion, indicates a mechanism for greater progression of high blood pressure and might explain the sex disparity in development of salt-sensitive hypertension.
doi:10.1016/j.genm.2012.06.001
PMCID: PMC3419837  PMID: 22795463
angiotensinogen; intrarenal RAS; mRNA; proteinuria; sex differences; Sprague-Dawley rats; systolic blood pressure
24.  Enhancement of Collecting Duct Renin in Angiotensin II—Dependent Hypertensive Rats 
Hypertension  2004;44(2):223-229.
Distal nephron renin may provide a possible pathway for angiotensin (Ang) I generation from proximally delivered angiotensinogen. To examine the effects of Ang II on distal nephron renin, we compared renin protein and mRNA expression in control and Ang II—infused rats. Kidneys from sham (n=9) and Ang II—infused (80 ng/kg per minute, 13 days, n=10) Sprague-Dawley rats were processed by immunohistochemistry, Western blot, reverse transcriptase—polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Ang II infusion increased systolic blood pressure (181±4 versus 115±5 mm Hg) and suppressed plasma and kidney cortex renin activity. Renin immunoreactivity was suppressed in juxtaglomerular apparatus (JGA) cells in Ang II—infused rats compared with sham (0.1±0.1 versus 1.0±0.1 relative ratio) but increased in distal nephron segments (6.4±1.4 versus 1.0±0.1 cortex; 2.5±0.3 versus 1.0±0.2 medulla). Tubular renin immunostaining was apically distributed in principal cells colocalizing with aquaporin-2 in connecting tubules and cortical and medullary collecting ducts. Renin protein levels were decreased in the kidney cortex of Ang II—infused rats compared with that of sham (0.4±0.2 versus 1.0±0.4) rats but higher in the kidney medulla (1.2±0.4 versus 1.0±0.1). In kidney medulla, RT-PCR and quantitative real-time PCR showed similar levels of renin transcript in both groups. In summary, the detection of renin mRNA in the renal medulla, which is devoid of JGA, indicates local synthesis rather than an uptake of JGA renin. In contrast to the inhibitory effect of Ang II on JGA renin, Ang II infusion stimulates renin protein expression in collecting ducts and maintains renin transcriptional levels in the medulla, which may contribute to the increased intrarenal Ang II levels in Ang II—dependent hypertension.
doi:10.1161/01.HYP.0000135678.20725.54
PMCID: PMC2628717  PMID: 15226276
renin; angiotensin II; angiotensinogen; immunohistochemistry; Western blot
25.  Endothelial Cell-specific Deficiency of AngII Type 1a Receptors Attenuates AngII-induced Ascending Aortic Aneurysms in LDL Receptor −/− Mice 
Circulation research  2011;108(5):574-581.
Rationale
Human studies and mouse models have provided evidence for angiotensin II (AngII)-based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that AngII infusion induces aneurysmal pathology in the ascending aorta.
Objective
The aim of this study was to define the role of angiotensin II type 1a (AT1a) receptors and their cellular location in AngII-induced ascending aortic aneurysms (AAs).
Methods and Results
Male LDL receptor −/− mice were fed a saturated fat-enriched diet for 1 week prior to osmotic mini-pump implantation and infused with either saline or AngII (1,000 ng/kg/min) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT1a receptor deficiency ablated AngII-induced ascending AAs (P<0.001). To determine the role of AT1a receptors on leukocytes, LDL receptor −/− x AT1a receptor +/+ or −/− mice were irradiated and repopulated with bone marrow-derived cells isolated from either AT1a receptor +/+ or −/− mice. Deficiency of AT1a receptors in bone marrow-derived cells had no effect on AngII-induced ascending AAs. To determine the role of AT1a receptors on vascular wall cells, we developed AT1a receptor floxed mice with depletion on either smooth muscle (SMC) or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT1a receptor deletion in SMCs had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology.
Conclusions
AngII infusion promotes aneurysms in the ascending aorta via stimulation of AT1a receptors that are expressed on endothelial cells.
doi:10.1161/CIRCRESAHA.110.222844
PMCID: PMC3076204  PMID: 21252156
ascending aneurysm; angiotensin II; AT1a receptor; Tek-cre

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