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

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

Background Previous studies demonstrated that angiotensin (Ang) III, not Ang II, is the predominant endogenous agonist for Ang type‐2 receptor (AT 2R)‐induced natriuresis in normal rats, and that hypertensive 12‐week‐old spontaneously hypertensive rats (SHR) lack natriuretic responses to Ang III. This study tested whether prehypertensive SHR already have defective Ang III‐induced natriuresis and determined possible mechanisms. Methods and Results Female and male normotensive 4‐week‐old SHR and Wistar Kyoto rats were studied after 24‐hour systemic AT 1R blockade. Left kidneys received 30 minute renal interstitial infusions of vehicle followed by Ang III (3.5, 7.0, 14, and 28 nmol/kg per min; each dose for 30 minutes). Right kidneys received vehicle infusions. In 4‐week‐old Wistar Kyoto rats, renal interstitial Ang III increased urine sodium (Na+) excretion but failed to induce natriuresis in 4‐week‐old SHR. Renal Ang III levels were similar between Wistar Kyoto rats and SHR, making increased Ang III degradation as a possible cause for defective natriuresis in SHR unlikely. In Wistar Kyoto rats, renal interstitial Ang III induced translocation of AT 2Rs to apical plasma membranes of renal proximal tubule cells. Simultaneously, Ang III induced retraction of the major Na+ transporter Na+‐H+ exchanger‐3 (NHE‐3) from apical membranes and internalization of Na+/K+ ATPase (NKA) from basolateral membranes of renal proximal tubule cells. Consistent with NHE‐3 and NKA retraction, Ang III increased pSer552‐NHE‐3 and decreased pSer23‐NKA. In contrast, in SHR, intrarenal Ang III failed to induce AT 2R translocation, NHE‐3 or NKA retraction, pSer552‐NHE‐3 phosphorylation, or pSer23‐NKA dephosphorylation. Conclusions These results indicate impaired Ang III/AT 2R signaling as a possible primary defect in prehypertensive SHR., See Editorial Zhuo and Li

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18–21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

Angiotensin III (Ang III) [des-aspartyl 1 -Ang II] is the predominant endogenous agonist for angiotensin type-2 receptor (AT 2 R)-induced natriuresis in rats. Hypertensive 12 wk-old SHR lack natriuretic responses to Ang III, and renal interstitial (RI) Ang III administration induces natriuresis in 4 wk-old Wistar-Kyoto rats (WKY), but not in 4 wk-old pre-hypertensive SHR. The defect in natriuresis could be explained by the concurrent observation that in WKY, but not SHR, Ang III induced AT 2 R translocation to renal proximal tubule apical plasma membranes and internalization/inactivation of Na + -transporters Na + - H + exchanger-3 (NHE-3) and Na + /K + ATPase (NKA). These findings suggest an Ang III/AT 2 R signaling defect in pre-hypertensive SHR. The present study sought to determine the cause of the defect: (1) accelerated intrarenal Ang III metabolism or (2) a primary AT 2 R (or post-receptor) signaling defect. Selective AT 2 R agonist Compound-21 (C-21) was infused interstitially [20, 40 and 60 ng/kg/min, each dose for 30 min] into experimental (left) kidneys of 4 wk-old WKY and SHR (N=7) pretreated for 24h with systemic infusion of Ang type-1 receptor (AT 1 R) antagonist candesartan (0.01 mg/kg/min by osmotic minipump); vehicle was infused into control (right) kidneys. Pre-infusion urinary Na + excretion (U Na V) was 0.022 ± 0.004 μmol/min in WKY and 0.024 ± 0.005 μmol/min in SHR (P=NS). In WKY, C-21 dose-dependently increased U Na V from 0.022 ± 0.004 to 0.058 ± 0.017, 0.078 ± 0.014 and 0.079 ± 0.079 μmol/min (P=0.01 from pre-infusion and time control kidneys). In contrast, SHR did not increase U Na V in response to C-21 [highest U Na V 0.033 ± 0.007 μmol/min (P=NS from pre-infusion and time control kidneys)]. Mean arterial pressure (MAP) was 64.6 ± 1.80 mmHg in WKY and 76.4 ± 2.2 mmHg (P2 R defect as responsible for increased Na + reabsorption in SHR.

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

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

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

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

In angiotensin type 1 receptor–blocked rats, renal interstitial (RI) administration of des-aspartyl 1 -angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III–induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor–blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III–induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP–dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.

OBJECTIVE This study aimed to examine the effects of salt loading, with or without simultaneous angiotensin receptor blocker (ARB) treatment, on the systemic and tissue renin-angiotensin system (RAS) in spontaneously hypertensive rats (SHRs). METHOD Evaluation was performed early (4 weeks) in the course of salt loading in order to examine initial mediating events of cardiovascular and renal damage produced by salt excess. Four groups of rats were studied. Group 1 received regular rat chow (normal-salt diet); group 2 received normal-salt diet and an ARB (losartan, 30 mg/kg per day); group 3 received high-salt (8%) chow; and group 4 received high-salt diet and losartan. RESULTS High-salt diet increased systolic pressure to 193±1 mmHg compared to 180±2 in normal-salt diet group. Losartan reduced SBP in SHRs fed normal-salt diet but did not reduce SBP in the SHRs fed high-salt diet (192±2 mmHg). High-salt diet markedly increased urinary protein excretion from 27±4 to 64±13 mg/day and this increase was ameliorated by losartan (40±9 mg/day). In SHRs on high-salt diet, plasma angiotensin II concentration increased three to four-fold, whereas urinary angiotensinogen excretion increased 10-fold; and these changes were significantly reduced by losartan. High-salt diet accelerated glomerular injury and interstitial fibrosis in SHRs which were reduced by losartan. CONCLUSION These results demonstrate that the activity of RAS was either not suppressed or, even augmented, after 4 weeks of salt loading despite high salt intake and increased SBP. The data suggest that an augmented intrarenal RAS during high-salt diet may contribute to the development of renal injury in this experimental model.

In 2-kidney 1-clip (2K1C) hypertension, intrarenal angiotensin II (Ang II) levels are increased in both kidneys, which are often implicated in the further augmentation of the intrarenal renin-angiotensin system and the development of hypertension and kidney injury. We recently reported that angiotensinogen (AGT) expression and secretion are increased only in the non-clipped kidneys (NCK). These findings provide the basis for the hypothesis that elevated Ang II levels, augmented AGT, and high arteriolar pressure in NCK synergistically cause greater kidney injury. Accordingly, we compared the degrees of kidney injury between clipped kidneys (CK) and NCK using a 2K1C model that maintains normal renal blood flow and GFR in the CK. Left kidneys of male rats were clipped for 21 days. Histological and immunohistological analyses were performed on both the CK and NCK. Twenty glomeruli and 20 microscope fields in the cortex and the medulla were examined for each group. The PAS-positive area in the glomeruli was higher in NCK compared with sham (33.9 ± 0.89 vs. 12.4 ± 0.51%) and CK (vs. 15.1 ± 0.58%). Similarly, the Masson's trichrome-positive area in the medullary region was greater in NCK compared with sham (2.21 ± 0.10 vs. 1.32 ± 0.05%) and CK (vs. 1.67 ± 0.10%), but the changes were not observed in the interstitial tissue of the cortex. Immunoreactivity for CD68, a marker of the macrophage and monocyte levels, was higher in NCK compared with sham (0.72 ± 0.07 vs. 0.36 ± 0.04%), but similar to that in CK. In contrast, accumulation of the immune cells in the glomeruli was greater in NCK compared with sham (8.99 ± 0.69 vs. 3.46 ± 0.46%) and CK (vs. 3.08 ± 0.24%). The proliferating cell nuclear antigen levels, a marker of cell proliferation, were greater in NCK (3.49 ± 0.09 %) but not in the CK. Levels of vimentin, a cell transformation and regeneration marker, were also higher in NCK compared with CK and sham. Increased vascular wall thickness (α-SMA) was observed in both kidneys. These results indicate that pathological factors associated with the high blood pressure are required for the development of renal injury in the 2K1C model including glomerular expansion, medullary fibrosis, immune cell infiltration in glomeruli and cell proliferation/transformation.

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459–6463). As these changes of temperature affect membrane lipid order and lipid–protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order–dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2°C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.

Alendronate, an aminobisphosphonate, produces as a side effect a topical (pill induced) esophagitis. To gain insight into this phenomenon, we assessed the effects of luminal alendronate on both esophageal epithelial structure and function. Sections of rabbit esophageal epithelium were exposed to luminal alendronate at neutral or acidic pH while mounted in Ussing chambers to monitor transmural electrical potential difference (PD), short-circuit current ( Isc), and resistance ( R). Morphological changes were sought by light microscopy in hematoxylin and eosin-stained sections. Impedance analysis was used for localization of alendronate-induced effects on ion transport. Luminal, but not serosal, alendronate (pH 6.9–7.2), increased PD and Iscin a dose- and time-dependent manner, with little change in R and mild edema of surface cell layers. The changes in Isc(and PD) were reversible with drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na channel activity was also supported by impedance analysis. Luminal alendronate at acidic pH was more damaging than either alendronate at neutral pH or acidic pH alone. These data suggest that alendronate stimulates net ion (Na) transport in esophageal epithelium by increasing apical membrane sodium channel activity and that this occurs with limited morphological change and no alteration in barrier function. Also alendronate is far more damaging at acidic than at neutral pH, suggesting its association with esophagitis requires gastric acid for expression. This expression may occur either by potentiation between the damaging effects of (refluxed) gastric acid and drug or by acid-induced conversion of the drug to a more toxic form.

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.

In angiotensin II (ANG II) infusion hypertension, there is an augmentation of intratubular angiotensinogen (AGT) and ANG II leading to increased urinary AGT and ANG II excretion rates associated with tissue injury. However, the changes in urinary AGT and ANG II excretion rates and markers of renal injury during physiologically induced stimulation of the renin-angiotensin system (RAS) by a low-salt diet remain unclear. Male Sprague-Dawley rats received a low-salt diet (0.03% NaCl; n = 6) and normal-salt diet (0.3% NaCl, n = 6) for 13 days. Low-salt diet rats had markedly higher plasma renin activity and plasma ANG II levels. Kidney cortex renin mRNA, kidney AGT mRNA, and AGT immunoreactivity were not different; however, medullary renin mRNA, kidney renin content, and kidney ANG II levels were significantly elevated by the low-salt diet. Kidney renin immunoreactivity was also markedly increased in juxtaglomerular apparati and in cortical and medullary collecting ducts. Urinary AGT excretion rates and urinary ANG II excretion rates were not augmented by the low-salt diet. The low-salt diet caused mild renal fibrosis in glomeruli and the tubulointerstitium, but no other signs of kidney injury were evident. These results indicate that, in contrast to the response in ANG II infusion hypertension, the elevated plasma and intrarenal ANG II levels caused by physiological stimulation of RAS are not reflected by increased urinary AGT or ANG II excretion rates or the development of renal injury.

We were pleased that our article titled, “Angiotensin II Type 1 Receptor–Mediated Augmentation of Urinary Excretion of Endogenous Angiotensin II in Val5-Angiotensin II–Infused Rats” generated sufficient interest to prompt van den Heuvel et al1 to suggest how we could distinguish between plasma- and kidney-derived angiotensin II (Ang II) in the urine. The focus of our study was to determine whether chronic Ang II infusion increased production of intrarenal Ang II as reflected by urinary excretion of endogenous Ang II.2 Importantly, during infusion of Val5-Ang II for 14 days, the plasma concentrations of Ile5-Ang II were markedly reduced compared with control rats3 because of the substantial reduction in plasma renin …

Rats infused chronically with Val 5 -Angiotensin (Ang) II exhibit increased urinary excretion of endogenous Ile 5 -Ang II by the 12th day of infusion, suggesting the stimulation of endogenous Ang II formation by Val 5 -Ang II infusion. The present study determined the time course of increased urinary Ang II excretion and the effects of Ang II type 1 receptor blockade (candesartan, 2 mg/kg per day) on the urinary excretion rates of Ile 5 -Ang II in Val 5 -Ang II-infused (80 ng/min) rats. Ile 5 -Ang II was separated from Val 5 -Ang II by high-performance liquid chromatography and measured by radioimmunoassay. Systolic blood pressure increased progressively (215±2 mm Hg) in Val 5 -Ang II-infused rats (n=5), whereas the candesartan-treated group (n=6) remained normotensive (124±3 mm Hg). Candesartan treatment significantly increased the level of plasma Ile 5 -Ang II (24.0±7.6 versus 156.9±24.6 fmol/mL; P 5 -Ang II content (357.9±76.6 versus 21.1±2.8 fmol/g; P 5 -Ang II excretion rates were elevated by day 9 (2185.7±283.2 fmol/24 hours) in Val 5 -Ang II-infused rats but not in candesartan-treated rats (740.6±110.3 fmol/24 hours). Thus, Ang II type 1 receptor blockade prevents the increase in urinary excretion of endogenous Ang II in rats subjected to chronic Ang II infusion. These data indicate that the increased urinary excretion of endogenous Ang II in Val 5 -Ang II-infused rats is primarily attributed to Ang II type 1 receptor-dependent secretion into and/or de novo formation of Ang II within the tubular lumen.

In angiotensin II (ANG II)-induced hypertension, intrarenal ANG II levels are increased by AT1 receptor-mediated ANG II internalization and endogenous ANG II generation. The objective of the present study was to determine the relative contribution of de novo formation of endogenous ANG II. Male Sprague-Dawley rats were divided into three groups: sham operated ( n = 6), Val5-ANG II infused ( n = 16), and Ile5-ANG II infused ( n = 6). Val5-ANG II and Ile5-ANG II were infused at 80 ng/min via subcutaneous osmotic minipump for 13 days, followed by harvesting of blood and kidney samples. In six Val5-ANG II-infused rats, urine was collected on the day before infusion and on day 12 of infusion. Extracted samples were subjected to HPLC to separate Val5-ANG II from Ile5-ANG II followed by RIA. Systolic blood pressure increased significantly from 121 ± 2 to 206 ± 4 mmHg in the Val5-ANG II-infused rats and from 124 ± 3 to 215 ± 5 mmHg in the Ile5-ANG II-infused rats. In the Val5-ANG II-infused rats, the plasma Ile5-ANG II levels increased 196.2 ± 70.1% compared with sham plasma Ile5-ANG II concentration. Val5-ANG II levels were 150.0 ± 28.2 fmol/ml which accounted for 53.5 ± 10.1% of the total ANG II in plasma. The kidney Ile5-ANG II levels in the Val5-ANG II-infused rats increased 69.9 ± 30.7% compared with sham kidney Ile5-ANG II concentrations. Intrarenal accumulation of Val5-ANG II accounted for 52.5 ± 5.3% of the total kidney ANG II during Val5-ANG II infusion while endogenous Ile5-ANG II accounted for 47.5 ± 8.6%. The urinary Ile5-ANG II excretion rate on day 12 increased 93.2 ± 32.1% compared with preinfusion level indicating increased formation of endogenous ANG II. Thus, the increases in intrarenal ANG II levels during chronic ANG II infusions involve substantial stimulation of endogenous ANG II formation which contributes to overall augmentation of intrarenal ANG II.

Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. However, this method only measures the sum of the activity of all plasma membrane-bound channels. Therefore, this measurement cannot discriminate between effects on channel density and individual channel activity. To address this shortcoming, we have developed a simple assay to detect changes of membrane-bound channel density in intact oocytes. This nonradioactive assay relies on specific antibody binding in whole live cells utilizing a simple spectrophotometric measurement. This assay is linear over a wide range of channel expression levels and provides a simple cost-effective way of monitoring changes of membrane-bound channel density. Moreover, when the heterologous proteins poorly express at the plasma membrane, this method becomes advantageous to complex biochemical cell fractionation.

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na+ channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 ± 12 μS ( n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts.

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459-6463). As these changes of temperature affect membrane lipid order and lipid-protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order-dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2 degrees C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.

The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.