There has been an explosive growth of interest in the multiple interacting paracrine systems that influence renal microvascular function. This review first discusses the membrane activation mechanisms for renal vascular control. Evidence is provided that there are differential activating mechanisms regulating pre- and postglomerular arteriolar vascular smooth muscle cells. The next section deals with the critical role of the endothelium in the control of renal vascular function and covers the recent findings related to the role of nitric oxide and other endothelial-derived factors. This section is followed by an analysis of the roles of vasoactive paracrine systems that have their origin from adjoining tubular structures. The interplay of signals between the epithelial cells and the vascular network to provide feedback regulation of renal hemodynamics is developed. Because of their well-recognized contributions to the regulation of renal microvascular function, three major paracrine systems are discussed in separate sections. Recent findings related to the role of intrarenally formed angiotensin II and the prominence of the AT1 receptors are described. The possible contribution of purinergic compounds is then discussed. Recognition of the emerging role of extracellular ATP operating via P2 receptors as well as the more recognized functions of the P1 receptors provides fertile ground for further studies. In the next section, the family of vasoactive arachidonic acid metabolites is described. Possibilities for a myriad of interacting functions operating both directly on vascular smooth muscle cells and indirectly via influences on endothelial and epithelial cells are discussed. Particular attention is given to the more recent developments related to hemodynamic actions of the cytochrome P-450 metabolites. The final section discusses unique mechanisms that may be responsible for differential regulation of medullary blood flow by locally formed paracrine agents. Several sections provide perspectives on the complex interactions among the multiple mechanisms responsible for paracrine regulation of the renal microcirculation. This plurality of regulatory interactions highlights the need for experimental strategies that include integrative approaches that allow manifestation of indirect as well as direct influences of these paracrine systems on renal microvascular function.

Abstract-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 (nϭ12) maintained on an 8% salt diet. Ang II was administered via osmotic minipumps (40 ng/min) to 1 group (nϭ6) while the remaining rats were sham-operated. A H/S diet alone did not alter systolic blood pressure in sham animals (109Ϯ6 mm Hg at day 12); however, Ang II infusions to the H/S rats significantly increased systolic blood pressure (167Ϯ7 at day 12) and intrarenal Ang II content (459Ϯ107 fmol/g versus 270Ϯ42) despite a marked suppression of plasma renin activity (0.9Ϯ0.2 ng Ang I ⅐ mL Ϫ1 ⅐ h Ϫ1 versus 2.8Ϯ1.3). Ang II infusions significantly increased kidney Ao mRNA compared with the H/S diet alone by 1.9Ϯ0.1-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 (1.9Ϯ0.1-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.

Abstract-Angiotensin II (AngII) infusions augment renal angiotensinogen mRNA and protein and urinary angiotensinogen excretion (U AGT ). Further experiments were performed in 4 groups of rats: normal salt diet with sham operation, NSϩSham, nϭ6; NS with AngII infusion at 40 ng/min via osmotic minipump, NSϩAngII(40), nϭ9; NS with AngII infusion at 80 ng/min, NSϩAngII(80), nϭ9; high-salt diet with deoxycorticosterone acetate salt pellet (100 mg), HSϩDOCA, nϭ4. These experiments sought to determine whether enhanced U AGT is specifically associated with increased kidney AngII levels or is a nonspecific consequence of the hypertension. Systolic BP (SBP) was significantly increased to 131Ϯ2 and 162Ϯ2 mm Hg at day 11 in NSϩAngII (40) n previous studies, we demonstrated that chronic angiotensin (Ang) II infusion results in significant increases in renal expression of angiotensinogen protein, 1 as well as angiotensinogen mRNA. 2 Furthermore, we recently showed that urinary excretion of angiotensinogen was significantly increased and was associated with enhanced intrarenal AngII levels in AngII-infused rats fed a high-salt diet. 3 These results prompted us to perform further experiments to evaluate the relationships between urinary excretion rates of angiotensinogen and intrarenal activity of the renin-angiotensin system (RAS), as well as blood pressure (BP), in AngII-induced hypertensive rats and in a volume-dependent model of hypertension induced by administration of a high-salt diet and deoxycorticosterone acetate salt (DOCA).This study was performed to address the following hypotheses: (1) urinary excretion of angiotensinogen during AngII infusions is enhanced in a dose-and time-dependent manner, (2) enhanced urinary excretion of angiotensinogen during AngII infusions is closely associated with increased kidney AngII levels, (3) enhanced urinary excretion of angiotensinogen is not primarily a consequence of the elevated arterial pressure or of hypertension-induced proteinuria, and (4) urinary excretion of angiotensinogen originates from the kidney and not the plasma. Methods Preparation of AnimalsThe experimental protocol was approved by the Tulane Animal Care and Use Committee. Male Sprague-Dawley rats (175 to 200g, Charles River, Wilmington, Mass.) were housed in wire metabolic cages and maintained, with free access to water, in a temperaturecontrolled room regulated on a 12-hour light/dark cycle. Rats (nϭ40) were fed a commercially available rat chow containing normal salt (NS, 0.6% sodium chloride, Harlan Teklad 170950, nϭ36) or high salt (HS, 8% sodium chloride, Harlan Teklad TD 79119, nϭ4) for 2 weeks. Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and an osmotic minipump (Alza) or a pellet of DOCA (100 mg, Innovative Research M-121) was implanted subcutaneously at the dorsum of the neck on day 0. Rats were selected at random from the NS group to serve as sham controls (nϭ10) or to receive AngII (Calbiochem-Novabiochem) infusion at a rate of 40 ng/min for AngII(40), nϭ9, or 80 ng...

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.

Abstract-This study was performed to examine whether there is an inappropriate regulation of intrarenal angiotensinogen in Dahl-salt sensitive rats (DS) fed a high salt diet (HS). Dahl salt-resistant rats (DR) and DS were maintained on HS (8% NaCl) or low salt diet (LS, 0.3% NaCl) for 4 weeks. Systolic blood pressure (SBP), measured by tail-cuff plethysmography, was unaltered in DR (DRϩHS, 127Ϯ3 mm Hg, nϭ5; DRϩLS, 126Ϯ3, nϭ5); however, SBP was significantly increased in DSϩHS (208Ϯ7, nϭ9) compared with DSϩLS (134Ϯ2, nϭ5 Key Words: angiotensin II Ⅲ angiotensinogen Ⅲ rats, Dahl Ⅲ kidney Ⅲ urine Ⅲ hypertension, sodium-dependent Ⅲ Western blot V arious epidemiological studies have showed a correlation of dietary salt intake with the prevalence and progression of hypertension. 1 Although the degree of salt sensitivity is variable, some individuals are particularly prone to have hypertension in response to an increased dietary salt intake. Subjects with essential hypertension have a higher frequency of salt sensitivity than is found in the normotensive population. 2 There is some evidence that salt sensitivity is associated with low plasma renin activity (PRA) and impaired renal sodium excretion. However, the mechanisms underlying this phenomenon are poorly understood. 3 Dahl salt-sensitive (DS) rats have been used as a model of human salt-sensitive hypertension because salt loading exaggerates the development of hypertension in strains that are genetically predisposed to hypertension. 4 Mature DS rats are reported to have low PRA levels, which has been interpreted as being indicative of an overall suppression of the renin-angiotensin system (RAS) 4 ; however, few studies of angiotensinogen (AGT) have been carried out in these rats. Although generally considered to be characterized by a low activity of circulating RAS, recent studies indicate that treatment with angiotensin (Ang) I-converting enzyme inhibitors or Ang II type I receptor antagonists reduces cardiac and/or renal dysfunction in DS rat fed a high salt diet (HS). [5][6][7][8][9][10] These findings suggest that the local RAS may be inappropriately activated and contribute to the development of hypertension in this animal model.Previous studies have demonstrated that Ang II infusions to normal rats result in paradoxical increases in renal expression of AGT mRNA 11,12 and protein. 13 Furthermore, urinary excretion of AGT was significantly increased in Ang II-infused rats, which was associated with an enhancement of intrarenal Ang II levels. 14 These results indicate that intrarenal AGT levels are not necessarily associated with increased plasma or renal renin levels. However, the extent to which this may occur in DS rats has not been determined. Therefore, this study was performed to determine if there is an inappropriate regulation of intrarenal AGT in DS rats fed HS and if enhanced

Abstract-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 (AT 1 ) receptors. Male Sprague-Dawley rats (200 to 220 g) were divided into 3 groups: sham surgery (nϭ10), subcutaneous infusion of Ang II (80 ng/min, nϭ11), and Ang II infusion plus AT 1 blocker (ARB), olmesartan (5 mg/d, nϭ12). Ang II infusion progressively increased systolic blood pressure (SBP) compared with sham (178Ϯ8 mm Hg versus119Ϯ4 at day 11). ARB treatment prevented hypertension (113Ϯ6 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 IIϩARB group had a significant increase in plasma Ang II compared with Ang II and sham groups (365Ϯ46 fmol/mL versus 76Ϯ9 and 45Ϯ14, respectively). Nevertheless, ARB treatment markedly limited the enhancement of kidney Ang II by Ang II infusion (65Ϯ17 fmol/g in sham, 606Ϯ147 in Ang II group, and 288Ϯ28 in Ang IIϩARB group). Ang II infusion significantly increased kidney angiotensinogen compared with sham (1.69Ϯ0.21 densitometric units versus 1.00Ϯ0.17). This change was reflected by increased angiotensinogen immunostaining in proximal tubules. ARB treatment prevented this increase (1.14Ϯ0.12). Urinary angiotensinogen excretion rates were enhanced 4.7ϫ in Ang II group (4.67Ϯ0.41 densitometric units versus 1.00Ϯ0.21) but ARB treatment prevented the augmentation of urinary angiotensinogen (0.96Ϯ0.23). These data demonstrate that augmentation of intrarenal angiotensinogen in Ang II-infused rats is AT 1 -dependent and provide further evidence that urinary angiotensinogen is closely linked to intrarenal Ang II in Ang II-dependent hypertension. Key Words: angiotensin II Ⅲ angiotensinogen Ⅲ receptors, angiotensin II Ⅲ rats Ⅲ kidney A ngiotensin II (Ang II) plays an important role in proximal tubular reabsorptive function primarily via activation of Ang II type 1 (AT 1 ) receptor at both basolateral and luminal membranes. 1 Although some Ang II type 2 receptors have been confirmed on proximal tubules, 2 most functional studies suggest that the major effects of Ang II on proximal tubules are via AT 1 receptors. 3 Several studies have suggested that Ang II also plays an important role in the regulation of distal tubular reabsorption rate. 4 This effect was blocked by either saralasin or losartan, indicating that this stimulation involves AT 1 receptor activation. 4 These findings, together with the demonstration that AT 1 receptor is present on the luminal membranes of distal nephron segments, 5,6 suggest that luminal Ang II plays an important role in the regulation not only of proximal reabsorption rate but also of distal tubular reabsorptive function. 7 Studies in isolated proximal tubular cells showed that Ang II stimulates Na ϩ /H ϩ exchanger via AT 1 ...

Abstract-Previous studies have indicated that angiotensin II (Ang II) concentrations in renal interstitial fluid are much higher than plasma levels. In the present study, we performed experiments to explore renal interstitial fluid concentrations of Ang I and Ang II further and to determine whether these levels are altered by acute arterial infusion of an ACE inhibitor (enalaprilat) or by volume expansion. Microdialysis probes (molecular weight cutoff point: 30 000 Da) were implanted in the renal cortex of anesthetized rats and were perfused at a rate of 2 L/min. Using relative equilibrium rates, the basal renal interstitial fluid Ang II concentration averaged 3.07Ϯ0.43 nmol/L, a value much higher than the plasma Ang II concentration of 107Ϯ8 pmol/L (nϭ7). Interstitial fluid Ang I concentrations (0.84Ϯ0.04 nmol/L) were consistently lower than the Ang II concentrations but higher than the plasma Ang I concentrations (112Ϯ14 pmol/L). Intra-arterial infusion of enalaprilat (7.5 mol/kg/min, nϭ5) for 120 minutes resulted in a significant decrease in mean arterial pressure (from 114Ϯ4 to 68Ϯ4 mm Hg) along with reductions in plasma and renal ACE activity (by Ϫ99% and Ϫ52%, respectively). Enalaprilat resulted in a significant increase in plasma Ang I from 133Ϯ21 to 1167Ϯ328 pmol/L and a decrease in plasma Ang II from 110Ϯ12 to 67Ϯ9 pmol/L. During enalaprilat infusion, interstitial fluid concentration of Ang I was significantly increased from 0.78Ϯ0.06 to 0.97Ϯ0.08 nmol/L; however, Ang II concentrations were not altered significantly

Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT1 immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.