Novel markers of graft outcome in a cohort of kidney transplanted patients: a cohort observational study.

Renal biopsy (RBx) informs about kidney transplantation (KTx) prognosis. In our observational study the prevalence of histological anomalies and the prognostic role of CD45, vimentin (VIM) and periostin (POSTN) in KTx-RBx have been evaluated. One hundred forty-six KTx-RBx (2009-2012) were analysed for general histology and in immunohistochemistry for CD45, VIM and POSTN. Clinical data of the 146-KTx patients were collected at the RBx time (T0), 6 and 12 months before and after RBx. Follow-up time was 21 ± 14 months. Glomerulosclerosis was 20% glomeruli/biopsy. Tubular atrophy (TA), Interstitial infiltrate (I-Inf) and interstitial fibrosis (IF) were slight in 21-18% and 25%, moderate in 22-30% and 26% and severe in 30-18% and 28% of patients. Fifty-eight percent of patients had lesions compatible with IF-TA. CD45, VIM and POSTN correlated to each-other and to TA, I-Inf and IF. VIM and POSTN correlated to GS. CD45 and VIM correlated directly to renal function (RF) and 25(OH)VitD, while POSTN inversely to 25(OH)VitD. Thirty patients restarted dialysis (HD+). HD+ had lower T0-eGFR, and higher CD45, VIM and POSTN than HD-. POSTN resulted the strongest in discriminate for HD+ . CD45, VIM and POSTN correlate to each-other and predict graft outcome. POSTN was the strongest in discriminate for HD+. 25(OH)VitD might influence inflammation and fibrosis in KTx.

[Hypertension-induced fibrosis: a balance story]. / La fibrose dans l'hypertension artérielle : une histoire d'équilibre.

Cardiac remodeling is a deleterious consequence of arterial hypertension. This remodeling results in cardiac transcriptomic changes induced by mechanical and hormonal factors (angiotensin II and aldosterone are the most important). The major features of cardiac remodeling are the hypertrophy of cardiomyocytes, interstitial and perivascular fibrosis, and microvascular rarefaction. Inappropriate stimulation of the renin-angiotensin-aldosterone system (RAAS) participates to the development of heart failure. The respective roles of angiotensin II and aldosterone in cardiac remodeling are poorly understood. The development of fibrosis in the heart depends of a balance between profibrotic (TGFß, CTGF, inflammation) and antifibrotic (BNP, ANP, BMP4 and BMP7) factors. The profibrotic and proinflammatory effects of angiotensin II and aldosterone are very well demonstrated; however, their actions on antifibrotic factors expression are unknown. In order to explore this, we used RenTgKC mice overexpressing renin into the liver, leading to an increased plasma angiotensin II and thus induction of severe hypertension, and AS mice overexpressing aldosterone synthase (AS) in cardiomyocytes which have a doubled intracardiac aldosterone concentration. Male AS mice have a dysfunction of the coronary arteries relaxation without structural and functional changes of the myocardium. Mice derived from a crossing between the RenTgKC and AS strains were used in this work. It is shown that angiotensin II induces the expression of BNP and BMPs which ultimately slows the progression of myocardial fibrosis, and that aldosterone inhibits the expression of these factors and thus worsens the fibrosis.

Reversal of renal disease: is it enough to inhibit the action of angiotensin II?

Over the last years, evidence emerged demonstrating that the progression of renal fibrosis is reversible in experimental models. The present review summarizes the new insights concerning the mechanisms of progression and regression of renal disease and examines this novel evidence under the light of feasibility and transfer to human nephropathies. The involved mechanisms are discussed with particular emphasis on the fibrotic role of vasoactive peptides such as angiotensin II and endothelin, and growth factors such as transforming growth factor beta (TGFbeta). The possibility of regression is introduced by presenting the in vivo efficiency of anti-hypertensive treatments and of systems that antagonize the fibrogenic action of TGFbeta such as bone morphogenic protein-7 (BMP-7) and hepatocyte growth factor. Finally, we provide a brief description of the promising future directions and clinical considerations about the applications of the experimental data to humans.

[Renal hemodynamics and development of renal fibrotic lesions during hypertension]. / Dissociation entre l'hémodynamique rénale et les lésions histologiques rénales induites par l'hypertension artérielle.

Hypertension is frequently associated with the development of renal fibrosis leading to chronic renal failure. The objective of the present study was to evaluate the role of blood pressure and renal hemodynamics on the development of renal lesions during hypertension. To this end, rats were treated with a NO synthase inhibitor, L-NAME, for 4 weeks. At this time point, systolic blood pressure reached 170 mmHg, renal blood flow dropped to 3.3 +/- 0.7 ml/min and kidneys displayed glomerular and tubulo-interstitial lesions as evidenced by histological analysis. Thereafter, L-NAME treatment was combined with an AT1 receptor antagonist, losartan (30 mg/kg/d), for an additional period of 4 weeks. Treatment with losartan for 4 additional weeks did not significantly modify hypertension (168 mmHg) either the degree of tubulo-interstitial lesions; in contrast, a significant regression of ischemic and sclerotic glomerular lesions was observed. In parallel, renal blood flow was significantly improved by losartan (5.2 +/- 0.8 ml/min). In addition a negative correlation was observed between renal blood flow and index of glomerulosclerosis (r = -0.82), whereas tubulo-intarstitial damage was positively correlated to systemic pressure (r = 0.93). In conclusion, inhibition of the local effects of angiotensin II alleviates the fall of renal blood flow consecutive to NO deficiency and reduces the morphological and functional lesions of glomeruli, independently of the changes in blood pressure. In contrast, tubulo-interstitial lesions are not correlated with the levels of renal blood flow and do not regress with the blockade of AT1 receptors when rats remain hypertensive.

Regression of renal vascular fibrosis by endothelin receptor antagonism.

In previous studies, we have observed that endothelin participates in the progression of renal vascular and glomerular fibrosis during hypertension by activating collagen I gene synthesis. The present study investigated whether administration of endothelin receptor antagonists leads to the regression of renal sclerotic lesions. Experiments were performed in transgenic mice harboring the luciferase gene under the control of the collagen I-alpha2 chain promoter. Hypertension was induced by long-term inhibition of nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (L-NAME); systolic pressure gradually increased, reaching a plateau of 165 mm Hg after 10 weeks of hypertensive treatment. At the same time, collagen I gene expression was increased 2- and 5-fold compared with control animals in afferent arterioles and glomeruli, respectively (P<0.01). This increase was accompanied by the appearance of sclerotic lesions within the renal vasculature. When renal vascular lesions had been established (20 weeks of L-NAME), animals were divided into 2 subgroups: the one continued to receive L-NAME, whereas in the other, bosentan, a dual endothelin antagonist, was coadministered with L-NAME for an additional period of 10 weeks. Bosentan coadministration did not alter the increased systolic pressure at 30 weeks; in contrast, collagen I gene activity returned almost to control levels in renal vessels and glomeruli. In this subgroup of animals, renal vascular lesions (collagen and/or extracellular matrix deposition) and mortality rates were substantially reduced compared with untreated mice. These data indicate that endothelin participates in the mechanism(s) of renal vascular fibrosis by activating collagen I gene. Treatment with an endothelin antagonist normalizes expression of collagen I gene and leads to the regression of renal vascular fibrosis and to the improvement of survival, thus providing a complementary curative approach against renal fibrotic complications associated with hypertension.

Mechanisms mediating the renal profibrotic actions of vasoactive peptides in transgenic mice.

Transgenic mice are useful tools to investigate the mechanisms of the renal profibrotic actions of endothelin and angiotensin II. The overexpression of angiotensinogen and renin genes induces renal sclerosis independently of changes in systemic hemodynamics. The same results are observed when the endothelin-1 gene is overexpressed. Transgenic mice harboring the luciferase gene, under the control of the collagen I alpha2 chain promoter, and made hypertensive by induction of a nitric oxide (NO) deficiency have been studied. In this strain of mice, luciferase activity is an early index of renal and vascular fibrosis. Luciferase activity was increased in preglomerular arterioles and glomeruli when mice were treated with N:(omega)-nitro-L-arginine methyl ester, an inhibitor of NO synthases. Bosentan (an endothelin receptor antagonist) was as efficient as losartan (an AT1 receptor antagonist) in preventing renal fibrosis, although it did not decrease BP. In short-term experiments, angiotensin II produced an increase in luciferase activity that was entirely prevented by losartan but also by bosentan. It can be concluded that, during chronic inhibition of NO, the collagen I gene is activated, which contributes to the development of nephroangiosclerosis and glomerulosclerosis. Angiotensin II plays a major role in this fibrogenic process, and its effect is at least partly independent of systemic hemodynamics and mediated by the profibrotic action of endothelin-1.

Angiotensin II activates collagen I gene through a mechanism involving the MAP/ER kinase pathway.

Vascular remodeling and rearrangement of the extracellular matrix formation are among the major adaptive mechanisms to chronic increase in blood pressure. In previous studies we have found that angiotensin II (Ang II) participates in the hypertension-associated aortic and renal vascular fibrosis by stimulating collagen type I formation. The purpose of the present study was to gain insight into the molecular events that lead from the Ang II receptor to collagen I gene activation. To this end, we used a novel strain of transgenic mice harboring the luciferase gene under the control of the collagen I-alpha(2) chain promoter [procolalpha(2)(I)]. Ang II produced an early (1 hour) 2- to 3-fold stimulation of procolalpha(2)(I) activity in freshly isolated aortas and renal cortical slices (P:<0. 01) followed by similar increase in procolalpha(2)(I) mRNA aortic levels. This effect of Ang II was inhibited by AT1-receptor antagonism (candesartan) and blockade of the MAPK/ERK cascade (PD98059); in contrast, inhibition of the P38 kinase pathway (SB202190) and blockade of the release of the transcription factor NFkappaB (PDTC) did not have any effect in the Ang II-induced activation of the collagen I gene. In addition, Ang II induced a rapid (5 minutes) increase of the MAPK/ERK activity that was accompanied by increased expression (3-fold) of the c-fos proto-oncogene. This increase of c-fos mRNA expression was blocked by PD98059; in addition, curcumin, a blocker of the transcriptional factor AP-1, canceled the effect of Ang II on the collagen I gene. Decorin, a scavenger of the active form of transforming growth factor-beta (TGF-beta), canceled the Ang II effect on collagen I gene, whereas inhibition of the MAPK/ERK pathway had no effect on the TGF-beta-induced activation of procolalpha(2)(I). These data indicate that the cellular events after AT1 receptor stimulation and leading to activation of collagen I gene expression require activation of both the MAPK/ERK and TGF-beta signaling pathways.

Endothelin and renal vascular fibrosis: of mice and men.

The present review focuses on recent data regarding the role of endothelin as a mediator of renal vascular fibrosis. Following a brief description of the endothelin system, the question of whether endothelin is involved in hypertensive mechanisms is examined in experimental, genetic and transgenic animal models. Evidence is provided that implicates endothelin as an important factor of the development of tissue fibrosis and end-organ damage associated with hypertension, with particular emphasis on renal vascular fibrosis. Data indicating that endothelin interacts with other vasoconstrictor systems, such as angiotensin II, are also considered. Finally, results from preliminary clinical studies using endothelin receptor antagonists to treat cardiac and renal pathologies are briefly discussed.

Impaired prostaglandin E(2)/prostaglandin I(2) receptor-G(s) protein interactions in isolated renal resistance arterioles of spontaneously hypertensive rats.

The protective effect of vasodilator agents linked to the cAMP pathway is less effective for buffering the vasoconstrictor effect of angiotensin II in young animals with genetic hypertension. To determine the underlying cellular mechanism, experiments were performed on freshly isolated preglomerular resistance arterioles obtained from kidneys of 7-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Specific high-affinity saturable binding of (3)H-prostaglandin (PG) E(2) revealed 1 receptor class in renal microvessels; PGE(2) receptor density was similar in SHR and WKY (106 versus 115 fmol/mg; P>0.8), as was receptor affinity (3.6 versus 3.5 nmol/L; P>0.7). Basal cAMP activity was similar in renal arterioles from SHR and WKY. A major finding was that PGE(2), PGI(2), and isoproterenol produced weaker stimulation of cAMP formation in arteriolar cells of SHR (P<0.02). In contrast, GTPgammas and forskolin stimulated cAMP generation to a similar degree in both rat strains, which suggests normal adenylate cyclase activity in hypertension-prone SHR. Immunoblots revealed the presence of 3 classes of G proteins (G(s), G(i), and G(q)) in preglomerular arterioles. The relative amounts of discernible G-protein alpha-subunits in renal resistance vessels did not differ between SHR and WKY. These results extend previous in vivo studies of abnormal renal vascular reactivity in SHR and more directly localize defective coupling of the prostaglandin and beta-adrenergic receptors to a stimulatory G protein and cAMP production in freshly isolated preglomerular arteriolar cells of young SHR. This dysfunction may be due to an abnormal interaction between prostaglandin receptors and G(s) protein that leads to inefficient coupling of initiating steps in the cAMP-protein kinase A cascade during the development of hypertension.

Angiotensin II activates collagen type I gene in the renal vasculature of transgenic mice during inhibition of nitric oxide synthesis: evidence for an endothelin-mediated mechanism.

BACKGROUND: Hypertension is frequently associated with renal vascular fibrosis. The purpose of this study was to investigate whether angiotensin II (Ang II) is involved in this fibrogenic process. METHODS AND RESULTS: Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha(2) chain promoter [procolalpha(2)(I)]. Hypertension was induced by chronic inhibition of NO synthesis (N(G)-nitro-L-arginine methyl ester, L-NAME). Procolalpha(2)(I) activity started to increase in the renal vasculature after 4 weeks of L-NAME treatment (P<0.01) and at 14 weeks reached 3- and 8-fold increases over control in afferent arterioles and glomeruli, respectively (P<0.001). Losartan, an AT(1) receptor antagonist, given simultaneously with L-NAME prevented the increase of procolalpha(2)(I) levels and attenuated the development of renal vascular fibrosis without normalizing systolic pressure increase. Because we found previously that endothelin mediated renal vascular fibrosis in the L-NAME model, the interaction between Ang II, endothelin, and procolalpha(2)(I) was investigated in ex vivo and short-term in vivo experiments. In both conditions, the Ang II-induced activation of procolalpha(2)(I) in renal cortex was blocked by an endothelin receptor antagonist. CONCLUSIONS: During chronic inhibition of NO, the collagen I gene becomes activated, leading to the development of renal vascular fibrosis. Ang II is a major player in this fibrogenic process, and its effect on collagen I gene is independent of systemic hemodynamics and is at least partly mediated by the profibrogenic action of endothelin.

Vascular endothelin-1 gene expression and synthesis and effect on renal type I collagen synthesis and nephroangiosclerosis during nitric oxide synthase inhibition in rats.

BACKGROUND: The progression of hypertension during NO deficiency is associated with renal vascular fibrosis due to increased extracellular matrix (mainly collagen I) formation. The purpose of the present study was to investigate whether endothelin-1 (ET-1) is involved in this pathophysiological process. METHODS AND RESULTS: Treatment of rats for 4 weeks with the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) 50 mg. kg-1. d-1 increased systolic blood pressure to 159+/-12 mm Hg. In animals treated with L-NAME, histological evaluation of renal sections revealed an increased formation of extracellular matrix (Masson's trichrome), and specifically of collagens (Sirius red). A part of this fibrosis was attributed to abnormal collagen I presence, because mRNA expression of the collagen I alpha1 chain (reverse transcription-polymerase chain reaction) and procollagen I formation (radioimmunoassay) were increased 3- and 2.5-fold, respectively, in the renal resistance vessels of hypertensive animals. In subsequent experiments, we examined whether ET-1 was involved in activation of collagen I formation. mRNA expression (RNase protection assay) of preproET-1 and ET-1 content (radioimmunoassay) were 10-fold and 3-fold increased, respectively, in renal microvessels of rats treated with L-NAME. Interestingly, in these vessels, ET-1 (immunostaining) was colocalized with sudanophilic lesions. Bosentan, an ET receptor antagonist (20 mg. kg-1. d-1), coadministered with L-NAME canceled the increased mRNA expression and synthesis of collagen I and attenuated the severity of renal vascular lesions without affecting L-NAME-induced high blood pressure. CONCLUSIONS: These data demonstrate that ET-1 synthesis is increased in renal microvessels when NO production is suppressed. In this model of hypertension, ET-1 is a major activator of collagen I formation in renal resistance vessels and participates in the development of renal fibrosis without affecting systolic blood pressure.

Mesangial AT1 receptors: expression, signaling, and regulation.

Mesangial cells are one of the main targets of angiotensin II (AngII) in the renal cortex. AngII receptors on mesangial cells are of high affinity (nanomolar range). They belong to the AT1 subtype as shown by the inhibitory effect of AT1 antagonists on [125I]-Sar1, Ala8 AngII binding and on all of the biologic effects mediated by AngII, such as cytosolic calcium stimulation, inositol phosphate formation, prostaglandin production, and cell contraction. AngII also exerts long-term effects on mesangial cells, including stimulation of cell growth and synthesis of a variety of proteins, essentially the components of the extracellular matrix (collagen, fibronectin) and the type 1 inhibitor of plasminogen activator. These effects are mediated, at least in part, by autocrine products, in particular endothelin, platelet-derived growth factor, and transforming growth factor-beta, whose synthesis is enhanced by AngII. Treatment by an AT1 receptor blocker of mice with experimental nephritis inhibits activation of type I collagen alpha2 chain promoter and prevents the development of glomerulosclerosis. AngII receptors in rat mesangial cells are equally distributed between the AT1A and AT1B isoforms. Treatment of these cells by AngII or losartan, an AT1 receptor blocker, has no effect on AT1A and AT1B receptor mRNA expression, whereas candesartan, another AT1 receptor blocker, increases and dexamethasone decreases this expression.

[Interactions between the renin-angiotensin system, nitric oxide and endothelin]. / Interactions entre le système rénine-angiotensine, le monoxyde d'azote et l'endothéline.

The renin-angiotensin system plays a major role in the regulation of blood pressure and sodium balance. Nitric oxide (NO) and endothelin (ET-1) are involved in the regulation of renin release and modulate the vasoconstrictive and fibrogenic effects of angiotensin II. the mechanisms that activate renin production are less effective when endogenous NO synthesis is inhibited. In the absence of NO, ET-1 prevents renin secretion. Angiotensin II stimulates the production of NO and ET-1 by endothelial cells. The vascular effects of angiotensin II are inhibited by NO reinforced by ET-1. The stimulation of ET-1 secretion could partly explain the long-term effects of angiotensin II on vascular remodelling.

Nitric oxide inhibition induces early activation of type I collagen gene in renal resistance vessels and glomeruli in transgenic mice. Role of endothelin.

Hypertension is often associated with the development of nephroangio- and glomerulo-sclerosis. This pathophysiological process is due to increased extracellular matrix protein, particularly type I collagen, accumulation. This study investigated whether nitric oxide (NO) synthesis is involved in the mechanism(s) regulating activation of the collagen I gene in afferent arterioles and glomeruli. Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha2 chain promoter [procolalpha2(I)]. Measurements of luciferase activity provide highly sensitive estimates of collagen I gene activation. NO synthesis was inhibited by NG-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg per day) for a period of up to 14 wk. Systolic blood pressure was increased after 6 wk of treatment (117+/-2 versus 129+/-2 mmHg, P < 0.01) and reached a plateau after 10 wk (around 160 mmHg). Luciferase activity was increased in freshly isolated afferent arterioles and glomeruli as early as week 4 of L-NAME treatment (150 and 200% of baseline, P < 0.01, respectively). The activation of procolalpha2(I) became more pronounced with time, and at 14 wk increased four- and tenfold compared with controls in afferent arterioles and glomeruli, respectively (P < 0.001). In contrast, luciferase activity remained unchanged in aorta and heart up to 8 wk and was increased thereafter. Increased histochemical staining for extracellular matrix deposition, and particularly of collagen I, was detected in afferent arterioles and glomeruli after 10 wk of L-NAME treatment. This fibrogenic process was accompanied by an increased urinary excretion rate of endothelin. In separate experiments, the stimulatory effect of L-NAME on collagen I gene activation was abolished when animals were treated with bosentan, an endothelin receptor antagonist. Similarly, bosentan reduced the increased extracellular matrix deposition in afferent arterioles and glomeruli during NO inhibition. Interestingly, bosentan had no effect on the L-NAME- induced increase of systolic pressure. These data indicate that NO inhibition induces an early activation of the collagen I gene in afferent arterioles and glomeruli. This activation in the kidney precedes the increase in blood pressure and the procolalpha2(I) activation in heart and aorta, suggesting a specific renal effect of NO blockade on collagen I gene expression that is independent of increased blood pressure and, at least partly, mediated through stimulation of the endothelin receptor. Use of procolalpha2(I) transgenic mice provides a novel and efficient model to study the pathophysiological mechanism(s) regulating renal fibrosis.

Activation of renin synthesis is dependent on intact nitric oxide production.

The present study investigated whether or not nitric oxide (NO) synthesis mediates mechanisms regulating activation of renin formation. Studies were performed on afferent arterioles freshly isolated from the rat kidney. We have shown previously that this preparation is a useful model to study regulation of renin synthesis and secretion. The expression of renin mRNA was assessed by ribonuclease protection assay, and total renin content and renin secretion by radioimmunoassay. In afferent arterioles isolated from rats treated with the angiotensin-converting enzyme inhibitor ramipril, renin mRNA levels, total renin content and renin secretion were increased threefold compared to untreated controls. Inhibition of NO-synthase by NG-nitro-L-arginine methyl ester (L-NAME) in the ramipril-treated rats, abolished the increase in renin mRNA levels, total renin content and renin secretion. In other animals furosemide, a diuretic acting on macula densa cells, activated renin synthesis to a level similar to that found in the ramipril-treated group. Addition of L-NAME to the furosemide-treated rats suppressed the increases in renin mRNA levels, total renin content and renin secretion, suggesting that NO acts on renin activation by a mechanism independent of angiotensin II. In separate experiments, the inhibitory effect of L-NAME on the activation of renin secretion was abolished when afferent arterioles were treated with nicardipine, an L-type Ca2+ channel blocker, suggesting that the suppression of renin activation during NO inhibition is due to increased Ca2+ entry. Since endothelin is a potent mediator of Ca2+ influx and an inhibitor of renin release, we tested whether or not endothelin could be involved in the inhibitory effect of L-NAME on renin secretion. Application of the endothelin receptor antagonist, bosentan, in vitro mimicked the effect of nicardipine. In addition, bosentan coadministered with L-NAME in vivo blunted the inhibitory effect of L-NAME and restored the increases in renin mRNA level, synthesis and secretion. These data indicate that the physiological mechanism(s) regulating activation of renin synthesis and secretion are impaired during NO inhibition, probably because of increased Ca2+ influx. This increase in calcium flux is mediated at least partially by the action of endothelin.

Regulation of angiotensin II receptor AT1 subtypes in renal afferent arterioles during chronic changes in sodium diet.

Studies determined the effects of chronic changes in sodium diet on the expression, regulation, and function of different angiotensin II (ANG II) receptor subtypes in renal resistance vessels. Rats were fed low- or high-sodium diets for 3 wk before study. Receptor function was assessed in vivo by measuring transient renal blood flow responses to bolus injections of ANG II (2 ng) into the renal artery. ANG II produced less pronounced renal vasoconstriction in rats fed a low- compared with high-sodium diet (16% vs. 56% decrease in renal blood flow, P < 0.001). After acute blockade of ANG II formation by iv enalaprilat injection in sodium-restricted animals, ANG II produced a 40% decrease in renal blood flow, a level between untreated dietary groups and less than high salt diet. Intrarenal administration of angiotensin II receptor type 1 (AT1) receptor antagonists losartan or EXP-3174 simultaneously with ANG II caused dose-dependent inhibition of ANG II responses. Based on maximum vasoconstriction normalized to 100% ANG II effect in each group, AT1 receptor antagonists produced the same degree of blockade in all groups, with an apparent maximum of 80-90%. In contrast, similar doses of the angiotensin II receptor type 2 (AT2) receptor ligand CGP-42112 had only a weak inhibitory effect. In vitro equilibrium-saturation 125I-ANG II binding studies on freshly isolated afferent arterioles indicated that ANG II receptor density was lower in the low- vs. high-sodium animals (157 vs. 298 fmol/mg, P < 0.04); affinity was similar (0.65 nM). Losartan and EXP-3174 displaced up to 80-90% of the ANG II binding; fractional displacement was similar in both diet groups. In contrast, the AT2 receptor analogues PD-123319 and CGP-42112 at concentrations < 10(-6) M had no effect on ANG II binding. RT-PCR assays revealed the expression of both angiotensin II receptor type 1A (AT(1A)) and angiotensin II receptor type 1B (AT(1B)) subtypes in freshly isolated afferent arterioles, while there was very little AT2 receptor expression. Total AT1 receptor mRNA expression was suppressed by low sodium intake to 66% of control levels, whereas it was increased to 132% of control by high-sodium diet, as indicated by ribonuclease protection assay. Receptor regulation was associated with parallel changes in AT(1A) and AT(1B) expression; the AT(1A)/AT(1B) ratio was stable at 3.7. We conclude that AT1 receptors are the predominant ANG II receptor type in renal resistance vessels of 7-wk-old rats. Chronic changes in sodium intake caused parallel regulation of expression and amount of receptor protein of the two AT1 receptor genes that modulate receptor function and altered reactivity of renal vessels to ANG II.

Protein kinase A activity modulates natriuretic peptide-dependent cGMP accumulation in renal cells.

The purpose of this work was to examine whether the level of cAMP accumulation and protein kinase A (PKA) activity influence atrial natriuretic factor (ANF)-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in two renal cell types: rabbit cortical vascular smooth muscle cells (RCSMC) and SV-40-transformed human glomerular visceral epithelial cells (HGVEC-SV1). N-[2-(p-bromocinnamylamino)ethyl]- 5-isoquinolinesulfonamide (H-89), a PKA inhibitor, decreased ANF-stimulated cGMP production in RCSMC in a time- and concentration-dependent manner. ANF-stimulated cGMP production was markedly inhibited after prolonged 9- and 18-h incubations with 25 microM H-89 (52 and 65%, respectively) but was not altered after exposure of cells to this agent for 1 h. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine and N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, protein kinase inhibitors not selective for PKA, did not reproduce the effect of H-89, even at higher concentrations (50 and 100 microM). Cycloheximide (10 microM), a protein synthesis inhibitor, limited the inhibitory effect of H-89, although alone it did not modify the ANF-stimulated cGMP production. H-89 did not affect cGMP production when it was stimulated by SIN-1, a nitric oxide donor. Prolonged incubation (18 h) with 8-bromo cAMP or cholera toxin, an activator of Gs protein resulting in adenylate cyclase stimulation, enhanced ANF-dependent cGMP production by 225 and 176%, respectively. This stimulatory effect was blocked by 25 microM H-89. 125I-ANF binding to RCSMC at 4 degrees C was not affected by preincubation of the cells with H-89. There was a 44% decrease in the expression of ANF C receptors measured as the ANF-(4-23)-displaceable 125I-ANF binding at 37 degrees C, which could not, however, explain the inhibitory effect of H-89 on cGMP production. Modulation of ANF- and C-type natriuretic peptide-dependent cGMP production by H-89 and cholera toxin was also found in HGVEC-SV1 with the same characteristics as in RCSMC. Taken together, these results suggest that PKA activity controls the function of natriuretic peptide guanylate cyclase-coupled receptors in the two cell types studied. PKA-dependent inhibition of a negatively regulatory protein distinct from the receptor itself seems necessary for a full cGMP response.