Sympathetic blockade prevents the decrease in cardiac VEGF expression and capillary supply in experimental renal failure.

Uremic cardiomyopathy of men and rodents is characterized by lower myocardial capillary supply that in rats could be prevented by central and peripheral blockade of the sympathetic nervous system. The underlying pathomechanisms remain largely unknown. We investigated whether alterations of cardiac vascular endothelial growth factor (VEGF) gene and protein expression were involved. In our long-term experiment, we analyzed whether VEGF gene and protein expression was altered in the heart of male Sprague-Dawley rats with either sham operation (sham, n=10) or subtotal nephrectomy (SNX, n=10). In our short-term experiment (17 sham, 24 SNX), the effect of a putative downregulation of sympathetic nervous activity by surgical renal denervation (interruption of renal afferent pathways) on cardiac gene expression of VEGF, flt-1, and flk-1 and on myocardial capillary supply was analyzed. In the long-term study, cardiac capillary supply and vascular endothelial growth factor gene and protein expression were significantly lower in SNX than in sham. In the short-term experiment, cardiac VEGF mRNA expression was significantly lower in untreated SNX (4,258±2,078 units) than in both sham groups (11,709±4,169 and 8,998±4,823 units); this decrease was significantly prevented by renal denervation (8,190±3,889, P<0.05). We conclude that cardiac VEGF gene and protein expression is reduced in experimental renal failure, and this may be considered as one potential reason for impaired myocardial adaptation under the situation of cardiac hypertrophy. The beneficial effect of sympathetic downregulation on cardiac structure and function in renal failure may be at least in part explained by increased cardiac VEGF gene expression.

Lack of nidogen-2 increases blood pressure, glomerular and tubulointerstitial damage in DOCA-salt hypertension.

BACKGROUND: Nidogen-2, an extracellular matrix protein, is ubiquitous in renal basement membranes linking the laminin and collagen IV networks. Nidogen-2-deficient (nidogen-2(-/-)) mice do not exhibit a phenotype, and renal basement membranes appear normal. The functional role of nidogen-2 in the adult kidney under pathological conditions however remains unclear. We tested the hypothesis that nidogen-2 mediated cell-matrix interactions are important to maintain glomerular integrity and structure in renal hyperperfusion and hypertension. MATERIALS AND METHODS: Two weeks after unilateral nephrectomy (UNX), desoxycorticosterone (DOCA)-salt hypertension was induced in nidogen-2(-/-) mice and their wild type littermates for 6 weeks. Renal damage was assessed by means of semiquantitative scoring, morphometric analysis, immunohistochemistry and measurement of serum creatinine and albumin excretion. RESULTS: UNX alone resulted in a very mild increase in renal damage in nidogen-2(-/-) mice compared to wild type animals. Following DOCA-salt treatment, blood pressure, serum creatinine and albumin excretion were significantly higher in nidogen-2(-/-) than in wild type mice. In addition, nidogen-2(-/-) mice showed increased mesangial cell hyperplasia and matrix expansion with higher expression of fibronectin and its receptor alpha8 integrin. Glomerular capillaries were significantly reduced in size and number. CONCLUSIONS: We demonstrate that in both mild and severe glomerular damage, lack of nidogen-2 is associated with: (i) increased mesangioproliferation; (ii) higher mesangial matrix expansion; and (iii) reduction in glomerular capillary supply. These findings suggest a critical role for nidogen-2 in the maintenance of glomerular structure in the diseased kidney.

Rapid development of severe end-organ damage in C57BL/6 mice by combining DOCA salt and angiotensin II.

The C57BL/6 mouse strain serves as the genetic background of many transgenic and gene knockout models; however, this strain appears to be resistant to hypertension-induced renal injury. We developed a new model of hypertensive end-organ damage in C57BL/6 mice by combining deoxycorticosterone acetate (DOCA) and salt with angiotensin II infusion. The systolic blood pressure (SBP) was significantly elevated in DOCA salt-angiotensin II mice compared to control mice or mice treated individually with DOCA salt or angiotensin II. Hypertensive glomerular damage, increased expression of profibrotic and inflammatory genes, albuminuria, tubular casts, increased plasma cholesterol, cardiac hypertrophy, and fibrosis were found in mice treated with DOCA salt-angiotensin II. The SBP in the angiotensin II-infused group was further increased by increasing the infusion rate; only mild injury was observed in these mice, suggesting that blood pressure was not a causal factor. Removal of DOCA and the angiotensin pump lowered blood pressure to normal; however, albuminuria along with the glomerular and cardiac damage did not completely resolve. Our study describes a new model of hypertensive end-organ damage and repair in C57BL/6 mice.

In-situ analysis of mast cells and dendritic cells in coronary atherosclerosis in chronic kidney disease (CKD).

AIMS: Mast cells (MC) and dendritic cells (DC) have immune modulatory function and can influence T-cell activity. Both cell types have been found in atherosclerotic plaques and are thought to play an important role for plaque stability. Compared to matched segments of the non-renal population, patients with chronic kidney disease (CKD) show a more pronounced and more aggressive course of atherosclerosis with higher plaque calcification and significantly higher complications rates. It was the aim of this study to analyze the number and localization of MCs and DCs, macrophages, T- and B-cells as well as the expression of markers of inflammation such as CRP and NFκB in calcified and non-calcified atherosclerotic plaques of patients with CKD and control patients. METHODS: Fifty coronary atherosclerotic plaques from patients with endstage CKD (CKD, n=25) and control (n=25) patients were categorized according to the Stary classification and investigated using immunohistochemistry (markers for MC, DC, T, B, macrophage and NFκB). Expression was analyzed separately for the complete plaque area as well as for the different plaque subregions and correlations were analyzed. RESULTS: We found only very few DCs and MCs per lesion area with slightly increased numbers in calcified plaques. MCs per plaque area were significantly more frequent in CKD than in control patients and this was independent of plaque calcification. MCs were most frequently found in the shoulder and basis of the plaque. DCs per plaque area were significantly less in calcified plaques of CKD compared to control patients. In control, but not in CKD patients, DCs were significantly more frequent in calcified than in non-calcified plaques. Within the plaques DCs were similarly distributed between all 4 subregions. CONCLUSIONS: Coronary atherosclerotic plaques of CKD patients showed a significantly higher number of MCs whereas DCs were less frequent compared to control patients particularly if plaques were calcified. These findings might indicate a potential proinflammatory role of MCs, but not of DCs in atherosclerotic lesions of CKD patients, adding another characteristic of advanced atherosclerosis in these patients.

Angiotensin II type 1 receptor blockade prevents lethal malignant hypertension: relation to kidney inflammation.

BACKGROUND: Angiotensin II is elevated in malignant hypertension. We tested the hypothesis that angiotensin II type 1 receptor blockade can prevent the development of malignant hypertension even in the absence of a blood pressure-lowering effect. METHODS AND RESULTS: Two-kidney, 1-clip rats were followed up for 28 days; blood pressure was measured by tail-cuff plethysmography and intra-arterially. After a 2-week run-in phase, rats received valsartan at a dose of 0.3 (n=14) or 3 (n=12) mg. kg(-1). d(-1) or solvent (n=27). Only the higher dose of valsartan, but not the lower dose, decreased blood pressure. Both doses of valsartan prevented the development of lethal malignant hypertension. Twenty of 27 solvent-treated renovascular hypertensive rats died, but only 3 of 14 rats treated with the low dose and 1 of 12 rats treated with the high dose of valsartan died. Histological signs of malignant nephrosclerosis were found in all rats examined that had died throughout the study and in 6 of 7 surviving solvent-treated renovascular hypertensive animals. Increased expression of monocyte chemoattractant protein-1 and prominent interstitial influx of macrophages occurred in the nonclipped kidneys exposed to high pressure in solvent-treated rats. These alterations were prevented by valsartan at both doses, irrespective of blood pressure effects. CONCLUSIONS: Angiotensin II type 1 receptor blockade by valsartan prevents lethal malignant hypertension independently of blood pressure. The results suggest that reduction of angiotensin-induced inflammation in the kidney may contribute to the protective effects of valsartan.

1166 A/C polymorphism of the angiotensin II type 1 receptor gene and the response to short-term infusion of angiotensin II.

BACKGROUND: Previous studies reported an association of the 1166 A/C polymorphism of the angiotensin II (Ang II) type 1 receptor gene with high blood pressure and cardiovascular disease. We tested the hypothesis that this polymorphism affects the blood-pressure, renal hemodynamic, and aldosterone response to infused Ang II. METHODS AND RESULTS: Young, male, white volunteers (n = 116) with normal (n = 65) or mildly elevated (n = 51) blood pressure on a high salt intake were genotyped for the 1166 A/C polymorphism. Two doses of Ang II (0.5 and 3 ng x kg(-1) x min(-1) over 30 minutes each) increased blood pressure, plasma aldosterone, glomerular filtration rate, and filtration fraction and decreased renal blood flow. The blood-pressure, renal hemodynamic, and aldosterone responses were not significantly different between subjects homozygous for the A allele (n = 56) and heterozygous subjects (n = 47) or subjects homozygous for the C allele (n = 13). Comparison of A allele homozygotes with all C allele carriers pooled (n = 60) or restriction of the analysis to normotensive volunteers also revealed no significant differences between genotypes. CONCLUSIONS: The 1166 C variant of the Ang II type 1 receptor does not lead to a greater blood-pressure, aldosterone, or renal vascular response to infused Ang II in young, male, white subjects. We conclude that the 1166 A/C polymorphism does not have a major effect on these actions of Ang II.

Aldosterone synthase (CYP11B2) -344 C/T polymorphism is associated with left ventricular structure in human arterial hypertension.

OBJECTIVES: This study examined the association between the -344 C/T polymorphism of the human aldosterone synthase promoter and left ventricular structure in arterial hypertension. BACKGROUND: Because of conflicting results from different studies, the mechanism of such an association, if any, has not been determined. METHODS: We examined the aldosterone synthase promoter genotype in 120 young (age: 26 +/- 3 years) male, white subjects with normal or mildly elevated blood pressure. Left ventricular structural parameters and urinary sodium excretion over 24 h before and after additional oral sodium load (6 g/day over 1 week) were determined. RESULTS: Hypertensive subjects with the CC genotype had a greater left ventricular end-diastolic diameter but smaller relative wall thickness than those with the TT genotype (54 +/- 2 vs. 50 +/- 4 mm, and 0.37 +/- 0.07 vs. 0.44 +/- 0.06 mm, respectively; p < 0.05). Hypertensive subjects with the TT genotype (n = 15) had a greater increase in urinary sodium excretion after oral sodium load than those with the CC genotype (n = 11) (135 +/- 95 vs. 24 +/- 133 mmol/liter/day; p < 0.05). Serum aldosterone levels were found to be decreased after oral sodium load in hypertensive subjects with the TT and CT genotypes only (-37 +/- 45 and -38 +/- 51 pg/ml, respectively; all p < 0.01) but not in those with the CC genotype (-12 +/- 30 pg/ml, n.s.). Such differences were not found in normotensive subjects. CONCLUSIONS: Hypertensive subjects with the -344 CC genotype of the aldosterone synthase promoter are characterized by a pattern of early eccentric left ventricular hypertrophy. Differences in renal sodium handling across the genotypes might contribute to this finding.

Coordinate expression of cyclooxygenase-2 and renin in the rat kidney in renovascular hypertension.

Prostaglandins contribute to the regulation of renin synthesis and secretion. We tested the hypothesis that the inducible isoform of prostaglandin G/H synthase, cyclooxygenase-2, contributes to the stimulation of renin synthesis in renovascular hypertension. The expression of cyclooxygenase-2 and renin was investigated in the kidneys of rats with two-kidney, one-clip renovascular hypertension or sham operation. Systolic blood pressure was increased 2 weeks after clipping (153+/-7 versus 112+/-4 mmHg in controls, n=6 each, P<.05) and continued to rise until 4 weeks. Cyclooxygenase-2 mRNA levels were increased in clipped kidneys but remained unchanged or slightly decreased in nonclipped kidneys. Cyclooxygenase-2 protein was expressed mainly in the macula densa and occasionally in distal tubular cells not associated with the macula densa. Two weeks after clipping, the percentage of juxtaglomerular apparatus staining positive for cyclooxygenase-2 was 27.8+/-3.6 in clipped kidneys, 3.1+/-0.4 in contralateral kidneys, and 8.0+/-1.3 in controls; the percentages for immunoreactive renin staining in the afferent arteriole were 33.6+/-2 in clipped kidneys, 1.9+/-0.5 in contralateral kidneys, and 12.4+/-4.0 in controls, respectively. Similar parallel changes in renin and cyclooxygenase-2 staining were observed 4 weeks after clipping. The percentage of cyclooxygenase-2-positive juxtaglomerular apparatus correlated positively with the percentage that was renin positive (r=0.78, P<.05). Double immunostaining showed coexpression of cyclooxygenase-2 and renin protein in the same juxtaglomerular apparatus. Our data are consistent with a role for macula densa cyclooxygenase-2 in the regulation of renin in renovascular hypertension.

Experimental studies on the role of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 in hypertensive nephrosclerosis.

T helper cells and macrophages infiltrate into the renal cortical interstitium during the course of hypertensive nephrosclerosis. To investigate the mechanisms of mononuclear cell infiltration, we examined the expression of the intercellular adhesion molecule-1 (ICAM-1) and its counterpart lymphocyte function-associated antigen-1 (LFA-1) in the progression of hypertensive renal injury. We studied nonclipped kidneys of two-kidney, one clip renovascular hypertensive and sham-operated control rats immunohistochemically at 4, 7, 14, and 28 days after clipping (n = 5 per group and time point). Systolic pressure was significantly elevated by day 7 (154 +/- 4 versus 117 +/- 6 mm Hg in sham, P < .05). The development of hypertension resulted in a progressive increase of ICAM-1 expression in the perivascular and interstitial areas of the renal cortex and on proximal tubular brush borders. Only a few glomeruli showed augmented ICAM-1 staining. Increased ICAM-1 was associated with an accumulation of LFA-1-positive mononuclear cells in the perivascular region (day 14: 15 +/- 4 versus 2 +/- 0.2 cells/mm2 in sham, P < .005) and intertubular region (127 +/- 11 versus 32 +/- 3 cells per millimeter squared in sham, P < .005). The maximum was obtained at day 14 and remained elevated until day 28. In addition, the number of interstitial LFA-1-positive infiltrating cells was related to the degree of interstitial and tubular ICAM-1 expression and correlated with blood pressure (r = .75, P < .001, n = 18). Our data suggest that ICAM-1 is involved in the recruitment of macrophages/lymphocytes via specific interaction of ICAM-1 and LFA-1 in this model of hypertensive target-organ damage.

Vascular renin in the guinea pig. Suppression by the renin inhibitor remikiren.

Angiotensin I and II are generated by the vascular wall. Whether this generation depends on renin or on other enzymes is debated. We tested the hypothesis that remikiren, a highly specific inhibitor of human and guinea pig renin, may inhibit the vascular renin-angiotensin system. Isolated hindquarters from guinea pigs were perfused with an artificial medium, and angiotensin I and II release was measured by high-performance liquid chromatography and radioimmunoassay. Guinea pig hindquarters released angiotensin I (23.8 +/- 5.6 fmol/30 min; n = 13) and angiotensin II (95.2 +/- 19 fmol/30 min; n = 13) spontaneously. Inhibition of the angiotensin I-converting enzyme by captopril (10 nmol/mL) suppressed angiotensin II by 85% and increased angiotensin I by 352% (n = 5, P < .05). Infusion of remikiren (1.6 nmol/mL) in addition to captopril decreased angiotensin I release by 68% (P < .05 versus captopril alone, n = 5 each). We conclude that renin generates angiotensin I in an isolated guinea pig resistance vessel bed. Our study demonstrates that renin rather than nonrenin enzymes is responsible for the major part of vascular angiotensin formation.

Angiotensin II facilitates sympathetic transmission in rat hind limb circulation.

We developed a novel method to stimulate the sympathetic innervation of the isolated, perfused rat hind limb to investigate whether a subpressor concentration of angiotensin II (Ang II) facilitates noradrenergic transmission in the vascular bed to skeletal muscle. We electrically stimulated the lumbar sympathetic trunk while perfusing the preparation with artificial medium. Seventy-five percent of the resulting frequency-dependent increases in perfusion pressure were mediated by alpha 1-adrenergic receptors. Ang II (10 nM) significantly enhanced the effects of nerve stimulation at 1 and 10 Hz (by 42% and 35%, respectively). At a supramaximal stimulation frequency (20 Hz), Ang II prolonged the duration of the response without changing the peak increase in pressure. The reuptake inhibitor cocaine did not influence the effects of Ang II at 1 and 10 Hz but blocked the effect at 20 Hz. To control for nonspecific synergism with norepinephrine, we compared Ang II with vasopressin. Both peptides potentiated the pressor response to exogenous norepinephrine; however, vasopressin did not change the pressor response to nerve stimulation at any frequency. We conclude that Ang II, but not vasopressin, facilitates noradrenergic transmission in skeletal muscle resistance vessels, independent of its direct vasoconstrictor activity. The neurovascular preparation we describe may be useful in addressing other hypotheses concerning sympathetic transmission in skeletal muscle resistance vessels.

Aberrant renal vascular morphology and renin expression in mutant mice lacking angiotensin-converting enzyme.

To determine whether angiotensin-converting enzyme plays a role in the development and maintenance of normal renal architecture, the renal morphology of 10- to 12-month-old female mice homozygous for a disruption of the converting enzyme gene was compared with that of age-matched wild-type mice. Tubular obstruction, dilatation, and atrophy were present in all kidneys from the homozygous mutant mice but absent in wild types; two kidneys from 4 mutant mice but none from the wild types were hydronephrotic. The entire arterial vascular tree, microdissected from mice with no converting enzyme, was grossly distorted in comparison to the vasculature of wild-type mice; all intrarenal arterial vessels were widened and thickened, including the terminal (afferent) arterioles. In wild-type mice kidneys, renin-positive cells were detected exclusively in a juxtaglomerular localization. In contrast, abnormal distribution of renin immunostaining was observed in mice without converting enzyme; scattered renin-positive cells were seen along the arterial vessels, often in a perivascular localization, and interstitial renin-positive cells surrounded glomeruli. Kidney renin mRNA was increased more than 32-fold in the mutant mice compared with wild types. Northern blot analysis revealed that this increase included the accumulation of large amounts of smaller renin RNA transcripts. In summary, mice lacking the converting enzyme exhibit abnormal renal vessels and tubules. Renin synthesis is increased, accompanied by the presence of small renin mRNA species, and renin is present mainly in interstitial and perivascular cells. We conclude that angiotensin-converting enzyme is necessary to preserve normal kidney architecture and the normal pattern of renin expression.

Angiotensinases restrict locally generated angiotensin II to the blood vessel wall.

We tested the hypothesis that angiotensinases limit the spillover of locally formed angiotensin II into the circulation. The release of angiotensin peptides from isolated rat hindquarters perfused with an artificial medium was measured by high-performance liquid chromatography and radioimmunoassay. The spontaneous release of angiotensins was increased by the angiotensinase inhibitors phenanthroline (850+/-195 versus 95+/-33 fmol of angiotensin I per 30 minutes in controls, P<.05, n=5 each) and amastatin (P<.05, n=5 each). Infusion of renin induced sustained local angiotensin I formation, which was also increased by phenanthroline. Stimulation of local angiotensin formation by renin infusion was compared with infusion of exogenous angiotensin II. Renin caused similar increases of perfusion pressure (11.1+/-2.2 versus 7.6+/-1.9 mm Hg after angiotensin II, P>.05) despite lower angiotensin II levels in the venous effluent than during infusion of exogenous angiotensin II (65+/-2 versus 482+/-33 fmol/mL, P<.05, n=7 each). Thus, renin must have caused higher angiotensin II tissue levels than indicated by the measurements in the venous effluent. The pressor response to renin was abolished by the type 1 angiotensin II receptor antagonist losartan. We conclude that the major part of locally generated angiotensins is not released into the circulation but degraded by angiotensinases within the tissue compartment.

Nerve-mediated antidiuresis and antinatriuresis after air-jet stress is modulated by angiotensin II.

A putative interaction between angiotensin II (Ang II) and the sympathetic nervous system within the kidney has been reported. We tested the hypothesis in conscious rats that endogenous Ang II modulates the renal effects of a stress-induced increase in sympathetic nerve activity. We recorded mean arterial blood pressure, heart rate, renal sympathetic nerve activity, renal hemodynamics, urine volume, and urinary sodium content in conscious rats. We used the Ang II type 1 receptor blocker ZD 7155 to inhibit the effects of endogenous Ang II. Ten minutes of air-jet stress increased renal sympathetic nerve activity by 98 +/- 4% (n = 6) without changing systemic hemodynamics. Air-jet stress reduced urine volume (from 31 +/- 3 to 8 +/- 4 microL/min per gram kidney weight, P < .05, n = 12) and sodium excretion (from 4.3 +/- 0.9 to 1.2 +/- 0.3 mumol/min per gram kidney weight, P < .05, n = 12). After renal denervation, air-jet stress had no effect on either parameter. Six micrograms of the Ang II type 1 receptor inhibitor ZD 7155 blunted the decrease in urine volume and sodium excretion in response to air-jet stress, although the increase in renal sympathetic nerve activity during air-jet stress and the pressor response to exogenous Ang II were not affected. Glomerular filtration rate and renal plasma flow were also not affected. Higher doses of 30 and 60 micrograms ZD 7155 inhibited the pressor response to exogenous Ang II and abolished the changes in urine volume and sodium excretion in response to air-jet stress. None of the ZD 7155 doses affected urinary sodium excretion permanently. Hence, the Ang II type 1 receptor antagonist ZD 7155 impaired or abolished the renal nerve-mediated antinatriuresis and anitidiuresis in response to air-jet stress. We conclude that endogenous Ang II modulates the renal effects of centrally mediated changes of sympathetic nerve activity in conscious rats.

Epicardial bradykinin B2 receptors elicit a sympathoexcitatory reflex in rats.

Bradykinin may be generated in the heart during ischemia and is involved in nociception. We tested the hypothesis that bradykinin elicits a sympathoexcitatory reflex in rats by stimulating cardiac afferent nerve fibers. Rats were implanted with femoral catheters for measurement of blood pressure and heart rate, a bipolar electrode for measurement of renal sympathetic nerve activity, and a pericardial catheter for intrapericardial injection of substances. Rats were slightly anesthetized with hexobarbital so pain reactions were prevented. Graded doses of bradykinin (2.5, 12, 25 micrograms) were injected intravenously or intrapericardially into control rats, intrapericardially after vagotomy, intrapericardially after intrapericardial pretreatment with the bradykinin B2 receptor antagonist Hoe 140, and intrapericardially after cardiac autonomic blockade (intrapericardial pretreatment with 10% procaine). For comparison, the serotonin 5-HT3 agonist phenylbiguanide, a substance known to elicit sympathoinhibitory reflexes by cardiac vagal afferents, and adenosine, putatively inducing sympathoexcitatory responses via the heart, were applied intrapericardially. Bradykinin increased blood pressure when administered intrapericardially but decreased blood pressure when injected intravenously; both intrapericardial and intravenous bradykinin increased renal sympathetic nerve activity. Intrapericardial adenosine had no effect on circulatory control. Intrapericardial pretreatment with the B2 receptor antagonist Hoe 140 completely inhibited the increases of blood pressure and renal sympathetic nerve activity in response to intrapericardial bradykinin but did not affect the responses to intrapericardial phenylbiguanide. Bilateral cervical vagotomy abolished the decreases of blood pressure, heart rate, and renal sympathetic nerve activity after intrapericardial phenylbiguanide but did not influence the responses to intrapericardial bradykinin. Cardiac autonomic blockade with intrapericardial procaine abolished all responses to bradykinin and phenylbiguanide. We conclude that cardiac bradykinin elicits a sympathoexcitatory reflex by epicardial B2 receptors in rats. The afferent portion of the reflex is most likely contained within sympathetic cardiac afferent fibers. Bradykinin may contribute to increased sympathetic nerve activity in pathophysiological situations of coronary artery disease and cardiac ischemia.

Expression of angiotensin-converting enzyme in renovascular hypertensive rat kidney.

We hypothesized that the gene expression of angiotensinogen, angiotensin-converting enzyme, and angiotensin II type 1 receptor, in addition to renin, is increased in kidneys after renal artery stenosis. Two-kidney, one clip renovascular hypertension was initiated in Sprague-Dawley rats by clipping of the left renal artery; control rats were sham operated. Blood pressure was not changed for the first 2 days after clipping but was elevated on day 4 (mean arterial pressure, 104 +/- 4 versus 87 +/- 2 mm Hg in sham-operated control rats, P < .002) and increased further during the next 24 days. Rats were killed 2, 4, 7, 14, and 28 days after clipping or sham operation, and poly(A)(+)-purified renal cortical RNA was analyzed by Northern blotting. Autoradiographs were quantitated by densitometry and normalized for the expression of a housekeeping gene. Renin expression was increased in the clipped kidney (by 149% on day 2) and decreased in the nonclipped kidney (by 82% on day 2), compared with kidneys of control rats. Expression of the angiotensin-converting enzyme was increased in clipped kidneys from the first day after clipping (158%) and throughout the experiment (66% on day 28), but was unchanged or slightly decreased in nonclipped kidneys. Angiotensinogen mRNA showed little change. Angiotensin II type 1 receptor expression was decreased in nonclipped kidneys but unchanged during the first 7 days in clipped kidneys. Our results show that components of the renin-angiotensin system other than renin are also differentially expressed in clipped kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the renin-angiotensin system upregulates cyclooxygenase-2 expression in the macula densa.

The expression of cyclooxygenase 2 (COX-2) in the late thick ascending limb, including the macula densa, is found to be upregulated in an activated renin-angiotensin system. How this upregulation is managed is not yet known. We therefore considered the possibility that the stimulation of COX-2 expression is triggered by the activation of the renin-angiotensin system. For this purpose, we treated male Sprague-Dawley rats with the angiotensin I-converting enzyme inhibitor ramipril (10 mg/kg per day), the angiotensin II type 1 (AT(1)) receptor blocker losartan (30 mg/kg per day), and the angiotensin II type 2 (AT(2)) receptor blocker PD123319 (6 mg/kg per day) for 4 days. We determined the expression of COX-2 mRNA and protein in the renal cortex. We found that ramipril and the AT(1) receptor blocker losartan increased COX-2 mRNA and COX-2 immunoreactivity in the macula densa approximately 4-fold, whereas the AT(2) blocker PD123319 showed no effect. A low-salt diet (0.02% wt/wt) stimulated COX-2 expression in the kidney cortex <2-fold. The combination of a low-salt diet with ramipril led to a further increase of COX-2 mRNA and COX-2 immunoreactivity compared with low salt or ramipril alone. These data indicate that endogenous angiotensin II apparently inhibits COX-2 expression in the macula densa via AT(1) receptors and can therefore not account for the stimulation of COX-2 expression associated with an activated renin-angiotensin system. Because macula densa-derived prostaglandins are considered stimulators of renin secretion and renin synthesis, inhibition of macula densa COX-2 by angiotensin II could form a novel indirect negative feedback control of the renin system.

Early interstitial changes in hypertension-induced renal injury.

To elucidate the mechanisms of hypertensive renal injury, we investigated the time course and extent of changes in matrix composition, as well as cell proliferation and infiltration in two-kidney, one clip rats. The nonclipped kidneys from hypertensive and sham-operated control rats (n = 5 to 10 in each group) were studied at 7, 14, 21, and 28 days after clipping. Systolic blood pressure was elevated by day 7 (154 +/- 3 versus 111 +/- 4 mm Hg in sham group, P < .001, n = 10 each). Hypertension resulted in an early expansion of the interstitial volume by 37%, whereas hypertensive vascular changes and glomerular injury did not become evident until day 21. Immunofluorescence studies revealed an early interstitial accumulation of collagens I, III, IV, V, VI, and fibronectin by day 7. In contrast, the glomeruli showed a mild to moderate increase in collagens I, III, IV, V, laminin, and fibronectin but not collagen VI later in the established phase of hypertension. Staining for proliferating cell nuclear antigen as a marker of cell replication was increased in tubular epithelial but not interstitial or glomerular cells. A progressive infiltration of macrophages (16 +/- 2 versus 9 +/- 1 ED1+ cells/mm2, P < .05, n = 6) and T lymphocytes (93 +/- 15 versus 74 +/- 7 CD4+ cells/mm2, n = 8) in the cortical interstitium had already occurred by day 7. On the other hand, only macrophages increased in number within the glomeruli. Thus, renovascular hypertension leads to an early tubular cell proliferation, mononuclear cell recruitment, and deposition of matrix proteins primarily within the interstitium. We conclude that the injury producing nephrosclerosis in this model extends far beyond the glomeruli. Both the tubules and the interstitium are actively involved and may be the more important initial sites of injury.

Impaired cardiovascular reflexes precede deoxycorticosterone acetate-salt hypertension.

We hypothesized that impaired cardiopulmonary reflexes but not altered baroreceptor reflexes precede deoxycorticosterone acetate (DOCA)-salt hypertension. Uninephrectomized rats were given either DOCA and 0.9% NaCl as drinking water, 0.9% NaCl alone, or tap water. We measured mean blood pressure, heart rate, and renal sympathetic nerve activity. After 8 days, mean blood pressure was not different in DOCA-salt and control rats. Volume-sensitive cardiopulmonary reflexes were tested by intravenous volume loading with saline (10% body weight in 15 minutes), which decreased renal sympathetic nerve activity without changing mean blood pressure or heart rate. This response was blunted in DOCA-salt rats. Chemosensitive cardiopulmonary reflexes were tested by 15-minute infusions of the serotonin 5-HT3 agonist phenylbiguanide, which decreased renal sympathetic nerve activity without changing mean blood pressure or heart rate. Sustained decreases in renal sympathetic nerve activity occurred during phenylbiguanide infusion in controls but were blunted over time in DOCA-salt rats. The arterial baroreflex responses to graded infusions of methoxamine and nitroprusside were analyzed by sigmoidal curve fitting. There were no differences in gain of renal sympathetic nerve activity or heart rate between the groups. Thus, DOCA-salt rats exhibit impaired cardiopulmonary reflexes before the onset of hypertension; the volume-sensitive reflexes are more severely affected than chemosensitive reflexes. The arterial baroreceptor reflex is unaltered. The decreased sensitivity of cardiopulmonary reflexes may contribute to DOCA-salt hypertension.

Increased vascular angiotensin formation in female rats harboring the mouse Ren-2 gene.

Rats harboring the mouse Ren-2 transgene develop hypertension despite low levels of plasma renin activity. We tested the hypothesis that these rats exhibit an increase in vascular angiotensin formation caused by the presence of the transgene. We measured the release of angiotensins I and II from isolated perfused hindquarters by high-performance liquid chromatography and radioimmunoassay. Female rats heterozygous for the transgene had significantly elevated mean arterial pressure compared with control rats (189.3 +/- 9.5 versus 110.0 +/- 5.4 mm Hg, p less than 0.05). Plasma angiotensin II was significantly decreased in transgenic rats. Transgenic rat hindquarters released more angiotensin I (121 +/- 37 versus 39 +/- 12 fmol/30 min, n = 7 each) and more angiotensin II (210 +/- 21 versus 62 +/- 12 fmol/30 min, p less than 0.05, n = 7 each) than control rat hindquarters. Captopril increased angiotensin I release and decreased angiotensin II values in both transgenic and control rat hindquarters. Bilateral nephrectomy 24 hours before hindquarter perfusion greatly reduced angiotensin release from control rat hindquarters but not from transgenic rat hind limbs. We also tested for the presence of Ren-2 messenger RNA in mesenteric and aortic tissue by RNase protection assay and Northern blot analysis. We found that Ren-2 messenger RNA was present in mesenteric and aortic tissue of transgenic but not of control rats. We conclude that the Ren-2 transgene is expressed in vascular tissue of transgenic rats and may be responsible for substantial increases in vascular angiotensin formation.