Karl F. Hilgers

Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C–dependent buffering mechanism

In salt-sensitive hypertension, the accumulation of Na+ in tissue has been presumed to be accompanied by a commensurate retention of water to maintain the isotonicity of body fluids. We show here that a high-salt diet (HSD) in rats leads to interstitial hypertonic Na+ accumulation in skin, resulting in increased density and hyperplasia of the lymphcapillary network. The mechanisms underlying these effects on lymphatics involve activation of tonicity-responsive enhancer binding protein (TonEBP) in mononuclear phagocyte system (MPS) cells infiltrating the interstitium of the skin. TonEBP binds the promoter of the gene encoding vascular endothelial growth factor-C (VEGF-C, encoded by Vegfc) and causes VEGF-C secretion by macrophages. MPS cell depletion or VEGF-C trapping by soluble VEGF receptor-3 blocks VEGF-C signaling, augments interstitial hypertonic volume retention, decreases endothelial nitric oxide synthase expression and elevates blood pressure in response to HSD. Our data show that TonEBP–VEGF-C signaling in MPS cells is a major determinant of extracellular volume and blood pressure homeostasis and identify VEGFC as an osmosensitive, hypertonicity-driven gene intimately involved in salt-induced hypertension.

Renin-angiotensin system and cardiovascular risk

The renin-angiotensin system is a major regulatory system of cardiovascular and renal function. Basic research has revealed exciting new aspects, which could lead to novel or modified therapeutic approaches. Renin-angiotensin system blockade exerts potent antiatherosclerotic effects, which are mediated by their antihypertensive, anti-inflammatory, antiproliferative, and oxidative stress lowering properties. Inhibitors of the system-ie, angiotensin converting enzyme inhibitors and angiotensin receptor blockers, are now first-line treatments for hypertensive target organ damage and progressive renal disease. Their effects are greater than expected by their ability to lower blood pressure alone. Angiotensin receptor blockers reduce the frequency of atrial fibrillation and stroke. Renin-angiotensin system blockade delays or avoids the onset of type 2 diabetes and prevents cardiovascular and renal events in diabetic patients. Thus, blockade of this system will remain a cornerstone of our strategies to reduce cardiovascular risk.

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.

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.

(Pro)renin receptor peptide inhibitor "handle-region" peptide does not affect hypertensive nephrosclerosis in Goldblatt rats.

The (pro)renin receptor [(P)RR], a new component the renin-angiotensin system, was cloned recently. The (P)RR promotes direct mitogen-activated protein kinase signaling and nonproteolytic prorenin activation. We investigated the role of a (P)RR blocker, a peptide consisting of 10 amino acids from the prorenin prosegment called the “handle-region” peptide (HRP), on target organ damage in renovascular hypertensive 2-kidney, 1-clip (2K1C) rats. Vehicle-treated 2K1C rats were compared with HRP-treated 2K1C rats (3.5 &mgr;g/kg per day) and sham-operated controls. Vehicle-treated 2K1C rats developed hypertension (186±17 mm Hg), cardiac hypertrophy (3.16±0.16 mg/g), renal inflammation, fibrosis, vascular, and tubular damage. Chronic HRP treatment did not affect blood pressure (194±15 mm Hg), cardiac hypertrophy (2.97±0.11 mg/g), or renal damage. Furthermore, we investigated the renal renin and (P)RR expression. The clipped kidney of 2K1C and HRP-treated 2K1C rats showed a higher renin expression and juxtaglomerular index compared with sham-operated kidneys. The unclipped kidney showed suppressed renin expression. In contrast, (P)RR mRNA expression was not altered in any group. Plasma renin activity and aldosterone were increased in 2K1C rats compared with sham controls. HRP-treated 2K1C rats tended to lower plasma renin activity but showed similar aldosterone levels as vehicle-treated 2K1C rats. Our results indicate that blockade of the (P)RR with HRP does not improve target organ damage in renovascular hypertensive rats.

Impaired Endothelial Function of the Retinal Vasculature in Hypertensive Patients

Background and Purpose— Arterial hypertension constitutes a central factor in the pathogenesis of stroke. We examined endothelial function of the retinal vasculature as a model of the cerebral circulation. Methods— Thirty-eight young subjects (19 hypertensive and 19 normotensive) were treated with the AT1-receptor blocker candesartan cilexetil and placebo, each over 7 days. Retinal capillary flow and blood flow velocity in the central retinal artery were assessed with scanning laser Doppler flowmetry and pulsed Doppler ultrasound, respectively. NG-mono-methyl-L-arginine (L-NMMA) was infused to inhibit nitric oxide (NO) synthesis. Diffuse luminance flicker was applied to stimulate NO release. Results— In normotensive subjects, L-NMMA decreased retinal capillary flow by 8.2%±13% (P < 0.05) and flickering light increased mean blood flow velocity in the central retinal artery by 19%±29% (P < 0.01). In contrast, no significant change to these provocative tests was seen in hypertensive subjects. Treatment with candesartan cilexetil restored a normal pattern of reactivity in retinal capillaries (L-NMMA: decrease in perfusion by 10%±17%, P < 0.05) and the central retinal artery (flicker: increase in mean blood flow velocity by 42%±31%, P < 0.001) in hypertensive patients. Conclusions— Endothelial function of the retinal vasculature is impaired in early essential hypertension but can be improved by AT1-receptor blockade.

Local Angiotensin II Generation in the Rat Heart Role of Renin Uptake

To elucidate the local effects of renin in the coronary circulation, we examined local angiotensin (Ang) I and II formation, as well as coronary vasoconstriction in response to renin administration, and compared the effects with exogenous infused Ang I. We perfused isolated hearts from rats overexpressing the human angiotensinogen gene in a Langendorff preparation and measured the hemodynamic effects and the released products. We also investigated cardiac Ang I conversion, including the contribution of non-angiotensin-converting enzyme-dependent Ang II-generating pathways. Finally, we studied Ang I conversion in vitro in heart homogenates. Renin and Ang I infusion both generated Ang II. Ang II release and vasoconstriction continued after renin infusion was stopped, even though renin disappeared immediately from the perfusate. In contrast, after Ang I infusion, Ang II release and coronary flow returned to basal levels. Ang I conversion (Ang II/Ang I ratio) was higher after renin infusion (0.109+/-0.027 versus 0.026+/-0.003, 15 minutes, P<.02) compared with infused Ang I. Remikiren added to the renin infusion abolished Ang I and II; captopril suppressed only Ang II, whereas an AT1 receptor blocker did not affect Ang I and II formation. All the drugs prevented renin-induced coronary flow changes. Total cardiac Ang II-forming activity was only partially inhibited by cilazaprilat (4.1+/-0.1 fmol x min(-1) x mg[-1]) and on a larger extent by chymostatin (2.6+/-0.3 fmol x min(-1) x mg[-1]) compared with control values (5.6+/-0.4 fmol x min(-1) x mg[-1]). We conclude that renin can be taken up by cardiac or coronary vascular tissue and induces long-lasting local Ang II generation and vasoconstriction. Locally formed Ang I was converted more effectively than infused Ang I. Furthermore, the comparison of in vivo and in vitro Ang I conversion suggests that in vitro assays may underestimate the functional contribution of angiotensin-converting enzyme to intracardiac Ang II formation.

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.

Vascular Angiotensin-Converting Enzyme Expression Regulates Local Angiotensin II

We tested the hypothesis that changes in angiotensin-converting enzyme (ACE) gene expression can regulate the rate of local vascular angiotensin II (Ang II) production. We perfused isolated rat hindlimbs with an artificial medium and infused renin and Ang I via the perfusate. Ang I and II were measured by radioimmunoassay. We then increased ACE gene expression and ACE levels in the rat aorta by producing two-kidney, one clip (2K1C) hypertension for 4 weeks. Gene expression was measured by RNAse protection assay, and ACE activity in the vessel wall was measured by the Cushman-Cheung assay. Angiotensin I infusion at 1, 10, 100, and 1000 pmol/mL led to 371 +/- 14 (+/-SEM), 3611 +/- 202, 44,828 +/- 1425, and 431,503 +/- 16,439 fmol/mL Ang II released, respectively, from the hindlimbs (r = .98, P < .001). Thus, the conversion rate did not change across four orders of magnitude, and the system was not saturable under these conditions. In 2K1C hindlimbs, Ang I infusion (0.5 pmol/mL) resulted in increased Ang II generation (157 +/- 16 versus 123 +/- 23 fmol/mL, P = .014 at minute 10) compared with controls. ACE gene expression and ACE activity were increased in 2K1C hindlimbs compared with controls (36 +/- 4 versus 17 +/- 1 mU/mg protein, P < .001). Ang II degradation in the two groups did not differ. To investigate the conversion of locally generated Ang I, we infused porcine renin (0.5 milliunits per mL) into 2K1C and control hindlimbs. Despite markedly higher Ang I release in sham-operated than in 2K1C rats (71 +/- 8 versus 37 +/- 6 pmol/mL, P = .008 at minute 12), Ang II was only moderately increased (36 +/- 3 versus 25 +/- 6 pmol/mL, P = .12 at minute 12). This difference between 2K1C rats and controls reflected a higher rate of conversion in 2K1C rats. Thus, Ang I conversion in the rat hindlimb is linear over a wide range of substrate concentrations and occurs at a fixed relationship. Nevertheless, increased ACE gene expression and ACE activity in the vessel wall lead to an increase in the conversion of Ang I to Ang II. We conclude that local ACE gene expression and ACE activity can influence the local rate of Ang II production.

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 < 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 < 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.