Sandford L. Skinner

Retinal Neovascularization Is Prevented by Blockade of the Renin-Angiotensin System

Both angiotensin II and vascular endothelial growth factor are angiogenic agents that have recently been implicated in the pathogenesis of proliferative diabetic retinopathy. In this study, retinal neovascularization was examined in a model of retinopathy of prematurity with the use of neonatal transgenic (mRen-2)27 rats, which overexpress renin in tissues, and Sprague-Dawley rats. Blockers of the renin-angiotensin system were administered during the neovascularization period. The ACE inhibitor lisinopril and the angiotensin type 1 receptor antagonist losartan both increased retinal renin levels and prevented inner retinal blood vessel growth. Quantitative in situ hybridization revealed that the expression of vascular endothelial growth factor and its type 2 receptor in the inner retina and proliferating blood vessels were increased in rats with retinopathy of prematurity. Lisinopril reduced both retinal vascular endothelial growth factor and its type 2 receptor mRNA in retinopathy of prematurity rats, whereas losartan had no effect. It is predicted that agents that interrupt the renin-angiotensin system may play an important role as retinoprotective agents in various forms of proliferative retinopathy.

Angiotensin and Bradykinin Peptides in the TGR(mRen-2)27 Rat

The transgenic TGR(mRen-2)27 rat, in which the Ren-2 mouse renin gene is transfected into the genome of the Sprague-Dawley rat, develops severe hypertension at a young age that responds to inhibitors of angiotensin-converting enzyme and to antagonists of the type 1 angiotensin II (Ang II) receptor. Despite this evidence that the hypertension is Ang II dependent, TGR(mRen-2)27 rats have suppressed renal renin and renin mRNA content, and there is controversy concerning the plasma levels of renin and Ang II in these rats. We investigated the effect of the transgene on circulating and tissue levels of angiotensin and bradykinin peptides in 6-week-old male homozygous TGR(mRen-2)27 rats. Systolic blood pressure of TGR(mRen-2)27 rats was 212 +/- 4 mm Hg (mean +/- SEM, n = 25) compared with 108 +/- 2 mm Hg (n = 29) for age- and sex-matched Sprague-Dawley rats. Compared with control rats, TGR(mRen-2)27 rats had increased plasma levels of active renin (4.5-fold), prorenin (300-fold), and Ang II (fourfold) as well as tissue levels of Ang II (twofold to fourfold in kidney, adrenal, heart, aorta, brown adipose tissue, and lung and 18-fold in brain). Plasma angiotensinogen levels were reduced to 73% of control, and plasma aldosterone levels were increased fourfold. Plasma angiotensin-converting enzyme was reduced to 64% of control. Compared with control rats, TGR(mRen-2)27 rats had increased bradykinin levels in brown adipose tissue (1.9-fold) and lung (1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

The Renin-Angiotensin System Influences Ocular Endothelial Cell Proliferation in Diabetes : Transgenic and Interventional Studies

Neovascularization in the retina and iris of diabetic patients is a major cause of severe visual loss. However, study of these lesions is compromised by the lack of a comparable diabetic rodent model. Because the vasoactive and angiogenic agent, angiotensin II, is involved in diabetic microvascular disease, we aimed to determine whether endothelial cell proliferation could be induced in the retinae and irides of hypertensive transgenic (mRen-2)27 rats that display an enhanced extra-renal renin-angiotensin system (RAS), including the eye. Six-week-old Ren-2, spontaneously hypertensive, and Sprague-Dawley rats received either streptozotocin or control vehicle and were studied for 36 weeks. Additional nondiabetic and diabetic Ren-2 rats were treated throughout with the angiotensin-converting enzyme inhibitor lisinopril (LIS) (10 mg/kg/day in drinking water). Endothelial cell proliferation was only observed in retinae and irides of diabetic Ren-2 rats and was reduced with LIS. In diabetic Ren-2, vascular endothelial growth factor (VEGF) and VEGFR-2 mRNA were increased in retinae and irides and reduced with LIS. Diabetes activated ocular renin in Ren-2 but not Sprague-Dawley rats. The diabetic Ren-2 rat is a model of intraocular endothelial cell proliferation that can be attenuated by RAS blockade via VEGF-dependent pathways. RAS blockade is a potential treatment for vision-threatening diabetic microvascular complications.

Haemodynamic and renal tubular effects of low doses of endothelin in anaesthetized rats.

1. Renal haemodynamic and tubular transport responses to low‐dose infusions (1 and 10 ng kg‐1 min‐1) of endothelin were investigated in anaesthetized rats. 2. Both doses caused transient increases in mean arterial blood pressure (17 +/‐ 5 mmHg, P < 0.05 at 1 ng kg‐1 min‐1) followed by sustained hypotension (‐14 +/‐ 5 mmHg, P < 0.05), reduced renal vascular resistance (‐42%, P < 0.05) and increased renal plasma flow (46%, P < 0.05). Glomerular filtration rate was unchanged. 3. Each dose caused profound diuresis and natriuresis. At 1 ng kg‐1 min‐1 urine flow rate and fractional water excretion increased 5‐fold and fractional sodium excretion 10‐fold. Fractional potassium excretion and solute‐free water clearance were unaltered. 4. End‐proximal fluid delivery estimated by lithium clearance doubled (P < 0.05) and fractional proximal and distal sodium reabsorption decreased 10‐20% (P < 0.05). Absolute proximal reabsorption also fell with the higher dose. 5. Hypotension and natriuresis persisted for 30 min after terminating infusions. Time‐control animals showed no changes in haemodynamics or renal tubular transport. 6. It is concluded that endothelin, at low concentrations, causes renal vasodilatation with concomitant natriuresis due to reduced sodium transport in proximal and distal nephron segments.

Characterization of angiotensin peptides in plasma of anephric man

Recent evidence suggests that a considerable proportion of plasma angiotensin is generated not in blood but in peripheral tissues. Through the measurement of angiotensin peptides and renin in the plasma of 11 anephric subjects, we have investigated whether kidney-derived renin, or some other tissue mechanism for angiotensin generation, is the major determinant of plasma angiotensin. Particular care was taken to prevent inadvertent activation of inactive renin and possible generation, conversion and metabolism of angiotensin peptides during processing of blood samples. Initial experiments revealed that plasma from anephric subjects contains high amounts of material which interferes in radioimmunoassays for angiotensin, even after high-performance liquid chromatography (HPLC). Therefore, in order to obtain an unambiguous identification of angiotensin peptides, a dual HPLC method was developed in which angiotensin peptides were first separated by HPLC, then acetylated and run again on HPLC before radioimmunoassay for angiotensin I and II (detection limits, 0.25 and 0.2 fmol/ml, respectively). The levels of angiotensin I and II were 1.2 +/- 1.6 and 0.7 +/- 0.5 fmol/ml (mean +/- s.d., n = 9-10), respectively, being 6% of levels in normal subjects, and were consistent with the active renin levels (1.8 +/- 1.7 muIU/ml, n = 11) which were 7% of levels in normal subjects. Artefactual activation of prorenin and angiotensin generation during sample processing were excluded as significant causes of the low levels of active renin and angiotensin I and II in anephric plasma. These data indicate that kidney-derived renin is the major determinant of angiotensin levels in normal human plasma. However, the present demonstration of low levels of active renin and angiotensin I and II in plasma of anephric subjects provides unequivocal evidence for a functional extrarenal renin-angiotensin system in man.

Alkalosis increases muscle K+ release, but lowers plasma [K+] and delays fatigue during dynamic forearm exercise

Alkalosis enhances human exercise performance, and reduces K+ loss in contracting rat muscle. We investigated alkalosis effects on K+ regulation, ionic regulation and fatigue during intense exercise in nine untrained volunteers. Concentric finger flexions were conducted at 75% peak work rate (∼3 W) until fatigue, under alkalosis (Alk, NaHCO3, 0.3 g kg−1) and control (Con, CaCO3) conditions, 1 month apart in a randomised, double‐blind, crossover design. Deep antecubital venous (v) and radial arterial (a) blood was drawn at rest, during exercise and recovery, to determine arterio‐venous differences for electrolytes, fluid shifts, acid–base and gas exchange. Finger flexion exercise barely perturbed arterial plasma ions and acid–base status, but induced marked arterio‐venous changes. Alk elevated [HCO3−] and P  CO 2, and lowered [H+] (P < 0.05). Time to fatigue increased substantially during Alk (25 ± 8%, P < 0.05), whilst both [K+]a and [K+]v were reduced (P < 0.01) and [K+]a‐v during exercise tended to be greater (P= 0.056, n= 8). Muscle K+ efflux at fatigue was greater in Alk (21.2 ± 7.6 µmol min−1, 32 ± 7%, P < 0.05, n= 6), but peak K+ uptake rate was elevated during recovery (15 ± 7%, P < 0.05) suggesting increased muscle Na+,K+‐ATPase activity. Alk induced greater [Na+]a, [Cl−]v, muscle Cl− influx and muscle lactate concentration ([Lac−]) efflux during exercise and recovery (P < 0.05). The lower circulating [K+] and greater muscle K+ uptake, Na+ delivery and Cl− uptake with Alk, are all consistent with preservation of membrane excitability during exercise. This suggests that lesser exercise‐induced membrane depolarization may be an important mechanism underlying enhanced exercise performance with Alk. Thus Alk was associated with improved regulation of K+, Na+, Cl− and Lac−.

The role of endogenous angiotensin II in the regulation of renal haemodynamics and proximal fluid reabsorption in the rat.

1. The influence of endogenous angiotensin II (AII) on renal haemodynamics and tubular function was examined by clearance and micropuncture methods in anaesthetized rats during AII receptor blockade with the non‐peptide antagonist DuP 753 (50 micrograms kg‐1 min‐1 i.v.). 2. Mean arterial pressure was reduced slightly (‐5 +/‐ 2 mmHg) while filtration fraction and glomerular filtration rate rose by 30% without changes in renal plasma flow (RPF) or renal vascular resistance (RVR). 3. Fractional proximal fluid reabsorption (calculated from lithium clearance) fell from 73 to 64% (P < 0.01) and fractional distal sodium reabsorption decreased from 98 to 94% (P < 0.01). 4. Urine flow rate more than doubled, sodium output increased 4‐fold and plasma renin concentration rose 8‐fold while potassium excretion remained unchanged. 5. Proximal tubular fluid reabsorption (Jv) as measured by shrinking split‐droplet micropuncture decreased by 21% (P < 0.01) during infusion of DuP 753 compared with 22.5% (P < 0.01) during converting enzyme inhibition by enalaprilat (MK422). 6. Responses to DuP 753 were similar to those previously documented with converting enzyme inhibitors except that DuP 753 failed to raise RPF. It is concluded that generation of intrarenal vasodilator paracrines has confounded conclusions about the renal action of converting enzyme inhibitors and we propose that in anaesthetized rats, endogenous angiotensin II (AII) has its major renal influences on glomerular filtration and proximal fluid reabsorption with little effect on renal vascular resistance.

Hypertension-accelerated atherogenesis in cholesterol-fed rabbits

Entry of 125I-labelled low density lipoprotein ([125I]LDL) into the aortic intima was studied over 6 hours in normotenisve and hypertensive rabbits fed a 1% cholesterol diet for 9 and 4 weeks respectively. Studies were also made in hypertensive and normotensive cholesterol-fed rabbits in which blood pressure was reduced acutely with parenteral hydralazine. In all groups the entry of E1125I]LDL was greatest in the aortic arch and significantly less in both the descending thoracic and abdominal regions. Lipoprotein entry into the aorta of cholesterol-fed rabbits was increased some 10-fold over the corresponding value previously found in rabbits fed a normal diet [1]. This increase was due to increased vascular permeability as well as to increased plasma LDL concentration. The hypertensive cholesterol-fed rabbits did not show significantly greater entry of [125I]LDL than the normotensive cholesterol-fed rabbits. Comparison of the rate of LDL entry over 6 house and the quanitity of cholesterol accumulated in the aortic segments over the period of cholesterol feeding indicated that lipoprotein fractions other than LDL must contribute singificant amounts of cholesterol to the developing lesion. The finding that LDL entry paralledled accumulation during cholesterol feeding, together with the finding that acute reversal of hypertension did not reduce the entry of [125I] LDL suggest that mechanisms other than increased filtration of plasma low density lipoprotein contribute significantly to the accelerated development of atherosclerosis in hypertension.

The effect of hypertension on the accumulation of lipids and the uptake of [3H]cholesterol by the aorta of normal-fed and cholesterol-fed rabbits

Abstract The relationship between blood pressure and arterial wall lipids was studied in normal-fed and cholesterol-fed rabbits with and without renal hypertension. 1.(a) Cholesterol feeding influenced neither the diastolic blood pressure of the normotensive rabbits nor the degree of diastolic hypertension developed by rabbits made hypertensive by cellophane perinephritis. 2.(b) Hypertension produced a significant increase in the cholesterol content of the intima of both normal-fed and cholesterol-fed rabbits, but did not affect the serum cholesterol levels of rabbits on either diet. The increase in intimal cholesterol with hypertension in rabbits on a normal diet was associated with a parallel rise in the intimal dry defatted weight. In cholesterol-fed rabbits, however, hypertension did not produce this effect on intimal weight. Hypertension also had no effect on the percentage cholesterol ester or cholesterol-phospholipid ratio in the intima or media of cholesterol-fed rabbits.In normal-fed animals hypertension had little effect on intimal phospholipid fatty acid composition, but was associated with a shift towards unsaturation in the fatty acid composition of intimal cholesterol ester and triglyceride similar to that produced by cholesterol feeding alone. In addition, cholesterol feeding alone produced a significant increase in the linoleic (18:2) and decrease in the arachidonic (20:4) acid components of the intimal phospholipids. In cholesterol-fed rabbits hypertension did not have any further effect on cholesterol ester and triglyceride fatty acids, but was associated with a significant increase in the palmitic (16: 0) and decrease in the arachidonic (20:4) acid components of the intimal phospholipids. 3.(c) Hypertension was associated with a significant increase in the influx of [ 3 H]cholesterol into the aortic intima in vivo in normal-fed rabbits but not in cholesterol-fed rabbits. However, in both normal-fed and cholesterol-fed rabbits there was a significant correlation between influx and the greater intimal cholesterol of hypertensive animals. 4.(d) The findings indicate that both increased entry and altered metabolism of intimal lipid occur during the acceleration of atherosclerosis by hypertension.

Granular juxtaglomerular cells and prorenin synthesis in mice treated with enalapril.

The short-term and long-term effects (for up to 98 days) of the angiotensin converting enzyme inhibitor enalapril were investigated in male and female BALB/c mice. In control animals, separate antisera to renin and its prosequence produced an identical pattern of staining in granular cells of the juxtaglomerular apparatus (JGA) a short distance from the glomerulus. After 1 day of the enalapril treatment there was a decrease in the number of JGA granular cells immunostained with antisera to both renin and its prosequence. Electron microscopy revealed degranulation of mature granules from JGA granular cells. Fusion of granules with the cell membrane was not observed, but numerous membrane-like structures (myelin figures) were identified in the cytoplasm and extracellular space, indicating possible secretion. In addition, the volume proportion of granulated cells in relation to the glomerular volume was decreased, as was renal renin content. With continuing enalapril treatment, separate antisera to renin and its prosequence stained the same granulated JGA cells with equal intensity. The cells so stained increased in number, extending down the wall of the afferent arteriole to cortical radial arteries (interlobular arteries) upstream from the glomerulus. Ultrastructural studies revealed a progressive development of cytoplasmic granulation in JGA granular cells and in smooth muscle cells extending into cortical radial arteries. Furthermore, the volume proportion of granulated cells in relation to the glomerular volume was significantly increased, as was renal renin content. Thus, short-term enalapril treatment in mice provoked rapid secretion of renin via degranulation of mature granules from JGA granular cells. In contrast, long-term enalapril treatment produced a continuing stimulus for renin synthesis, secretion and storage, resulting in an increased thickness of the afferent arteriolar wall. The mechanism for this change appears to be hypertrophy and hypergranulation of granular JGA cells and neogranulation of smooth muscle cells upstream from the glomerulus. Identification of the intrarenal mediators that induce these phenotypic changes presents an interesting challenge.