Paul C. Churchill

Genetics of Cd36 and the clustering of multiple cardiovascular risk factors in spontaneous hypertension

Disorders of carbohydrate and lipid metabolism have been reported to cluster in patients with essential hypertension and in spontaneously hypertensive rats (SHRs). A deletion in the Cd36 gene on chromosome 4 has recently been implicated in defective carbohydrate and lipid metabolism in isolated adipocytes from SHRs. However, the role of Cd36 and chromosome 4 in the control of blood pressure and systemic cardiovascular risk factors in SHRs is unknown. In the SHR. BN-Il6/Npy congenic strain, we have found that transfer of a segment of chromosome 4 (including Cd36) from the Brown Norway (BN) rat onto the SHR background induces reductions in blood pressure and ameliorates dietary-induced glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. These results demonstrate that a single chromosome region can influence a broad spectrum of cardiovascular risk factors involved in the hypertension metabolic syndrome. However, analysis of Cd36 genotypes in the SHR and stroke-prone SHR strains indicates that the deletion variant of Cd36 was not critical to the initial selection for hypertension in the SHR model. Thus, the ability of chromosome 4 to influence multiple cardiovascular risk factors, including hypertension, may depend on linkage of Cd36 to other genes trapped within the differential segment of the SHR. BN-Il6/Npy strain.

Genetic susceptibility to hypertension-induced renal damage in the rat. Evidence based on kidney-specific genome transfer.

To test the hypothesis that genetic factors can determine susceptibility to hypertension-induced renal damage, we derived an experimental animal model in which two genetically different yet histocompatible kidneys are chronically and simultaneously exposed to the same blood pressure profile and metabolic environment within the same host. Kidneys from normotensive Brown Norway rats were transplanted into unilaterally nephrectomized spontaneously hypertensive rats (SHR-RT1.N strain) that harbor the major histocompatibility complex of the Brown Norway strain. 25 d after the induction of severe hypertension with deoxycorticosterone acetate and salt, proteinuria, impaired glomerular filtration rate, and extensive vascular and glomerular injury were observed in the Brown Norway donor kidneys, but not in the SHR-RT1.N kidneys. Control experiments demonstrated that the strain differences in kidney damage could not be attributed to effects of transplantation-induced renal injury, immunologic rejection phenomena, or preexisting strain differences in blood pressure. These studies (a) demonstrate that the kidney of the normotensive Brown Norway rat is inherently much more susceptible to hypertension-induced damage than is the kidney of the spontaneously hypertensive rat, and (b) establish the feasibility of using organ-specific genome transplants to map genes expressed in the kidney that determine susceptibility to hypertension-induced renal injury in the rat.

Identification of renal Cd36 as a determinant of blood pressure and risk for hypertension

To identify renally expressed genes that influence risk for hypertension, we integrated expression quantitative trait locus (QTL) analysis of the kidney with genome-wide correlation analysis of renal expression profiles and blood pressure in recombinant inbred strains derived from the spontaneously hypertensive rat (SHR). This strategy, together with renal transplantation studies in SHR progenitor, transgenic and congenic strains, identified deficient renal expression of Cd36 encoding fatty acid translocase as a genetically determined risk factor for spontaneous hypertension.

Genetic Isolation of a Chromosome 1 Region Affecting Susceptibility to Hypertension-Induced Renal Damage in the Spontaneously Hypertensive Rat

Linkage studies in the fawn-hooded hypertensive rat have suggested that genes influencing susceptibility to hypertension-associated renal failure may exist on rat chromosome 1q. To investigate this possibility in a widely used model of hypertension, the spontaneously hypertensive rat (SHR), we compared susceptibility to hypertension-induced renal damage between an SHR progenitor strain and an SHR congenic strain that is genetically identical except for a defined region of chromosome 1q. Backcross breeding with selection for the markers D1Mit3 and Igf2 on chromosome 1 was used to create the congenic strain (designated SHR.BN-D1Mit3/Igf2) that carries a 22 cM segment of chromosome 1 transferred from the normotensive Brown Norway rat onto the SHR background. Systolic blood pressure (by radiotelemetry) and urine protein excretion were measured in the SHR progenitor and congenic strains before and after the induction of accelerated hypertension by administration of DOCA-salt. At the same level of DOCA-salt hypertension, the SHR.BN-D1Mit3/Igf2 congenic strain showed significantly greater proteinuria and histologically assessed renal vascular and glomerular injury than the SHR progenitor strain. These findings demonstrate that a gene or genes that influence susceptibility to hypertension-induced renal damage have been trapped in the differential chromosome segment of the SHR.BN-D1Mit3/Igf2 congenic strain. This congenic strain represents an important new model for the fine mapping of gene(s) on chromosome 1 that affect susceptibility to hypertension-induced renal injury in the rat.

Novel therapeutics acting via purine receptors

A recent conference entitled Purines in Cell Signalling: Targets for New Drugs, held in Rockville, Maryland, in September, 1989, was one indication of the increasing interest in developing agonists and antagonists of P1-(adenosine) and P2-(ATP) purinoceptors [1] as potential therapeutic agents. Extracellular adenosine, acting at its membrane bound A1 and A2 receptors, is a ubiquitous modulator of cellular activity. The purine can arise from several sources including ATP hydrolysis by ectokinase activity in the region of the nerve terminal [2] and from S-adenosylhomocysteine [3] and ATP within the cell. Together with its more stable analogs, adenosine is a potent inhibitor of neurotransmitter release in both the central and peripheral nervous systems, and in cardiac, adipose and other tissues. Adenosine can also affect blood pressure and heart rate as well as modulate the function of the immune, inflammatory, gastrointestinal, renal and pulmonary systems, either via its effects on transmitter release or directly via receptor mechanisms altering intracellular transduction processes.

Change of tubular reabsorption of sodium and water after denervation

1. Renal function was compared in dogs before and after denervation, with normal or reduced glomerular filtration rate (GFR). GFR was reduced by one of two means, aortic clamping or injection of plastic microspheres into one renal artery.

Effect of diltiazem, a calcium antagonist, on renin secretion from rat kidney slices

Abstract The purpose of these experiments was to characterize the effects of diltiazem on renin secretion from rat renal cortical slices. Incubation of slices in 60 versus 4 mM K medium almost completely abolished renin secretion. Diltiazem antagonized the inhibitory effect in a concentration-dependent manner but had no effect on secretion of slices incubated in 4 mM K medium. Lowering extracellular Ca enhanced the efficacy of diltiazem. These observations demonstrate that Ca influx through voltage-sensitive Ca channels mediates the inhibitory effect of depolarization and further demonstrate that such channels are not open in the basal state of this preparation. In the presence of a concentration of diltiazem which blocked the inhibitory effects of depolarization, both angiotensin II and antidiuretic hormone (ADH) still inhibited secretion. Therefore, both these peptides inhibit secretion by mechanisms which are independent of the voltage-sensitive Ca channels. These observations confirm and extend previous observations suggesting that Ca plays an inhibitory coupling role in the control of renin secretion.

Substantial cardiac parasympathetic activity exists during heavy dynamic exercise in dogs

We investigated the extent of functional parasympathetic and sympathetic activity to the heart at rest and during mild to heavy dynamic exercise in conscious dogs. The animals were chronically instrumented to monitor mean arterial pressure (MAP), heart rate (HR), and terminal aortic blood flow (TAQ) and trained to run on a motor-driven treadmill. MAP, HR, and TAQ were monitored at rest and during steady-state dynamic exercise ranging from mild [3.2 kilometers per hour (kph), 0% grade] to heavy exercise (8 kph, 15% grade). Experiments were performed before and after blocking the effects of either the parasympathetic nerves (atropine 0.2 mg/kg iv) or sympathetic nerves (atenolol 2.0 mg/kg iv) to the heart. In addition, blood samples were taken at rest and at steady state during exercise, and plasma levels of vasopressin and renin activity were assessed. At rest and during all levels of exercise, muscarinic cholinergic receptor blockade caused a marked increase in HR over control (saline treated) levels with little effect on MAP or TAQ. β-Adrenergic receptor blockade had no significant effect on HR at rest and during mild exercise. At moderate to heavy workloads, β-receptor blockade significantly reduced MAP, HR, and TAQ and increased plasma vasopressin levels. We conclude that, even during heavy dynamic exercise, significant functional parasympathetic tone to the heart exists. Thus, over a wide range of exercise workloads, HR is under the tonic control of both sympathetic and parasympathetic nerves.

Effect of Chromosome 19 Transfer on Blood Pressure in the Spontaneously Hypertensive Rat

Linkage studies in the spontaneously hypertensive rat (SHR) have suggested that a gene or genes regulating blood pressure may exist on rat chromosome 19 in the vicinity of the angiotensinogen gene. To test this hypothesis, we measured blood pressure in SHR progenitor and congenic strains that are genetically identical except for a segment of chromosome 19 containing the angiotensinogen gene transferred from the normotensive Brown Norway (BN) strain. Transfer of this segment of chromosome 19 from the BN strain onto the genetic background of the SHR induced significant decreases in systolic and diastolic blood pressures in the recipient SHR chromosome 19 congenic strain. To test for differences in angiotensinogen gene expression between the congenic and progenitor strains, we measured angiotensinogen mRNA levels in a variety of tissues, including aorta, brain, kidney, and liver. We found no differences between the progenitor and congenic strains in the angiotensinogen coding sequence or in angiotensinogen expression that would account for the blood pressure differences between the strains. In addition, no significant differences in plasma levels of angiotensinogen or plasma renin activity were detected between the 2 strains. Thus, transfer of a segment of chromosome 19 containing angiotensinogen from the BN rat into the SHR induces a decrease in blood pressure without inducing any major changes in plasma angiotensinogen levels or plasma renin activity. These results indicate that the differential chromosome segment trapped in the SHR chromosome 19 congenic strain contains a quantitative trait locus that influences blood pressure in the SHR but that this blood pressure effect is not explained by differences in plasma angiotensinogen levels or angiotensinogen expression.

Adenosine and Renal Function

Exogenous adenosine has been shown to affect nearly all aspects of renal function: renal blood flow and its distribution within the kidney, glomerular filtration rate, renin secretion, urine flow, sodium excretion, transmitter release from renal efferent nerves, and the activity of renal afferent nerves. Many of these effects are produced by adenosine receptors, since the effects are antagonized by alkylxanthines and mimicked by adenosine analogs that act as adenosine receptor agonists. The orders of potency of agonists in producing some of these effects have been determined, and therefore the subclasses of adenosine receptors that are involved have been established. These observations, taken together with the observation that kidneys produce and release adenosine into extracellular fluids, suggest that variations in the concentration of endogenously released adenosine could play important roles in renal function and/or dysfunction. Indeed, it has been postulated that adenosine is the mediator of several physiological and pathophysiological phenomena: the autoregulation of renal blood flow and glomerular filtration rate, the tubuloglomerular feedback response, the effect of macula densa cells on the adjacent reninsecreting juxtaglomerular cells, the hemodynamic changes in acute renal failure, and hypertension in some experimental animal models.