Monique C. Churchill

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.

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.

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.

Separate and combined effects of ouabain and extracellular potassium on renin secretion from rat renal cortical slices

1. Renin secretion of rat renal cortical slices was measured as a function of extracellular K and ouabain concentrations in the incubation medium.

Pertussis toxin reverses adenosine receptor-mediated inhibition of renin secretion in rat renal cortical slices.

Adenosine analogs selective for the A1 subclass of adenosine receptors, such as N6-cyclohexyladenosine (CHA), inhibit renin secretion in in vitro preparations. Ca chelation blocks the inhibitory effect, consistent with mediation by increased intracellular free Ca2+, and it has been suggested that intracellular Ca2+ could increase as a result of receptor-induced inhibition of adenylate cyclase followed by decreased Ca efflux from the renin-secreting cells. Pertussis toxin blocks receptor-induced inhibition of adenylate cyclase in many cells, and in others, it blocks receptor-induced phosphotidylinositol response. In the present studies, pertussis toxin treatment stimulated the basal renin secretory rate of rat renal cortical slices and blocked the inhibitory effect of CHA but not the inhibitory effect of K-depolarization. These data support the hypothesis that a pertussis toxin substrate, such as Ni, is involved in CHA-, but not in K-depolarization, -induced inhibition of renin secretion.

Kidney transplants in cyclosporine-treated Sprague-Dawley rats.

Previous studies by others have shown that transplanted rat kidneys have abnormally low clearances of paraaminohippuric acid, inulin, and creatinine, due to rejection and/or to warm-ischemia-induced injury. In the present studies, randomly bred Sprague-Dawley rats were used as donors and recipients. The left kidneys of recipients were removed, and the right kidneys were left intact. Donor kidneys were flushed with an ice-cold hypertonic solution (150 mM NaCl, 200 mM mannitol, pH 6.4), and the kidneys were kept cold during surgery. Renal function was assessed 1 week later. The left transplanted kidneys in untreated recipients exhibited morphologic evidence of rejection, and the clearances of PAH and inulin were approximately 50% of those of the right native kidneys. CsA-treated rats did not reject the transplants, and the PAH and inulin clearances of the left transplanted kidneys were identical to those of the right native kidneys. Untreated and CsA-treated rats with both native kidneys intact served as controls. The amount of CsA given during the 7-day period produced no measurable change in renal function. This is the first demonstration of virtually normal hemodynamics in transplanted rat kidneys when randomly bred animals are used as donors and recipients. Moreover, the results indicate that if both rejection and warm ischemia are avoided, the presence of a functioning native kidney does not have a detrimental effect on the function of a transplanted kidney.

Effect of melittin on renin and prostaglandin E2 release from rat renal cortical slices.

1. The present experiments were designed to determine the effect of melittin on renin secretion. Melittin is a polypeptide component of bee venom which stimulates phospholipase A2 activity, thereby increasing arachidonic acid release and prostaglandin (PG) synthesis, and which inhibits protein kinase C activity. Either of these actions might be expected to stimulate renin secretion, since renin secretion is stimulated by arachidonic acid and by several PGs, and since renin secretion is inhibited by several activators of protein kinase C. 2. In rat renin cortical slices incubated at 37 degrees C in a buffered and oxygenated physiological saline solution, 0.1‐10 microM‐melittin produced a concentration‐dependent stimulation of both prostaglandin E2 (PGE2) synthesis and renin secretion. However, melittin‐stimulated renin secretion is independent of melittin‐stimulated phospholipase A2 activity, arachidonic acid release, and PG synthesis, since 20 microM‐quinacrine (a phospholipase A2 antagonist) and 50 microM‐meclofenamate (a cyclooxygenase antagonist) antagonized basal and melittin‐stimulated PGE2 synthesis but had no effects on basal or melittin‐stimulated renin secretion. 3. Furthermore, melittin‐stimulated renin secretion is not produced by inhibition of protein kinase C, since an activator of protein kinase C (12‐O‐tetradecanoylphorbol 13‐acetate, TPA), enhanced rather than antagonized melittin‐stimulated renin secretion. Ouabain partially antagonized, but did not completely block, melittin‐stimulated renin secretion. 4. Thus, melittin‐stimulated phospholipase A2 activity probably accounts for stimulated PGE2 production, but not for stimulated renin secretion. The mechanism of melittin‐stimulated renin secretion is unclear; an effect on protein kinase C does not appear to be involved, and in contrast to the stimulatory effects of a variety of other substances, melittin‐stimulated renin secretion is only partially antagonized by ouabain.

Cyclosporine a inhibits prostaglandin E2 release, and has no effect on renin secretion, from rat renal cortical slices

These experiments were designed to test the hypothesis that cyclosporine A (CSA) inhibits renin secretion and stimulates renal prostaglandin E2 (PGE2) release in vitro. In rat renal cortical slices incubated at 37 degrees C in a buffered and oxygenated physiological saline solution containing 4 mM KCl, CSA concentrations ranging from 1 to 30 microM had no significant effect on renin secretion. Furthermore, partial depolarization of the cells, produced by increasing extracellular KCl concentration to 20 mM, failed to reveal any latent inhibitory or stimulatory effects of CSA on renin secretion. On the other hand, PGE2 release was significantly inhibited by CSA over the same range of concentrations. This inhibitory effect might be explained by the previous findings of others, that CSA inhibits phospholipase A2 activity, thereby decreasing arachidonic acid production, the rate-limiting step in PG synthesis. In conclusion, CSA inhibits PGE2 release but fails to affect renin secretion in vitro. These results suggest that the occasional effects of CSA on renin secretion in intact animals must be attributable to indirect and/or chronic effects.