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  • Renal Nerves Modulate Renin Gene Expressionin the Developing Rat Kidney with Ureteral Obstruction

    Samir Sayem El-Dahr,* R. Ariel Gomez,* Mark S. Gray,* Michael J. Peach,* Robert M. Carey,' Robert L. Chevalier*With the technical assistance of Marjorie J. Garmey* and Barbara A. Thomhill*Departments of*Pediatrics, tPharmacology, and lInternal Medicine, University of Virginia, Charlottesville, Virginia 22908

    Abstract

    Chronic unilateral ureteral obstruction (UUO) in newborn ratsactivates renin gene expression in the obstructed kidney, andincreases renin distribution along afferent glomerular arteri-oles in both kidneys. To investigate the role of the renal nervesin this response, 2-d-old Sprague-Dawley rats were subjectedto UUO or sham operation. Chemical sympathectomy was per-formed by injection of guanethidine, whereas, control groupsreceived saline vehicle. At 4-5 wk, renal renin distribution wasdetermined by immunocytochemistry, and renin mRNA levelswere determined by Northern blot hybridization. Compared tothe saline-treated rats with UUO, renin remained localized tothe juxtaglomerular region in both kidneys of rats with UUOreceiving guanethidine (P < 0.05). Moreover, renin mRNA lev-els were eightfold lower in obstructed kidneys of rats receivingguanethidine than in those receiving saline. Additional groupsof rats with UUO were subjected to unilateral mechanical renaldenervation: renin gene expression in the obstructed kidneywas suppressed by ipsilateral but not by contralateral renaldenervation. These findings indicate that either chemical ormechanical denervation suppressed the increase in renin geneexpression of the neonatal kidney with ipsilateral UUO. Weconclude that the renal sympathetic nerves modulate renin geneexpression in the developing kidney with chronic UUO. (J.Clin. Invest. 1991. 87:800-810.) Key words: renal nerves * gua-nethidine * sympathectomy - renin * messenger RNA * immuno-cytochemistry * ureteral obstruction * newborn

    Introduction

    There are close links between renal noradrenergic neurons andjuxtaglomerular (JG)' cells which synthetize, store, and release

    Portions of this study were published in abstract form (1990. KidneyInt. 37:354A; 1990. Pediatr. Res. 27:328A).

    Address reprint requests to Robert L. Chevalier, M.D., Departmentof Pediatrics, MR4/2034, University of Virginia Health SciencesCenter, Charlottesville, VA 22908.

    Received for publication 5 February 1990 and in revisedform 11October 1990.

    1. Abbreviations used in this paper: AA, afferent arteriolar segment; JG,juxtaglomerular; MAP, mean arterial pressure; RAS, renin-angioten-sin system; UUO, unilateral ureteral obstruction.

    renin. Anatomically, the sympathetic nerve terminals arefound in close association with the cells ofthe afferent arterioleand the granular JG cell (1, 2). On a functional basis, renalsympathetic nerve activity modulates renin release indepen-dent of the baroreceptor and macula densa mechanisms, pre-sumably by an action mediated by B, adrenergic receptors lo-cated on JG cells (3). What is not known, however, is the role ofthe sympathetic nerves in the modulation ofrenin gene expres-sion. Of particular interest is the contribution of the renalnerves in the modulation of renin gene expression in physio-logic or pathophysiologic states characterized by enhanced in-trarenal renin production. Of equal importance is the interac-tion between the renal nerves and the intrarenal renin-angio-tension system (RAS) at the level of gene expression duringearly development, a time in which intrarenal renin expressionand synthesis are markedly enhanced (4, 5), whereas functionalinnervation of the kidneys is incomplete (6). Physiologic stud-ies indicate that the intrarenal RAS is activated in animalssubjected to chronic unilateral ureteral obstruction (UUO) (7-12). In contrast to the adult rat kidney, in which renin geneexpression is not activated by chronic UUO (13), 1-mo-old ratssubjected to UUO since day 2 of life manifest a significantincrease in renin content and in the percent ofJG apparatusescontaining renin and its mRNA in the obstructed kidney ascompared with sham-operated controls (14). Interestingly, thedistribution of immunoreactive renin also was found to be in-creased in the intact opposite kidney, suggesting communica-tion between the two kidneys (14). The factors that modulaterenin gene expression in response to UUO and the mechanismsunderlying the interactions between the two kidneys have notbeen investigated previously.

    The present study was designed to investigate the role ofthesympathetic renal nerves in modulating renin gene expressionin response to chronic UUO in the newborn rat. We reasonedthat given the close anatomical and functional relationshipsbetween the renal nerves and renin-containing JG cells, activa-tion ofrenin gene expression in chronically obstructed kidneyscould be modulated by the renal nerves.

    To test this hypothesis, newborn rats subjected to UUO orsham-operation were treated chronically with guanethidine orsaline injections. At 1 mo of age, the intrarenal distribution ofimmunoreactive renin and renin mRNA levels were assessedand the effects of sympathetic denervation (guanethidine) orvehicle (saline) administration were compared among the dif-ferent groups. In addition, to assess whether the effects of gua-nethidine on renin gene expression were the result of renaldenervation and not due to other systemic or local effects ofthedrug, kidney renin mRNA levels were examined in similarlyobstructed rats subjected to mechanical or sham denervationof either the obstructed or intact opposite kidneys.

    800 El-Dahr et al.

    J. Clin. Invest. The American Society for Clinical Investigation, Inc.0021-9738/91/03/0800/11 $2.00Volume 87, March 1991, 800-810

  • Methods

    Protocol No. 1: systemic sympathectomy with guanethidine. These ex-periments were performed on 59 2-d-old Sprague-Dawley rats whichwere divided into four groups. Two groups were subjected to shamoperation and received saline or guanethidine (Sham + S, n = 13; andSham + G, n = 12; respectively). Two additional groups were subjectedto left complete ureteral obstruction and received saline or guanethi-dine (UUO + S, n = 12; and UUO + G, n = 22; respectively). Underhalothane and oxygen anesthesia, a small incision was made in the leftlower quadrant ofthe abdomen and the distal ureter was ligated (UUO)or left undisturbed (Sham) (14). The abdominal wall then was closed inone layer and the animal was allowed to recover from anesthesia. Sub-sequently, the animals were returned to their mothers until weaning at21 d ofage, after which the rats were fed regular rat chow (Purina 5012,Ralston-Purina, St. Louis, MO) and were allowed free access to drink-ing water.

    On day 7 of life (5 d after UUO or sham operation), guanethidine orsaline injections were begun. Guanethidine monosulfate (CIBA-GEIGY Pharmaceuticals, Summit, NJ) was diluted in 0.9% saline (5-10 gl/g body weight) and was given in a dose of 50 mg/kg per d, 5 d aweek for 3 wk by subcutaneous injections (a total of 15 injections)according to the method ofJohnson et al. (15). Equal amounts of0.9%saline were administered in a similar fashion to saline-treated animals.

    Protocol No. 2: mechanical renal denervation. 15 rats were used forthese experiments. The rat pups were subjected to left UUO at day 2 oflife as described above. At 25-27 d ofage, under pentobarbital anesthe-sia, left renal denervation (n = 5), right renal denervation (n = 6), orsham renal denervation (n = 4) were performed. Renal denervationwas accomplished as described previously ( 16, 17). Under microscopicvisualization, the renal artery was carefully stripped of its adventitiaand subsequently coated with a solution of 10% phenol in absolutealcohol. During this procedure, the kidney and surrounding tissueswere carefully protected from exposure to phenol. Sham renal denerva-tion was accomplished by left or right abdominal incisions, visualiza-tion of the kidneys, but the renal pedicles were left undisturbed. It hasbeen shown that this denervation procedure results in nearly total elimi-nation of renal tissue catecholamines within 72 h (17).

    At 4-5 wk of age, after an overnight fast with free access to water,the rats in protocols 1 and 2 were anesthetized with intraperitonealpentobarbital sodium (Abbott Laboratories, Chicago, IL) (4 mg/100 gbw) and placed on a thermostatically-controlled heating table to main-tain rectal temperature at 370.5C. The right carotid artery was can-nulated with polyethylene tubing (PE 50, Clay Adams, Parsippany, NJ)and mean arterial pressure (MAP) was recorded continuously bymeans of a Statham 23 ID pressure transducer (Gould Inc., Oxnard,CA) coupled to a Hewlett-Packard 7754 B recorder. The left ureteraldiameter was measured with calipers midway between the renal pelvisand the bladder. The rats were sacrificed with saturated potassium chlo-ride solution injected through the carotid catheter. The kidneys wereremoved, decapsulated, weighed, and processed for immunocytochem-istry and mRNA analysis as described below.

    Renin, beta-tubulin, and tyrosine hydroxylase immunocytochemis-try. Seven Sham + S, 5 Sham + G, 5 UUO + S, and 6 UUO + G ratswere studied. Immunocytochemical localization ofrenin, beta-tubulin,and tyrosine hydroxylase was performed using the ABC immunoper-oxidase technique (4, 5, 13, 14). The primary antibodies used in thepresent study included (a) a specific polyclonal antirat renin antibodyraised in the rabbit which binds to both renin and prorenin (diluted1:1,000, gift of Dr. T. Inagami, Department of Biochemistry, Vander-bilt University) (18, 19), (b) a specific monoclonal antibody againstclass III beta-tubulin isotype (neuron-associated), diluted 1:200 (gift ofDr. A. Frankfurter, University of Virginia Health Sciences Center)(20), and (c) a specific po