comparison of converting enzyme inhibitor and calcium...

452

Comparison of Converting Enzyme Inhibitorand Calcium Channel Blocker inHypertensive Glomerular Injury

Jonathan P. Tolins and Leopoldo Raij

The protective effect of converting enzyme inhibitors in experimental hypertensive glomerularinjury is associated with decreased systemic arterial and glomerular capillary pressure.Although calcium channel Mockers effectively lower systemic blood pressure, their effect onglomerular capillary pressure and on hypertensive glomerular injury is uncertain. Wecompared equihypotensive treatment with the calcium antagonist TA 3090 or the convertingenzyme inhibitor captopril in post-salt hypertensive Dahl salt-sensitive (DS) rats for up to 5weeks after five sixths nephrectomy. Before the nephrectomy, all rats demonstrated hyperten-sion (mean 177 mm Hg), proteinuria (mean 175 mg/day), and mild glomerulosclerosis (meaninjury score 35). Rats treated with captopril or TA 3090 demonstrated a significant andequivalent decrease in systolic blood pressure compared with untreated rats at 2,3, and 5 weeksafter five sixths nephrectomy; however, only captopril reduced proteinuria. Final proteinuriawas actually increased in rats treated with TA 3090 compared with untreated rats. Glomerularinjury score was significantly decreased in captopril-treated compared with untreated rats at 2weeks (33±9 versus 117±10, p<0.05) and 5 weeks (46±9 versus 94±24, p<0.05), whereastreatment with TA 3090 delayed but did not prevent progressive glomerular injury (2-weekscore 35±7,p<0.05 versus untreated; 5-week score 109±19,p=NS versus untreated). Thus, inhypertensive DS rats after subtotal nephrectomy, treatment with a converting enzyme inhibitorreduced systemic blood pressure, proteinuria, and glomerulosclerosis. However, equihypoten-sive treatment with a calcium channel blocker did not reduce proteinuria and delayed but didnot prevent glomerulosclerosis. Thus, in the rat similar reductions in systemic blood pressurewith these two classes of agents have disparate effects on the progression of chronic renalfailure. {Hypertension 1990;16:452-461)

Systemic hypertension complicates the course ofmost patients with chronic renal failure.1 Ithas been recognized for some time that

uncontrolled hypertension may accelerate deteriora-tion of renal function in these patients.2 On the otherhand, adequate blood pressure control has clearlybeen shown to slow the rate of disease progression.3

Although the mechanisms by which systemic hyper-tension accelerates glomerular injury are difficult todetermine in humans, a large body of experimental

From the Department of Medicine, University of MinnesotaSchool of Medicine and Veterans Administration Medical Center,Minneapolis, Minn.

Results of this study were presented, in part, at the NationalMeeting of the American Federation for Ginical Research, May1989, Washington, DC.

Supported by a grant from Marion Laboratories, Kansas City,Mo., and funds from the Veterans Administration.

Address for reprints: Jonathan P. Tolins, MD, Assistant Profes-sor of Medicine and Staff Nephrologist, Veterans AdministrationMedical Center, Renal Section 111-J, 1 Veterans Drive, Minne-apolis, MN, 55417.

Received January 30,1990; accepted in revised form May 2,1990.

evidence in various animal models of hypertension4-7

now suggests that hypertensive glomerular injuryresults from free transmission of elevated systemicpressures to the glomerulus and that it is elevatedglomerular capillary hydraulic pressure, rather thansystemic hypertension itself, that induces progressiveinjury.89 Effective autoregulation of the preglomeru-lar resistance vessels, preventing transmission of ele-vated systemic pressures to the glomerular capillar-ies, underlies the observation that glomerular injurydoes not invariably develop in the face of systemichypertension.710

Early workers1112 demonstrated that after theremoval of about three quarters of the renal mass inthe rat, the initially normal remaining nephronsdeveloped progressive glomerulosclerosis and therats developed proteinuria and azotemia. Micro-puncture studies in this species have revealed thatthe remnant nephrons undergo early functionalchanges consisting of increases in glomerular capil-lary pressures and plasma flows.4 In the remnantkidney model in the rat, treatment with converting

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Tolins and Raij Antihypertensive Therapy and Renal Injury 453

enzyme inhibitor (CEI) results in normalization ofboth systemic blood pressure and glomerular capil-lary pressure.13-14 These hemodynamic effects areassociated with decreased proteinuria and less evi-dence of glomerulosclerosis. Because treatment withCEI lowers not only systemic blood pressure butglomerular capillary pressure as well, it is possiblethat these agents will be more effective in preventingthe progression of chronic renal failure than otherantihypertensive agents that do not have this specificrenal hemodynamic effect. Preliminary studies haveindeed suggested that the CEIs may have particularadvantages over other antihypertensive regimens inslowing or arresting progressive renal failure inrats15-17 and patients,18 perhaps because of thesebeneficial hemodynamic effects.

The calcium channel blockers (CCBs) are in wide-spread clinical use as antihypertensive agents indiverse patient groups including those with chronicrenal failure. These drugs are effective antihyperten-sive agents in patients with renal impairment andhave not been demonstrated to have adverse short-term effects on renal function.19 However, the effectof long-term calcium channel blockade on the courseof progressive renal failure in experimental models orpatients is currently unknown and is a question ofpotential clinical importance. Experimentally, theCCBs have been demonstrated to have unique renalhemodynamic effects. Moderate reductions in sys-temic blood pressure by these agents are associatedwith maintenance of or increases in glomerular fil-tration rate (GFR) due to specific vasodilatation ofthe preglomerular or afferent resistance vessels.19

Consequently, at least in the rat, some investigatorshave reported that glomerular capillary pressure isnot reduced when the calcium antagonists are giveneither acutely20 or chronically.21 In these reports,reduction in systemic pressure was balanced withafferent vasodilatation20 and glomerular capillarypressure maintained. However, preliminary reportsby other investigators in other experimentalmodels22-23 have noted a decrease in glomerularcapillary pressure when CCBs are given acutely orchronically. Thus, at this time, the effects of theCCBs on glomerular capillary pressure remain uncer-tain. A further consideration in the use of CCBs inthe setting of hypertensive glomerular injury is thatthe normal renal autoregulatory response to changesin renal perfusion pressure, a potentially protectivemechanism, is impaired by these agents.24 Given theabove discussion, it is apparent that the hemody-namic profile of the calcium channel blockers is suchthat it is possible that they would not be effective inprotecting against hypertensive glomerular injury.

The effects of CCBs on glomerular injury in exper-imental models of hypertension have been controver-sial. Harris and coworkers25 demonstrated increasedmetabolic stresses in remnant nephrons after subtotalrenal ablation in the rat, and postulated that thesestresses may be detrimental and contribute to progres-sive injury. In this model, decreasing hypermetabolism

by treatment with a CCB appeared to protect thekidney. This same group26 treated rats with nonhy-potensive doses of verapamil for up to 15 weeks afterrenal ablation and noted improved survival. Althoughthe degree of glomerulosclerosis was diminished inrats receiving verapamil, proteinuria was not reducedand renal function, other than cxeatinine clearance,was not reported. Other investigators27-28 have beenunable to demonstrate a long-term protective effect ofCCBs against progressive glomerular injury in theremnant kidney model.

The effect of adequate, chronic antihypertensivetreatment with CCBs on the course of glomerularinjury in animal models has been difficult to studybecause of a lack of an effective means of drugdelivery. Rats will not drink water to which CCBshave been added, and the addition of these agents tothe food leads to decreased food intake and inaccu-rate drug dosing (J.P. Tolins and L. Raij, unpub-lished observations). We have developed a techniquefor antihypertensive drug delivery in the rat thatallows the administration of multiple daily doses in acarefully controlled manner. This technique, com-bined with the availability of very potent and long-lasting CCBs, has recently made it technically possi-ble to adequately study the effect of long-termadministration of CCBs on progressive renal injury.

After subtotal renal ablation, the Dahl salt-sensitive (DS) rat with post-salt hypertension rapidlydevelops progressive renal injury characterized bypronounced hypertension, heavy proteinuria, declinein glomerular filtration rate, and histological evi-dence of glomerulosclerosis.29 Using this model ofaccelerated hypertensive glomerular injury, we com-pared the effects of long-term treatment with equi-hypotensive doses of the converting enzyme inhibitorcaptopril or the CCB TA 3090. The latter agent is a1,5-benzothiazepine calcium antagonist that hasincreased potency and duration of hypotensiveaction,30 thus allowing sustained blood pressure con-trol in this experimental model.

MethodsStudies were performed on male DS rats

(Brookhaven National Laboratories, Upton, N.Y.).Weanling rats were allowed to equilibrate for 2weeks on standard rat chow (Rodent Lab Chow,Purina Mills, St. Louis, Mo.) and tap water adlibitum; after which time all rats were given chowsupplemented with 4% NaCl (Purina Mills) for 8weeks. At this time, after administration of brevitalanesthesia (100 mg/kg i.p.), rats underwent surgicalremoval of the right kidney and infarction of approx-imately two thirds of the left kidney by ligation of twoor three branches of the left renal artery. The rightkidney was processed for morphological studies asdescribed below. A gastrostomy tube was placed atthe same time for subsequent antihypertensive drugadministration. A length of PE-100 polyethylenetubing with a flared end was secured in the stomachwith a purse-string suture, and the exit site from the


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454 Hypertension Vol 16, No 4, October 1990

stomach was wrapped with a small square of sterilegauze to ensure adhesion to the parietal peritoneum.The tubing was tunneled through the abdominal walland subcutaneousry to exit the skin between thescapulae. The tube was flushed with water (1 ml)after placement and after each use. Rats wereallowed to recover for 3 days while consuming ratchow that contained 6% protein (ICN NutritionalBiochemical Lab., Cleveland, Ohio) ad libitum, andserum creatinine measured on distal tail vein bloodsamples (Creatinine Analyser 2, Beckman Instru-ments Inc., Fullerton, N.J.). Rats were randomlyassigned into experimental groups matched forserum creatinine to ensure equivalent reduction inrenal mass, placed on standard rat chow and tapwater ad libitum, and housed in individual cages forthe duration of the study.

Study 1Three groups of rats were studied (n=8 in each

group). Group 1 rats received tap water (2.5 ml) bygastric tube at 8:00 AM and 5:00 PM. Group 2 ratsreceived captopril (generous gift of ER Squibb &Sons, Princeton, N.J.) (50 mg/kg in 1.5 ml, followedby 1.0 ml water), and group 3 rats received TA 3090(generous gift of Marion Laboratories, Kansas City,Mo.) (100 mg/kg in 1.5 ml, followed by 1.0 ml water)at 8:00 AM and 5:00 PM. Drug therapy was begun onday 4 after renal ablation, after rats had been ran-domly assigned into experimental groups. Doses ofTA 3090 and captopril that conferred strictly equiv-alent reductions in systolic blood pressure in thismodel, as measured in the awake rat by the tail-cuffmethod, were determined in pilot studies (data notshown). Doses of antihypertensive agents were thenspecifically chosen to ensure equivalent reduction insystemic blood pressure during the experimentalperiods in the two treatment groups. After 8 weeks ofhigh salt diet (before renal ablation), and again 10days after starting drug treatment (13 days after renalablation), rats had awake systolic blood pressuresmeasured by the tail-cuff method (ProgrammedElectro-Sphygmomanometer, Narco Bio-Systems,Inc., Houston, Tex.). All blood pressures were mea-sured before the morning dose of antihypertensivemedication or vehicle; thus, these measurementsrepresent the nadir of drug effect and presumably thehighest blood pressure of the previous 24-hourperiod. Therefore, the blood pressures in the varioustreatment groups are directly comparable. More fre-quent blood pressure measurements were not possi-ble because of the stress of this procedure (warmingand restraint) with demonstrated loss of severalanimals during our pilot studies. At these same timepoints, rats were placed in metabolic cages for 24-hour urine collection. Urinary total protein excretionwas assayed by the Coomassie dye method (Bio-RadLaboratories, Richmond, Calif.). After overnightfast, tail vein blood was assayed for cholesterol andtriglycerides by standard laboratory techniques.Fourteen days after beginning drug treatment, renal

hemodynamics were measured by clearance tech-niques, as described below, and the remnant kidneywas weighed and processed for histological evalua-tion of glomerular injury.

Study 2Three groups of DS rats (n=8 or 9 in each group)

with post-salt hypertension underwent subtotal renalablation and placement of a gastric tube as describedabove. Twice daily rats were given water (group 4),captopril (group 5), or TA 3090 (group 6) asdescribed above, starting on day 4 after ablation.Systolic blood pressure was determined beforenephrectomy and 3 and 5 weeks after nephrectomy.Urinary protein excretion rate was measured beforenephrectomy and 3 weeks after nephrectomy. Ratswere examined daily and, when clinical signs ofimpending death were noted (ruffled fur, listlessness,lack of movement, weight loss, poor food intake), ratswere killed and the remnant kidney weighed andprocessed for histological evaluation. In our experi-ence with this model, these clinical signs are excellentpredictors of impending death. All surviving ratswere similarly killed 5 weeks after renal ablation at atime when two thirds of the group 4 rats had died. Instudies 1 and 2, all gastric tubes functioned wellwithout complication for the initial 2-3 weeks. Afterthis time, some rats required surgical revision of thegastric tube with either local (1% lidocaine) orgeneral (Brevital) anesthesia.

Clearance StudiesRats in study 1 underwent clearance studies for

determination of GFR and renal plasma flow (RPF)14 days after drug treatment was begun, as previouslydescribed.31 Briefly, surgical preparation was per-formed on a heated table, with rectal temperaturemaintained between 37.0 and 37.5° C. A trache-ostomy was performed after administration of anes-thesia (Inactin, 100 mg/kg i.p.). A double-lumenPE-50 catheter was placed in the left internal jugularvein for infusion. A PE-50 catheter was inserted intothe left femoral artery for blood pressure monitoring(transducer model TNf, Gould Inc., Oxnard, Calif.)and blood sampling. The left ureter was cannulatedwith a PE-10 catheter and timed urine samplescollected in preweighed plastic vials for gravimetricdetermination of urine flow rate. To determine GFRand RPF, a solution of normal saline containing[3H-methoxy]-inulin (4 /iCi/ml) and para-aminohippurate (PAH) (20 mg/ml) was infused at arate of 1.2 ml/hr after a priming dose of 0.5 ml. Tomaintain euvolemia, 5% bovine serum albumin innormal saline was given at 0.5 ml/hr after a primingdose of 1% of body weight. After surgical prepara-tion, rats were allowed to equilibrate for 30 minutesbefore two clearance periods were begun. Two 50 iAblood samples were drawn at the midpoint of each20-minute clearance period. Radioactivity was deter-mined in serum and urine by liquid scintillationcounting, and PAH levels in serum and urine were


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TABLE 1. Physical and

Group

Group 1 (control)Group 2 (captopril)Group 3 (TA 3090)

Biochemical Parameters

3dScr(mg/dl)

1.52±0.131.49±0.13130±0.14

of Rats in

Body wt(g)

367±12418±7*398±7'

Study 1

Hct(%)

34±341±1*40±l*

Right kidney wt(g)

1.15±0.131.34±0.101.00±0.08

Remnant kidneywt/body wt

(xlO3)

3.16±0.38

3.22±0.25

2.50±0.18t

3d Scr, serum creatinine 3 days after renal ablation and before initiation of antihypertensive treatment; Body wt, finalbody weight; Hct, hematocrit; remnant kidney wt, remnant kidney weight.

*p<0.05 versus group 1.tp<0.05 group 3 versus group 2.

determined by autoanalyzer (Technicon Corp., Tar-rytown, N.Y.). GFR and RPF were calculated bystandard formulas. Renal blood flow (RBF) wascalculated by dividing RPF by 1-hematocrit andrenal vascular resistance by dividing mean arterialpressure (MAP) by RBF.

MorphologyThe renal tissue obtained for histopathology was

fixed by immersion in buffered formaldehyde solution,sectioned at 3 .̂m and stained with the periodicacid—Schiff technique. All tissues were evaluated by asingle investigator (L.R.) without knowledge of thegroup to which the rat belonged. Glomerulosclerosiswas defined as the disappearance of cellular elementsfrom the tuft, collapse of capillary lumens, and foldingof the glomerular basement membrane with theentrapment of amorphous material. A semiquantita-tive scoring technique evaluating the percentage ofglomeruli with sclerosis and the extent of involvementof each glomerulus was used as previously described.7

Mean glomerular volume for each sample was deter-mined by the point counting technique of Weibel.32

Statistical AnalysisData are presented as mean±SEM. Parameters

between groups were compared by analysis of vari-ance and subsequent Scheffe's test, and parameterswithin one group at different time points were com-pared by analysis of variance for repeated measures(STATVIEW 512, Brainpower Inc., Calabasas, Calif.).Differences were considered significant for p< 0.05.

ResultsStudy 1

Rats tolerated the surgical procedures well, and ah1

gastric tubes functioned normally during the 14-daytreatment period. One control rat (group 1) died atthe time of the final clearance study. Two TA 3090-treated rats (group 3) died, one shortly after randomassignment to the group and the other at the time ofthe final clearance. All rats treated with captopril(group 2) survived. Physical and biochemical param-eters of rats in study 1 are shown in Table 1. Therewas no difference in serum creatinine, measured 3days after renal ablation and before beginning treat-ment, among the three groups, implying equivalentreduction in renal mass at the start of the experimen-

tal period. Rats treated with either antihypertensivedrug had a higher final body weight and hematocritthan untreated rats. Remnant kidney weight wasnumerically increased in captopril-treated rats com-pared with untreated and TA 3090-treated rats, thelatter difference achieving borderline statistical sig-nificance (significant at /?<0.05 by post hoc Fishertest). When factored for body weight, it can be seenthat treatment with calcium channel blockade tendedto reduce the renal hypertrophic response to subtotalrenal ablation, whereas captopril was without effecton this parameter.

Systolic blood pressures measured immediatelybefore subtotal renal ablation and 10 days afterrandom assignment into experimental groups areshown in Figure 1. As can be seen, all rats had asimilar degree of hypertension before renal ablation.Treatment with either captopril or TA 3090 by twicedaily gastric instillation resulted in significant, sus-tained reduction of systolic blood pressure comparedwith untreated rats. Furthermore, the hypotensiveeffect was identical with either drug. Because theseblood pressures were measured before the morningdrug dose, they reflect blood pressure in the presenceof trough drug levels and thus, presumably, thehighest blood pressure attained in a given 24 -hourperiod. It is clear that the experimental protocolachieved the goal of equivalent reduction of bloodpressure in both treatment groups.

Urinary excretion rates for total protein beforerenal ablation and 10 days after random assignment

250-1

Ore** IGr«4>tlOrtKjp III

pre-oblotton 10 d

FIGURE 1. Bar graph showing awake systolic blood pressurebefore renal ablation and 10 days after initiation of antihyper-tensive treatment in rats of study 1. Rats were treated withwater vehicle (Group I), captopril (Group II), or TA 3090(Group III). p<0.05 versus water vehicle group.


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0r«4>lGr*upllO-CK4> II

pre-oblation 10 d

FIGURE 2. Bar graph showing urinary protein excretion ratein rats of study 1 before renal ablation and 10 days afterinitiation of antihypertensive treatment. Rats were treated withwater vehicle (Group I), captopril (Group II), or TA 3090(Group III). *p<0.05 versus water vehicle group. tp<0.05captopril-treated versus TA 3090-treated group.

into experimental groups are shown in Figure 2.After induction of post-salt hypertension, beforerenal ablation, the DS rat develops pronouncedproteinuria, which was present to a similar degree inall three groups. Untreated rats had persistent mas-sive proteinuria despite high grade renal ablation,implying an increase in leakage of protein perremaining nephron. As can be seen, captopril treat-ment (group 2) markedly decreased urinary proteinexcretion. Despite equivalent reduction in systemicblood pressure in rats treated with TA 3090 (group3), proteinuria was not decreased compared withuntreated rats. Thus, equihypotensive treatment withconverting enzyme inhibitor or CCB had markedlydifferent effects on proteinuria: urinary proteinexcretion rates were decreased only with the formeragent.

Renal hemodynamic parameters, measured 14days after random assignment into experimentalgroups, are shown in Table 2. MAP measured in theanesthetized rat after surgical preparation was signif-icantly decreased in captopril-treated rats (group 2)compared with untreated rats (group 1). Rats receiv-ing TA 3090 (group 3) had a numerically decreasedMAP (19 mm Hg) under these conditions, comparedwith untreated rats, but this did not achieve statisticalsignificance. As described above, systolic blood pres-sure in the awake, unoperated rat was significantlyand equivalentry reduced in both treatment groups.Thus, in the setting of a clearance preparation, thegreater reduction in MAP in the captopril-treated

group most likely reflects the importance of therenin-angiotensin II system in the response to surgi-cal trauma. Captopril-treated rats had significantpreservation of GFR compared with untreated rats,whereas treatment with TA 3090 resulted in a modestbut not significant increase in GFR. RPF, as esti-mated by the clearance of PAH, was significantlyincreased, to an equivalent degree, in rats receivingeither captopril or TA 3090. As can be seen, treat-ment with either captopril or TA 3090 resulted inrenal vasodilatation, as reflected in the markedlydecreased RVR, compared with untreated rats.Thus, in the hypertensive DS rat with subtotal renalablation, reduction in systemic blood pressure withcaptopril resulted in significant preservation of GFRand renal vasodilatation. Equivalent reduction insystemic blood pressure with TA 3090 resulted insimilar directional changes in renal hemodynamics;however, the effect on GFR was proportionately notas great as the vasodilatory effect.

Nephrectomy specimens taken at the time of renalablation showed histological changes that primarilyaffected the glomeruli and were characterized bymild-to-moderate increases in mesangial matrix. Inaddition, some glomeruli were affected by focal areasof glomerulosclerosis (mean glomerular injury scoreof 35 ±2, n=24). The tubules and interstitium ap-peared well preserved. Proteinaceous casts were pre-sent in occasional tubules, mostly at the corticomed-ullary junction. The intrarenal vessels appearedthickened. Glomerular injury scores for study 1 rats14 days after renal ablation are shown in Figure 3A.The kidneys of untreated (group 1) rats showeddiffuse histological changes that affected glomeruli,tubules, and interstitium. The glomeruli showed dif-fuse mesangial expansion and widespread areas ofglomerulosclerosis (injury score 117±10). Tubuloint-erstitial infiltrates consisting of mononuclear cellswere easily detectable, particularly surrounding areasof tubular atrophy. The intrarenal vessels were mark-edly thickened. In group 2 rats, treated with capto-pril, the renal structures were well preserved. Mesan-gial expansion appeared less prominent than inglomeruli of the companion nephrectomy specimenstaken at the time of renal ablation. The capillaryloops were open and dilated. Focal areas of glomer-ulosclerosis were observed in few glomeruli. Theglomerular injury score was significantly reducedcompared with untreated rats (33±9, p<0.05). The

TABLE 2. Renal Hemodynamic Parameters of Rati In Study 1,Drug Treatment

Group

Group 1 (control)Group 2 (captopril)Group 3 (TA 3090)

MAP(mmHg)

177±9136±5*158 ±7

GFR(ml/min)

- 0.42±0.130.84±0.09*0.63 ±0.08

14 Days After Initiation of Antihypertensrve

RPF(rnVmin)

1.83±0.604.21 ±0.42*3.82±0.33'

FF

0.24+0.020.20±0.020.17±0.02*

RVR(ram Hg-min/ml)

123±3822±3*26±2*

MAP, mean arterial pressure; GFR, glomerular filtration rate; RPF, renal plasma flow, FF, filtration fraction; RVR,renal vascular resistance.

•p<0.05 versus group 1.


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B

FIGURE 3. Bar graphs showing renal morphological evalu-ation in rats of study 1: Glomerular injury score (panel A) andglomerular volume (panel B). *p<0.05 versus water vehicle(Group 1). fp<0.05 captopril-treated (Group II) versus TA3090-treated (Group III) group.

tubules were intact and there were no interstitialcellular infiltrates. Thus, captopril arrested the pro-gression of glomerulosclerosis after renal ablation. Inrats treated with TA 3090 (group 3) histologicalevidence of glomerular injury was absent to the samedegree as in group 2 (injury score 35 ±7, p<0.05versus group 1, p=NS versus group 2). Thus, at 14days after renal ablation, calcium channel blockadeprovided equivalent protection to captopril, despitethe lack of effect of TA 3090 on proteinuria.

Glomerular volumes for the rats in study 1,14 daysafter renal ablation, are shown in Figure 3B. As canbe seen, captopril had no effect on glomerular vol-ume compared with untreated rats. On the otherhand, treatment with TA 3090 significantly decreasedglomerular volume compared with both untreated(group 1) and captopril-treated (group 2) rats. Thus,the protective effect of TA 3090 on glomerular injury

14 days after renal ablation was associated withdecreased renal hypertrophy and glomerular volume.

Study 2In this study, we evaluated the effect of long-term

treatment with strictly equihypotensive doses of cap-topril or TA 3090 on survival and glomerular injury inhypertensive DS rats after subtotal renal ablation.Physical and biochemical parameters of rats in study2 are shown in Table 3. Body weights initially (beforeablation) were similar in all three experimentalgroups. By 4 weeks after initiation of treatment,untreated rats (group 4) had lost a significant amountof weight. Rats receiving TA 3090 (group 6) main-tained before-ablation weight, whereas in captopril-treated rats (group 5) body weight significantlyincreased over this time period. Final remnant kid-ney weights, determined at the end of the study ortime of death, were similar in all three treatmentgroups (group 4 versus group 5 versus group 6:2.70±0.33 versus 2.87±0.24 versus 3.24±0.14 g kid-ney wt/g body wt x 103, respectively, />=NS). Glomer-ular volumes, determined on these same kidneyspecimens, were also similar in all three groups(group 4 versus group 5 versus group 6: 1.78±0.20versus 1.83±0.18 versus 1.74±0.15 ^m3xl0*, respec-tively, p=NS). Thus, the reduction in kidney weightsand glomerular volumes in rats treated with CCBsobserved at 14 days after ablation was not sustainedduring long-term treatment with TA 3090.

Serum cholesterol and trigrycerides were measuredafter overnight fast, before beginning high salt diet,before ablation, and at 4 weeks after random assign-ment into experimental groups. As can be seen,serum cholesterol was similar in all three groupsbefore induction of hypertension and increasedslightly, although not significantly, after 8 weeks of4% salt diet. At 4 weeks after renal ablation, choles-terol had increased significantly compared with ini-tial values in all groups. This increase was modest inuntreated rats (group 4) and rats receiving captopril(group 5), and there was no difference between thesetwo groups. Rats receiving TA 3090 had a dramaticincrease in serum cholesterol to a level significantlyhigher than in groups 4 or 5. Triglycerides were notdifferent among the three groups at any time pointduring the experimental protocol. Serum creatinine 3days after renal ablation was similar in all three

TABLE 3.

Group 4Group 5Group 6

Physical and

(control)(captopril)(TA3O90)

Biochemical Parameters of Rats in Study 2Body wt

Prenephrectomy

386±11386±12393±8

(8)4-wk

323±22*424±14*t380±26

Serum cholesterol

Initial Preablation

65±2 73±260±2 72±359±2 65±2

(mg/dl)

4-wk

110±21*108±12«204±13*t*

3d Scr (mg/dl)

1.8±031.8±0.11.7±0.1

Final Scr(mg/dl)

2.0±0.21.6±0.21.7±0.2

Body wt, body weight; 3d Scr, serum crcatinine 3 days after renal ablation.*p<0.05 versus initial or preablation.1p<0.05 versus group 4.&><0.05 versus group 5.


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5u

2 0 0 -

100

• Group IV• Group V• Group VI

pre-abiatlon 3 wk 5 wk

FIGURE 4. Bar graph showing awake systolic blood pressurein rats of study 2 before renal ablation and after 3 and 5 weeksof antihypertensive treatment Rats were treated with watervehicle (Group IV), captopril (Group V), or TA 3090 (GroupVI). *p<0.05 versus water vehicle group.

groups, reflecting equivalent reduction in functioningrenal mass. At 4 weeks after random assignment intoexperimental groups serum creatinine in survivingrats tended to be highest in untreated rats (group 4)and lowest in rats receiving captopril (group 5), withTA 3090-treated rats (group 6) having intermediatevalues; however, these differences did not achievestatistical significance.

As shown in Figure 4, all three groups of rats hada similar degree of systolic hypertension before renalablation. At 3 and 5 weeks after initiation of drugtherapy, rats receiving either captopril (group 5) orTA 3090 (group 6) demonstrated significant andequivalent reductions in blood pressure comparedwith untreated rats. Thus, both treatment regimensachieved the goal of equivalent control of systemichypertension when chronically administered by gas-tric instillation twice daily.

When the experiment was ended, 5 weeks afterrandom assignment into experimental groups, six ofnine (67%) untreated rats had died. These ratsbecame progressively ill and were killed when theywere obviously moribund by clinical observation. Inrats receiving TA 3090, one rat of eight died (12.5%),and this death was within 24 hours of gastric tuberevision and not associated with preoperative signs ofclinical illness. In rats treated with captopril, three ofeight rats died (37.5%), and again all three deathswere associated with surgical revision of the gastros-tomy tube. Thus, all deaths in the rats receivingantihypertensive drug therapy were postoperativeand unrelated to the natural course of this diseasemodel. It is clear that treatment with either captoprilor TA 3090 significantly improved survival to anequivalent degree when compared with untreatedrats.

Urinary protein excretion rates for rats in study 2,measured before nephrectomy and 3 weeks afterrandom assignment into experimental groups, areshown in Figure 5. As in study 1, all rats developedsignificant proteinuria during the period when hyper-tension was induced by feeding the rats a high saltdiet. There was no difference among the groups. At 3weeks, untreated rats (group 4) maintained a high

Group IVGroup VGroup VI

i Pre-ablation 3 wkFIGURE 5. Bar graph showing urinary protein excretion ofrats in study 2 before renal ablation and 3 weeks afterinitiation of antihypertensive treatment Rats were treated withwater vehicle (Group IV), captopril (Group V), or TA 3090(Group VI). *p<0.05 versus water vehicle group. fp<0.05captopril-treated versus TA 3090-treated group.

level of proteinuria (180±23 mg/day) despite reduc-tion in nephron number. In rats receiving captopril(group 5), proteinuria was markedly decreased at 3weeks (54±16 mg/day, / J<0 .05 versus prenephrec-tomy,/><0.05 versus group 4). Group 6 rats, treatedwith TA 3090, markedly increased urinary proteinexcretion rates to levels significantly higher than bothuntreated and captopril-treated rats (3-week proteinexcretion rate, 333±64 mg/day,p<0.05 versus group4 and group 5). Thus, reduction in systemic bloodpressure by treatment with a CCB was associatedwith an actual increase in urinary protein excretion,whereas equivalent reduction in blood pressure witha converting enzyme inhibitor was associated with apronounced and significant fall in proteinuria.

Results from morphological analysis of renal tissuefrom rats in study 2 are shown in Figure 6. As in study1, untreated rats demonstrated pronounced glomer-ulosclerosis (glomerular injury score 94 ±24). Ratsreceiving captopril demonstrated a significantdecrease in glomerular injury score (46 ±9, /?<0.05versus group 4) 5 weeks after random assignmentinto treatment groups. However, treatment with TA3090 for this same time period did not significantlyprevent glomerulosclerosis: the glomerular injuryscore was similar to that seen in untreated rats

125 -

Group IV Group V Group VI

FIGURE 6. Bar graph showing glomerular injury score after 5weeks of antihypertensive treatment in rats of study 2. *p<0.05versus water vehicle group (Group IV). tp<0.05 captopril-treated (Group V) versus TA 3090-treated (Group VI) group.


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(109±19,/?<0.05 versus group 5,p=NS versus group4). Thus, it can be seen that the protective effect ofTA 3090 observed in study 1, 2 weeks after renalablation, was not sustained during long-term follow-up despite a reduction in blood pressure equivalentto that seen in captopril-treated rats.

DiscussionDS rats are genetically predisposed to develop

hypertension when fed a high salt diet.33-34 Micro-puncture studies in intact, hypertensive DS rats havedemonstrated an elevated single nephron GFR dueto an increase in glomerular capillary plasma flowand an increase in glomerular capillary hydraulicpressure.35-36 Thus, in this strain of rat, the elevatedsystemic blood pressure is freely transmitted to theglomerulus, and development of progressive glomer-ular injury due to glomerular capillary hypertensionwould be predicted.8 In the current study, systemichypertension in the DS rat was associated withpronounced proteinuria and definite structural evi-dence of glomerular injury consisting of mild mesan-gial expansion and focal areas of glomerulosclerosis.These changes were present in the intact animals atthe time of renal ablation and reflect the geneticvulnerability of this strain of rats to hypertensiveglomerular injury.37

The course of renal disease in the hypertensive DSrat after subtotal renal ablation has not been previ-ously reported. We have demonstrated that, after highgrade renal ablation, the untreated DS rat developspronounced systemic hypertension, continued massiveproteinuria, and by 14 days after ablation, impairedrenal function with widespread areas of glomerulo-sclerosis (study 1). When followed for up to 5 weeksafter renal ablation (study 2), untreated rats havepersistent hypertension and proteinuria. These ratshad a high mortality rate, with two thirds of the ratsdying by 5 weeks. Although severe glomerular injurywas present in the untreated rats of study 2 at the timethey were killed, final serum creatinine was not mark-edly elevated. Therefore, it is unlikely that theuntreated rats were dying of complications of renalfailure and uremia. Postmortem examination was notdone, other than evaluation of renal morphology;however, the high mortality rate most likely reflectsthe adverse systemic effects of prolonged uncontrolledhypertension, such as stroke.38 Thus, subtotal renalablation in the DS rat with post-salt hypertensionrepresents a model of accelerated hypertensive glo-merular injury characterized by proteinuria,depressed renal function, glomerulosclerosis, and inthe untreated rat, a high mortality rate.

We used this model to directly compare the potentialrenal protective effects of equihypotensive regimens oftwo classes of antihypertensive agents in common clin-ical use: CEIs and CCBs. We administered the antihy-pertensive agents by gastric instillation twice dairy,assuring accurate drug delivery when compared withproviding the drugs in food or drinking water. Bloodpressure was measured in conscious rats approximately

15 hours after the evening dose and thus at a time ofnadir drug effect. Doses of captopril and TA 3090 werespecifically chosen, based on results from pilot studies,to achieve significant and strictly equivalent reductionsin systolic blood pressure in the treatment groupscompared with untreated rats. This equivalent reduc-tion in blood pressure was maintained over the courseof the experimental period as determined by measure-ments at 2, 3, and 5 weeks. Therefore, any differencesbetween the two agents would be unrelated to varia-tions in degree of blood pressure control, a confound-ing factor in other studies using similar agents.27-28

In the hypertensive DS rat after renal ablation,control of systemic hypertension with captopril wasassociated with maintenance of general health com-pared with untreated rats, as demonstrated by weightgain, maintenance of hematocrit, and increased sur-vival. Rats receiving captopril had markedlydecreased proteinuria compared with untreated ratsat 10 and 21 days after nephrectomy, and this wasassociated with preservation of GFR and renal vas-odilatation, measured 14 days after ablation. Fur-thermore, histological evaluation of renal tissue 2and 5 weeks after ablation revealed a significantreduction in the extent of glomerulosclerosis com-pared with untreated rats. Thus, captopril treatmentwas associated with a renal protective effect, consis-tent with the effect of CEIs in the remnant kidneymodel in other strains of rats.13141617

In the current study, the effects of captopril weredirectly compared with those of the CCB TA 3090.Delivery of the CCB by gastric instillation twice dailyinsured accurate delivery of the drug dose andenabled us to obtain a reduction in systemic bloodpressure strictly equivalent to that obtained withcaptopril throughout the course of the study. After 2weeks of treatment, TA 3090-treated rats had a bodyweight and hematocrit similar to those of rats receiv-ing captopril. Long-term treatment with TA 3090 wasassociated with improved survival but impairedweight gain. Improved survival in hypertensive DSrats during treatment with oral CCB has beenreported previously.38 The effects of calcium channelblockade on proteinuria were markedly differentfrom those observed with converting enzyme inhibi-tion. At 10 days after renal ablation, rats receivingTA 3090 had a urinary total protein excretion rateequivalent to that observed in untreated rats. By 21days after ablation, proteinuria was significantlyincreased compared with both untreated rats andrats receiving captopril. This disparate effect onproteinuria of CEI and calcium antagonist has beenpreviously reported in rats27-28 and patients.39

As discussed above, the renal hemodynamic pro-files of these two classes of antihypertensive agentsare distinctly different. In particular, although it isclear that CEIs reduce glomerular capillary pressurein addition to systemic blood pressures,1314 equiva-lent reduction in systemic pressure with a CCB maynot lower glomerular capillary pressure2021 becauseof a concomitant reduction in preglomerular resist-


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ances19 and impairment of renal autoregulatoryresponses.22 It is possible that these disparate effectson glomerular capillary pressure underlie the dif-ferent effects on urinary protein excretion rates;however, without direct measurements of glomerularhemodynamics this proposed mechanism mustremain speculative.

Evaluation of glomerular morphology also revealeddifferences between the two treatment groups. At 14days after ablation, TA 3090 rats had a significantreduction in glomerular injury score compared withuntreated rats. However, by 5 weeks after ablation ratsreceiving TA 3090 demonstrated a level of glomerularinjury that was similar to that seen in untreated rats.Thus, treatment with a calcium antagonist delayed butdid not prevent the development of severe glomerulo-sclerosis in this model. Interestingly, Anderson andcoworkers15 found a similar pattern of early protectionbut ultimate lack of effectiveness in a study comparingtreatment with "triple therapy" or converting enzymeinhibition on the course of progressive renal injury indiabetic rats. In this study, despite equivalent reductionin systemic blood pressure with triple therapy or con-verting enzyme inhibition, only the latter reduced glo-merular capillary pressure.

Given the lack of effect on proteinuria, the earlyprotective effect of TA 3090 is interesting and possi-bly unrelated to glomerular hemodynamic factors.The CCBs have potentially beneficial metaboliceffects that may be important early in the course ofdevelopment of glomerulosclerosis. Raij and Keane40

showed that, in rats, verapamil was as effective assaralasin in reducing the increased mesangial trafficof macromolecules induced by subpressor doses ofangiotensin II. Recent studies have also shown thatCCBs inhibit mesangial cell proliferation in responseto agonists such as platelet-derived growth factor orthrombin.41 CCBs have also been demonstrated toinhibit the generation of the inflammatory mediator,platelet activating factor, by endothelial cells.42

Because increased mesangial traffic, inflammation,and cellular proliferation are potentially importantprocesses in the development of hypertensive glo-merular injury, it is possible that such metabolicactions of the CCBs on these cell types may bebeneficial. Indeed, in a preliminary report, Dworkinand coworkers21 demonstrated that nifedipine pre-vented glomerular injury without reducing glomeru-lar pressure in rats with deoxycorticosterone-salt-induced hypertension, presumably by a beneficialmetabolic action.

Recent studies suggest that compensatory renalhypertrophy may contribute to progressive glomeru-lar injury independently of adaptive changes in glo-merular hemodynamics.43'44 At 14 days after abla-tion, the protective effect of TA 3090 was associatedwith reduced compensatory renal hypertrophy asdemonstrated by a decreased kidney-to-body weightratio and a decreased glomerular volume. However,this antihypertrophic effect was not maintained dur-ing long-term treatment with CCB. Thus, modulation

of renal hypertrophy may contribute to the earlyprotective effect of calcium channel blockade seen inthe current study.

Based on observations in experimental models,hyperlipidemia has been proposed to be a contribut-ing factor to the progression of chronic renalfailure.45 In the current model, serum cholesterol andtriglycerides were measured during the developmentof hypertension and 4 weeks after renal ablation.Triglyceride levels were not different between thethree experimental groups at any time during theexperimental protocol. Because the degree of renalinjury was dramatically different, it is apparent thattriglyceride levels were not important determinantsof progression of glomerular injury in this model.During the development of hypertension, cholesterollevels were not markedly elevated in any groupdespite the development of proteinuria and mildglomerular injury. By 4 weeks after renal ablation,cholesterol levels had increased modestly in all ratsbut were similar in untreated rats and those treatedwith captopril despite pronounced differences indegree of glomerulosclerosis between these twogroups. It is thus apparent that the level of serumcholesterol is not correlated with glomerular injury inthis model. The rats receiving TA 3090 had the highestcholesterol levels, most likely reflecting the high levelof proteinuria seen in these rats. The current studydoes not support a primary role for hyperlipidemia inthe progression of glomerular injury.

In summary, the hypertensive DS rat with subtotalrenal ablation represents a model of acceleratedglomerular injury. Treatment with GEI reduced sys-temic blood pressure, proteinuria, and glomerulo-sclerosis. However, equihypotensive treatment with aCCB had no effect on proteinuria and delayed butultimately did not prevent glomerulosclerosis.Whether these two classes of antihypertensive drugshave disparate effects on the course of progressiverenal failure in patients is an important question thatwarrants clinical investigation. Furthermore, giventhe differences in the potential mechanisms of actionof these two classes of agents, it is possible that CEIsand CCBs may have a complementary and perhapssynergistic effect on the kidney.

AcknowledgmentsWe thank Bich Ha, Karen Coffee, and Linda

Hartich for excellent technical assistance and DianaJacobsen for assistance with manuscript preparation.

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KEY WORDS • renal hypertension • glomerular functionangiotensin converting enzyme inhibitors • glomerulosclerosiscalcium channel blockers • proteinuria


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J P Tolins and L Raijglomerular injury.

Comparison of converting enzyme inhibitor and calcium channel blocker in hypertensive

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1990 American Heart Association, Inc. All rights reserved.

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