Abstract We examined the effect of long-term enalapril treatment on renal function and histology in the monogenetically hypertensive TGR(mRen2)27 rat strain. Untreated transgenic rats had significantly (P<.01) higher blood pressures than treated transgenic and control animals throughout the study. Urinary nitric oxide metabolite excretion was significantly lower in young transgenic rats and rose with enalapril, suggesting abnormal TGR nitric oxide production and its correction by enalapril. Converting enzyme inhibition produced preferential preglomerular vasodilatation and increased renal blood flow (6.5±0.5 versus 9.0±0.7 mL/min per gram kidney weight, P<.05) without altering whole-kidney and single-nephron glomerular filtration rates in TGR(mRen2)27. Glomerular capillary pressure fell modestly in treated transgenic animals (54±1 versus 50±1 mm Hg, P<.05). These hemodynamic changes were associated with reductions in albuminuria (59±6 versus 9±2 mg/d, P<.01) and glomerulosclerosis in TGR. However, urinary albumin excretion (15±3 versus 3±1 mg/d, P<.05) and glomerulosclerosis also declined in treated control animals in the absence of significant alterations in glomerular hemodynamics. The mechanism of the beneficial effect of enalapril on the TGR(mRen2)27 kidney is unclear but could involve either control of hypertension or suppression of the intrarenal renin-angiotensin system.
The renin-angiotensin system plays an important role in the regulation of BP and kidney function. The recently developed transgenic rat strain TGR(mRen2)27, produced by inserting the mouse Ren-2 renin gene complete with flanking control regions into the rat genome, provides a unique opportunity for examining relationships among angiotensin, hypertension, and renal injury.1 These transgenic animals develop high BP before 8 weeks of age, with the severity of their hypertension being dependent on the transgene dose. Systolic BPs of rats heterozygous for the mouse renin transgene are approximately 200 mm Hg, whereas pressures of homozygous animals approach 300 mm Hg.2 Because hypertension and other phenotypic differences in this strain can be clearly related to the presence or absence of the mouse renin gene, the physiological consequences of a specific genetic change can be accurately studied in vivo. This situation contrasts with that in other genetically hypertensive strains whose high BP is polygenic. In these other animals, it is often difficult to determine whether particular genetic loci or physiological traits are causally related to hypertension.3
In an earlier study, we described various aspects of renal function in 2- to 8-month-old male rats heterozygous for the mouse renin transgene.4 We found that transgenic rats had significantly higher BPs than control animals at all ages but that the severity of their hypertension diminished over time. Albuminuria and glomerulosclerosis appeared early in life but did not lead to renal insufficiency. This lack of progressive renal injury was associated with elevated preglomerular vascular resistance that protected glomeruli from severe systemic hypertension. In the present study, we determined the effect of long-term treatment with the angiotensin-converting enzyme inhibitor enalapril on kidney function and histology in these animals.
We performed experiments on male transgenic rats heterozygous for the mouse Ren-2 gene (TGR) and age-matched male Sprague-Dawley Hannover rats (CON) obtained from Moellegaard Breeding Center GmbH, Schoenwalde, Germany. Animals were maintained on standard 24% protein rat chow and water ad libitum. Some rats received enalapril (100 mg/L drinking water) beginning at 3 months of age. The study protocol was approved by our institution’s laboratory animal care committee.
Indirect systolic BP was measured monthly in unanesthetized rats by a tail-cuff method (Narco Biosystems). Urine was collected from animals kept in metabolic cages for 16 hours with free access to water but deprived of food. Blood samples were obtained from the tail vein at the end of collection periods. Hematocrit; plasma sodium, total protein, albumin, creatinine, cholesterol, high-density lipoprotein, and triglyceride concentrations; and urinary sodium, creatinine, albumin, and NOM excretion, a measure of endogenous nitric oxide production,5 were determined from these samples.
Nonfasted 8-month-old TGR and CON rats were anesthetized with thiobutabarbital (80 mg/kg IP) and prepared for micropuncture studies as previously described.4 Isotonic saline containing tritiated inulin (1 mCi/mL) and PAH (0.1 g/dL) were given as a bolus injection (0.5% body weight) followed by continuous infusion at 0.6 mL · h−1 · 100 g body wt−1. After 30 minutes of equilibration, two clearance periods each approximately 45 minutes long were performed for each experiment. During each experiment, timed (3 to 5 minutes) collections of tubular fluid were made in four to six proximal tubules after the tubule lumen was blocked with Sudan black–stained castor oil to determine SNGFR. Four free flow pressures (FFP) and stop flow pressures (SFP) were measured in the earliest convolutions of proximal tubules with a 3- to 7-μm pipette connected to a servonull micropressure system (World Precision Instruments). SFP was determined by blocking the tubule with Sudan black–stained castor oil contained in a 12- to 14-μm pipette. Four hydraulic pressures in efferent star vessels were also measured. Renal vein samples were taken with a 30-gauge needle at the end of clearance periods to determine the extraction ratio for PAH. Heart and kidney weights were measured at the end of each experiment.
RPF was estimated from the clearance of and extraction ratio for PAH. RBF was calculated by dividing RPF by 1−arterial hematocrit. RVR was calculated by MAP/RBF. PGC was determined indirectly from SFP+Arterial Plasma Colloid Osmotic Pressure (COPA). Plasma colloid osmotic pressure was determined with 1.886C+0.206C2+0.005C3, where C is the plasma protein concentration. The ΔP was calculated as PGC−FFP. SNPF was estimated by dividing SNGFR by whole-kidney filtration fraction (GFR/RPF). Mean effective ultrafiltration pressure (PUF) was calculated by subtracting [(COPA−COPE)/2] from ΔP, where COPE was the colloid osmotic pressure in the efferent arteriole, the protein concentration of which was estimated from the arterial plasma protein concentration divided by 1−Whole-Kidney Filtration Fraction. Kf values were determined by dividing SNGFR by PUF. RA was calculated using [(MAP−PGC)/ SNBFA]×7.962×1010, where SNBFA was determined by dividing SNPF by 1−Arterial Hematocrit. RE was calculated using [(PGC−PE)/SNBFE]×7.962×1010, where PE was the pressure in the efferent star vessel and SNBFE was calculated as SNBFA−SNGFR.
We used a microcontinuous gradient gel electrophoresis procedure for the separation and quantification of proteins in plasma and urine.6 Tritiated samples of plasma, urine, and tubular fluid were placed in scintillation fluor and counted in a scintillation counter. PAH and creatinine concentrations were analyzed using previously described methods.4 Plasma lipid concentrations were measured using a commercial kit (Boehringer-Mannheim Diagnostics). Sodium concentrations were determined by flame photometry. NOM concentrations were measured as described by Feld et al.5
Kidney tissue samples were fixed in buffered formalin and embedded in paraffin. Sagittal sections were cut from the middle of each kidney and stained with periodic acid–Schiff. A total of 100 glomeruli were examined by light microscopy in each rat. Each glomerulus was graded from 1 to 4 using a previously described glomerular injury score7 : grade 1, normal glomerulus; grade 2, involvement of up to one third of the glomerulus; grade 3, involvement of one third to two thirds of the glomerulus; and grade 4, two thirds to global sclerosis. Each score was then calculated according to the formula Glomerular Injury Score=[(1×No. of Grade 2 Glomeruli)+(2×No. of Grade 3 Glomeruli)+(3×No. of Grade 4 Glomeruli)]×100/(No. of Glomeruli Evaluated).
Results are expressed as mean±SE. All data obtained from an individual animal during micropuncture experiments were averaged, and this single average represented that animal. Data for GFR and RBF were normalized for the weight of both kidneys. Data for SNGFR and SNPF were normalized for left (micropunctured) kidney weight. Differences between group means were analyzed by ANOVA with the Scheffé post hoc test or by Student’s t test and considered significant at a value of P<.05.
BPs, Body Weights, and Organ Weights
Table 1⇓ shows BP, body weight, and organ weight values. Awake systolic BP of untreated TGR rats was significantly greater than that of CON rats throughout the study. Enalapril produced a significant systolic BP decrease in both TGR and CON rats. Body and kidney weights among age-matched treated and untreated TGR and CON rats did not differ throughout the study. Heart weights were significantly increased in untreated TGR rats compared with age-matched CON rats and were significantly reduced by enalapril in TGR rats.
Plasma and Urine Composition of Conscious Animals
Table 1⇑ also shows the composition of plasma and urine in conscious animals. Hematocrit and plasma sodium, total protein, albumin, cholesterol, and high-density lipoprotein concentrations did not differ among age-matched treated and untreated TGR and CON rats during the study. Plasma creatinine concentrations were significantly greater in untreated TGR rats and decreased with enalapril therapy. Compared with levels in untreated CON rats, fasting plasma triglyceride levels of untreated TGR rats were elevated at 3 and 8 months of age. The use of enalapril did not alter triglyceride levels in either group. Urinary sodium excretion of 8-month-old TGR rats was lower than in CON rats. Enalapril did not change this difference. Endogenous creatinine clearance did not differ in TGR and CON rats at 3 months but fell in TGR relative to CON rats at 8 months of age. Enalapril did significantly affect creatinine clearance in either TGR or CON rats. Untreated TGR rats had significantly higher urinary albumin excretion than CON rats at age 3 and 8 months. Enalapril significantly reduced the amount of albuminuria in both TGR and CON rats. Urinary NOM excretion was lower in TGR than CON rats at 3 months of age. At 8 months, urinary NOM excretion did not differ in TGR and CON rats. Enalapril significantly increased NOM excretion in TGR but not CON rats. To reexamine these changes in urinary NOM excretion, we analyzed frozen samples from an earlier study.4 NOM excretion was also lower in TGR (n=7) than CON (n=7) rats at 4 months (43±14 versus 125±23 nmol/h, P<.05) and similar at 8 months of age (97±30 versus 114±39 nmol/h). Urine flow rate and sodium excretion did not differ significantly in TGR and CON rats in this group of animals.
Table 2⇓ shows whole-kidney function in TGR and CON rats. Consistent with their awake systolic BP, untreated anesthetized 8-month-old TGR rats had significantly higher MAP than untreated CON rats, and enalapril significantly lowered their MAP. MAP values did not differ among untreated CON rats, treated CON rats, and treated TGR rats. Urine flow rate, GFR, RBF, and RVR did not differ in treated and untreated CON rats. In TGR rats, enalapril produced a significant fall in urine flow rate and RVR, significantly increased RBF, but did not alter GFR.
Table 3⇓ shows glomerular hemodynamics of TGR and CON rats. No significant differences in SNGFR, SNPF, PGC, ΔP, Kf, or arteriolar colloid osmotic pressures or resistances were detected in untreated and treated CON rats. Treatment with enalapril significantly increased SNPF without altering SNGFR or Kf in TGR rats. Efferent arteriolar colloid osmotic pressure fell in treated TGR rats, reflecting their lower filtration fraction. Enalapril significantly lowered PGC, ΔP, RA, RE, and RA/RE of TGR rats.
The glomerular injury score of untreated TGR rats was approximately eight times greater than that of TGR rats receiving enalapril (22.7±6.6 versus 2.6±0.8, P<.01) and mainly affected deep cortical nephrons. The glomerular injury score of untreated CON rats was 0.8±0.4. No sclerotic glomeruli were found in enalapril-treated CON rats. There was a significant positive correlation between the incidence of glomerulosclerosis and the magnitude of albuminuria (r=.85, n=22, P<.001).
Although the mechanisms responsible for elevating BP in TGR(mRen2)27 rats are not completely understood, the hypertension of these animals clearly relates to overexpression of the mouse Ren-2 renin transgene. For this reason, medications that interrupt the renin-angiotensin system are ideal antihypertensive agents for this strain. Consistent with short-term studies using captopril and lisinopril, we found that long-term treatment with enalapril controlled hypertension in TGR rats.1 8 We cannot exclude the possibility that modulation of kinins, prostaglandins, or nitric oxide by converting enzyme inhibition influenced the results to some degree.9 Differences in urinary NOM excretion found during this study suggest nitric oxide system dysfunction in TGR rats and its correction by enalapril. However, we recognize that diet and renal function can influence NOM excretion independent of changes in systemic nitric oxide production.10 11 This issue will require further study.
The presence of the mouse renin transgene and use of enalapril did not change somatic growth as judged by absolute body and kidney weights. Increased cardiac weight has been previously reported in this and other genetically hypertensive rat strains.6 8 12 Its regression with enalapril potentially reflects not only lower BP but also a tissue-specific effect because normotension achieved by hydralazine does not reverse cardiac hypertrophy and angiotensin II receptor blockade reduces heart weight without altering BP.8 13
On the basis of elevated serum urea and creatinine concentrations, Lee et al8 noted that heterozygous TGR rats develop renal insufficiency that can be prevented by lisinopril. Enalapril had a similar beneficial effect on TGR serum creatinine concentrations in the present study; however, inulin clearances did not confirm either age-related GFR loss or alteration by enalapril. Acute administration of captopril and losartan to conscious TGR rats has been reported to cause a reduction in sodium excretion and creatinine clearance after salt or water loading.14 We did not perform the experiments needed to determine whether this response persists with long-term use of these medications. Treatment of 3-month-old TGR rats for 9 weeks with an angiotensin II receptor antagonist has been associated with decreased urine volume and sodium excretion.13 Long-term enalapril therapy did not cause these changes in the current study.
We previously reported a significant increase in fasting plasma triglyceride concentrations in transgenic rats.4 This finding was confirmed in the present study. We had speculated that the mouse renin transgene might alter some aspect of triglyceride metabolism because of its increased expression in adipose tissue; however, the inability of enalapril to significantly lower triglyceride levels suggests that neither angiotensin-converting enzyme nor angiotensin is involved in this alteration.4 15
In terms of renal function in anesthetized rats, enalapril significantly reduced urine flow rate in TGR rats. We assume that this reduction reflects a pressure-diuresis response and was responsible for the somewhat higher hematocrit values in untreated TGR rats. Enalapril did not affect whole-kidney and single-nephron filtration rates in CON or TGR animals. Whole-kidney blood flow and SNBF increased only in enalapril-treated TGR rats and were associated with lower filtration fraction, RVR, and arteriolar resistances. Given the significant decrease in RA/RE, this rise in RBF was primarily caused by preglomerular vasodilatation. Prior investigations of the effect of renin-angiotensin system inhibitors on TGR(mRen2)27 renal function have been limited to the acute administration of these medications. In their studies of pressure-natriuresis during which renal perfusion pressures were controlled, Gross et al16 found that treatment of TGR rats with captopril and an angiotensin II receptor blocker increased RBF without changing GFR. In contrast, Mitchell and Mullins17 reported that the angiotensin II antagonist L158,809 reduced GFR without altering effective RPF. Interpretation of this latter study is confounded by evidence that TGR rats appear to autoregulate poorly in the face of abrupt falls in renal perfusion pressure.18 The fact that mechanical reduction of renal perfusion pressure lowers both GFR and RBF while L158,809 only decreases GFR supports the blood flow–enhancing effect of angiotensin inhibitors in TGR rats.17
Renal injury, manifested by albuminuria and glomerulosclerosis, appears early in the life span of TGR(mRen2)27 rats.4 Treatment with converting enzyme inhibitors prevented these abnormalities and was associated with mildly diminished glomerular capillary pressure. It is generally accepted that sustained hypertension damages the kidney by preglomerular vascular injury leading to glomerular ischemia or by transmission of increased systemic pressure to glomerular capillaries.19 We have previously shown that renal injury in TGR rats evolves, at least initially, without evidence of glomerular ischemia or hypertension in superficial nephrons.4 Thus, either these mechanisms are not solely responsible for renal injury or they occur predominantly in deeper nephrons. Histological studies indicate that renal injury in TGR rats begins in the inner cortex and then progresses superficially.4 This pattern of injury is similar to that previously documented in spontaneously hypertensive rats, a strain in which progressive proteinuria and sclerosis also develop despite the absence of substantial reduction in superficial glomerular blood flow or elevation in superficial glomerular capillary pressure before 15 to 18 months of age.6 7 20 Because measurement of juxtamedullary glomerular hemodynamics is not feasible in the intact kidney, it is impossible to determine whether glomerular ischemia or hypertension exists in inner cortical nephrons.
Alternatively, the benefit of converting enzyme inhibitors and related medications might not depend on systemic/glomerular normotension. Despite only modest declines in awake systolic BP and no alterations in MAP or glomerular capillary pressure, our enalapril-treated control rats had minimal albuminuria and no glomerulosclerosis. This renal protective effect of chronic converting enzyme inhibition has been previously documented in aging rodents and suggests a mechanism that does not involve hemodynamic changes.21 22 Supporting this hypothesis are studies by Remuzzi et al23 dissociating the antihypertensive and renal protective effects of lisinopril in aging Munich-Wistar rats and by Bohm et al13 showing that low-dose treatment with an angiotensin II receptor antagonist ameliorates kidney damage without substantially lowering systemic BP in TGR(mRen2)27 rats. Suppression of the intrarenal renin-angiotensin system and its stimulation of cell proliferation, production of transforming growth factor-β, and synthesis of matrix protein could therefore play important roles in preserving function in hypertensive renal disease even when glomerular hemodynamics remain unchanged.24 The status of the intrarenal renin-angiotensin system in heterozygous TGR rats is currently unclear. Although functional studies suggest heightened activity,15 16 kidney angiotensin II levels are significantly lower compared with levels in normotensive Sprague-Dawley control rats.7 Regardless, angiotensin inhibition is clearly not a panacea for all forms of hypertension-associated kidney damage. Chronic treatment of spontaneously hypertensive rats with enalapril, which normalizes systemic BP and presumably suppresses glomerular barotrauma and intrarenal angiotensin II production, only postpones the eventual development of renal injury that is as severe as that found in their untreated hypertensive counterparts.25
In summary, long-term use of enalapril in TGR(mRen2)27 rats controls hypertension and cardiac hypertrophy, increases RBF by preferentially decreasing preglomerular vascular resistance, and prevents glomerulosclerosis and pathological proteinuria. The mechanism of its beneficial effect on the kidney is unclear. Potential explanations include reduction of glomerular capillary pressure and suppression of the intrarenal renin-angiotensin system. These animals represent a unique model for examining the effects of renin-angiotensin system dysfunction on BP regulation and the kidney. Future studies that compare varying doses of angiotensin II antagonists or renin inhibitors with other antihypertensive medications and employ telemetry to assure equivalent levels of BP control should prove useful in separating hypertensive from nonhypertensive actions of the mouse renin transgene on the kidney.
Selected Abbreviations and Acronyms
|GFR||=||glomerular filtration rate|
|MAP||=||mean arterial pressure|
|NOM||=||nitric oxide metabolite (nitrite+nitrate)|
|ΔP||=||glomerular transcapillary hydraulic pressure difference|
|PGC||=||glomerular capillary hydraulic pressure|
|RA||=||afferent arteriolar resistance|
|RBF||=||renal blood flow|
|RE||=||efferent arteriolar resistance|
|RPF||=||renal plasma flow|
|RVR||=||renal vascular resistance|
|SNBFA||=||afferent single-nephron blood flow|
|SNBFE||=||efferent single-nephron blood flow|
|SNGFR||=||single-nephron glomerular filtration rate|
|SNPF||=||single-nephron plasma flow|
This study was supported by a Grant-in-Aid from the American Heart Association, New York State Affiliate, Inc, and by the Office of Research and Development, Department of Veterans Affairs. We thank Susan Bemben, Sherry Davies, and Nancy Manz for their excellent technical assistance.
Reprint requests to James E. Springate, MD, Division of Nephrology, The Children’s Hospital, 219 Bryant Street, Buffalo, NY 14222.
Portions of this work were presented at the 1995 Annual Meeting of the Society for Pediatric Research, San Diego, Calif, May 9, 1995, and published in abstract form (Pediatr Res [1995;37:371A]).
- Received January 9, 1997.
- Revision received February 17, 1997.
- Accepted March 12, 1997.
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