1 Department of Pediatric Surgery, Erasmus University Medical School, Rotterdam, The Netherlands and 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Surgery was performed and L-NAME treatment (50 or 150 mg/l) was started at the age of 15 weeks. Systolic blood pressure (SBP) and urinary albumin excretion (UaV) were measured at 6 and 12 weeks post-UNx, followed by autopsy to determine the incidence of focal glomerulosclerosis (FGS). Using lisinopril (LIS) and L-NAME, another group of rats was evaluated at 12, 18, and 24 weeks after start of treatment.
Results. At similar L-NAME intake, F, rats developed more severe hypertension and more UaV than ACI rats. The increase in UaV per mmHg increase in SBP was fivefold higher in F1 compared with ACI rats. In F1 rats, the increase in UaV per percentage incidence increase in FGS was three times higher. In LIS treated F1 rats, no significant UaV or FGS was measured at low blood pressure levels, indicating that renal damage in hypertensive F1 rats is not a direct effect of L-NAME, but the result of the high blood pressure or another action of the reninangiotensin system.
Conclusion. We conclude that heterozygosity for the genes influencing the development of renal damage in the FHH strain increases the susceptibility of the kidney to develop damage after UNx combined with systemic hypertension.
Keywords: fawn-hooded rats; genetic susceptibility; glomerulosclerosis; hypertension; renal damage
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic factors influencing the development and progression of renal damage are apparent in FHH rats. Using a back-cross of (FHHx AugustxCopenhagen Irish (ACI)) F1xFHH rats, we mapped two quantitative trait loci (QTLs) on chromosome 1, influencing proteinuria and structural renal damage [5]. These genes were denoted Rf-1 and Rf-2. Rf-1 appeared at least partly independent from blood pressure. In a subsequent study, the development of renal damage after UNx was assessed in a (FHHxACI) F2 progeny [6]. Linkage analysis in this F2 cross not only confirmed the importance of Rf-1 and Rf-2, but also revealed the presence of three additional QTLs (Rf-3, Rf-4, and Rf-5) influencing the development of proteinuria. Thus, susceptibility to renal damage after UNx is influenced by at least five susceptibility genes.
Previously we have studied the effects of hypertension induced by chronic nitric oxide (NO) inhibition with N-nitro-L-arginine methyl ester (L-NAME) on the development of renal damage in F1 (FHHxACI) rats and in both parental strains with two kidneys. It was shown that despite similar levels of systolic blood pressure (SBP), severe UaV, and FGS were present in FHH rats, while ACI rats developed hardly any renal damage [7]. The most important finding in the F1 rats was that, although these hybrids developed less renal damage compared with FHH rats, being heterozygous for the susceptibility genes did not completely protect the kidney from developing UaV and FGS. Compared with ACI rats, the severity of UaV and FGS increased, especially at SBP levels above 180 mmHg.
The purpose of the present study was to assess the genetic susceptibility to develop renal damage after UNx in combination with L-NAME-induced hypertension. The changes in UaV and SBP at 6 and 12 weeks after UNx and L-NAME-induced hypertension were assessed in F1(FHHxACI) and ACI rats. At 12 weeks after surgery, rats were sacrificed, creatinine clearance was measured, and morphological studies were performed in kidney tissue to determine structural renal damage. FHH rats were not included in these studies because of their high susceptibility and mortality in response to chronic L-NAME treatment [7], especially in combination with UNx [4].
A second study was carried out under a similar protocol to exclude the possibility of renal damage being a direct toxic effect of L-NAME. In this study, F1 rats were treated with L-NAME and the angiotensin-converting enzyme inhibitor (ACEI) lisinopril, to keep SBP at a low concentration. The SBP and UaV levels were measured 12, 18, and 24 weeks after UNx in control and treated groups.
![]() |
Subjects and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
UNx
The right kidney was removed using ethyl ether (Sigma Chemical Co., St Louis, MO) anaesthesia, dministered through an evaporation system connected to an airtight box. Rats were allowed to recover from surgery in a warmed cage for 12 h.
Design study 1
Chronic nitric-oxide inhibition was started after the rats had recovered from surgery for 34 days using L-NAME, (Sigma Chemical Co., St Louis, MO) dissolved in the drinking water at a concentration of 50 or 150 mg/l to obtain various blood pressure elevations, and was based on previous experiments [7]. This dose was slightly lower compared with previous studies in two-kidney rats, because some F1 animals died in that study as a result of L-NAME-induced complications. Control animals were provided with normal drinking fluid.
Animals from each strain were divided into three groups, and were age- and body weight-matched. The F1 groups (n=29) consisted of nine control rats, 10 rats treated with 50 mg L-NAME, and 10 rats treated with 150 mg L-NAME. The ACI groups (n=26) consisted of 10 control rats, six rats treated with 50 mg L-NAME, and 10 rats treated with 150 mg L-NAME. Actual L-NAME intake was calculated in mg/kg from the metabolic data at 6 and 12 weeks after UNx.
Design study 2
Blood pressure was maintained at low levels by chronic angiotensin-converting enzyme inhibition (ACE-i), using lisinopril (LIS) (Novatec®, Merck Sharp & Dome, Haarlem, The Netherlands), that was administered through the drinking water at a concentration of 50 mg/l. This dose was used because it had previously proven to provide adequate blood pressure reduction in FHH rats [8]. As in study 1, hypertension was induced using L-NAME. The F1 rats were divided into three age- and body weight-matched groups, and were measured 12, 18, and 24 weeks after UNx. The end-point of study 1 was taken as a starting-point for this study to obtain a longer follow-up period in F1 rats with different blood pressure levels.
The control and L-NAME groups consisted of eight rats. The third group (NAME+LIS) consisted of nine rats that were simultaneously treated with L-NAME and LIS. Treatment was started after the rats had recovered from surgery for 34 days and continued until the final measurement at 24 weeks after UNx. Also in this study, actual L-NAME intake was calculated in mg/kg from the metabolic data at 12, 18, and 24 weeks after UNx.
SBP and metabolic measurements
SBP was measured as previously described [7]. The week before the measurement, animals were allowed to adapt to the equipment and the procedure to minimize stress-induced artefacts. Although SBP was measured every 3 weeks after UNx, only the values obtained at the time of the metabolic measurements will be presented. Urine excretion, food and water intakes were determined. gravimetrically in two 24-h collections at 6 and 12 weeks after UNx in study 1 and 12, 18, and 24 weeks after UNx in study 2, as described previously [7]. Rats were allowed to adapt to the metabolic cages over the weekend.
Terminal analysis
Autopsy of the rats, processing of kidneys were performed as described previously [7]. The criteria for the assessment of the incidence of FGS were adhesion of the glomerulus to Bowman's capsule, periodic acid-Schiff-positive material in the mesangial region, and folding of the glomerular basement membrane with entrapment of amorphous material. Final creatinine clearance as a measure of glomerular filtration rate (GFR) was calculated from the plasma creatinine concentrations from blood obtained at autopsy and from the urinary creatinine concentration obtained at the last metabolic measurement shortly before autopsy.
Analytic procedures
Plasma and urinary albumin concentration was measured with bromocresol green (Merck, Darmstadt, Germany), and creatinine with the Jaffé method without deproteinization using the semi-automatic ELAN system (Merck-Eppendorf, Hamburg, Germany).
Statistics
Data are presented as mean±SEM in the figures, text, and tables. Differences in mean values between groups were compared by ANOVA and a subsequent StudentNewmanKeuls test to identify the groups that were different. In case of a non-normal data distribution, groups were compared using the MannWhitney rank sum test. In all tests, statistical significance was defined as P<0.05.
The relationships between SBP and UaV, and between the level of UaV at week 12 and the incidence of FGS was assessed by linear regression analysis. All tests were performed using the Primer of Biostatistics (S. E. Glantz) software package.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Structural renal damage and final GFR
The level of structural damage, as indicated by FGS, is shown in Table 3. Both strains showed a dose-dependent increase in FGS. The incidence of FGS in the F1-150 group was significantly higher compared with all other groups. The 50 mg-treated groups of both strains showed similar FGS incidence.
|
Renal damage in groups with similar SBP levels
The groups that develop similar levels of SBP appeared to be the FI-CON and the ACI-50, and the F1-50 and the ACI-150 groups respectively. The SBP level in these groups averaged 132±1 and 139±1, and 165±1and 160±1 mmHg, averaged over all measured time-points. The UaV level in the F1 rats over the same period was higher, although not statistical significantly different from ACI rats, due to the large variation. However, when we compared the groups that received the same amount of L-NAME (Table 1), the level of UaV in the F1-150 group was significantly higher compared with every ACI group at 6 and 12 weeks post UNx.
Comparison of strains by linear regression analysis
To determine whether a difference in the relationship between SBP and UaV was present between F1 and ACI rats after UNx, linear regression analysis was performed on the data obtained at 6 and 12 weeks after start of the experiment. All control, 50-, and 150-mg treated ACI and F1 rats were included in the regression analysis, which is shown in Figure 1A,B
respectively. Already at 6 weeks, the slope of the best-fit regression line was significantly higher in F1 compared with ACI rats, and averaged 0.17±0.01 (r=0.397, P=0.033) and 0.10±0.01 (r=0.709, P<0.01) mg/24 h per mmHg. At week 12 after UNx, the increase in UaV per mmHg increase in SBP was five times more in F1 than in ACI rats. Values averaged 1.24±0.04 (r=0.807, P<0.001) and 0.25±0.01 (r=0.70, P<0.001) mg/24 h per mmHg respectively, and indicate that the amount of functional damage between week 6 and 12 was sevenfold in F1, and almost threefold in ACI.
|
Study 2
Blood pressure and albuminuria
The actual L-NAME intakes of the F1 rats used in this study are also given in Table 1, and showed no significant differences between the F1 groups.
Longitudinal values for the tail-cuff SBP and UaV at 12, 18, and 24 weeks after UNx are shown in Figure 2A,B
respectively. Also in this study, SBP in L-NAME-treated rats was significantly higher than that of the control group. In contrast, SBP in the L-NAME+LIS rats was significantly lower compared with the control rats. The SBP in the L-NAME group increased to around 200 mmHg at week 24 after UNx. The SBP levels in the control and L-NAME+LIS group remained stable around 135 and 100 mmHg, respectively.
|
Final GFR and structural renal damage
Final creatinine clearance as an indicator of final GFR were equal in the NAME and NAME+LIS groups and averaged 1.21±0.07 and 1.2±0.1 ml/min respectively. The clearance in the control group was slightly, but not significantly, higher compared with the other groups and averaged 1.42±0.07 ml/min. Final GFR in the CON group was slightly, but not significantly, higher than in the NAME and NAME+LIS groups. The level of structural damage, indicated by the incidence of FGS was significantly higher in the NAME group compared with the other groups, and averaged 27.1±2.7%. The incidence of sclerosis was significantly higher in the control compared with the NAME+LIS group and averaged 16.3±2.9, and 4.8±0.8% respectively. Long-term concomitant ACEI treatment completely prevented the development of FGS after UNx and L-NAME treatment. Furthermore, as evidenced by the significantly lower incidence in the NAME+LIS group, ACEI also reduced the incidence of FGS when compared with the control group. Thus the lower SBP levels in NAME+LIS-treated F1 rats appears to prevent the development of both functional (UaV) and structural (FGS) renal damage.
Regression analysis study 2
Linear regression analysis using the SBP and UaV data obtained at 12, 18, and 24 weeks after UNx showed significant relationships between these parameters (r=0.787, P<0.001; r=0.828, P<0.001; r=0.825, P<0.001). Furthermore, a significant correlation between FGS and UaV was present at 24 weeks after UNx (r=0.805, P<0.001). This indicates that in this group an even stronger correlation was present between the SBP level and UaV compared with study 1.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By comparing the creatinine clearance in the present study with those obtained in a previous study performed in F1 and ACI rats with two kidneys (2K) after similar L-NAME intake [7], we were able to calculate the compensatory increases in GFR after UNx. It appears that the compensatory increase in F1 rats was significantly higher compared with ACI rats. In control F1 rats, the final GFR after UNx on average increased by 59% compared with 2K-F1 rats. In control ACI rats, the level after UNx was only 17% higher than in 2K-ACI rats. This data indicates that F1 rats were less efficiently protected from an increase in GFR than ACI and might indicate differences in renal haemodynamic regulatory mechanisms between the strains. Our data support the view that, at low-dose L-NAME, the renal vasoconstriction caused by L-NAME leads to an increase in filtration and an increase in FGS due to a larger haemodynamic burden compared with the control groups in ACI and F1 rats. At high dose L-NAME, increasing constriction causes a decrease in filtration and a further increase in FGS due to ischaemic factors. The presence of an increase in filtration solely due to the reduction of renal mass might also play an additional role in this process [9,10].
The male FHH rat, the parental strain of the F1 rats used in the present study, develops mild systolic, but marked glomerular hypertension, and is extremely susceptible to develop renal damage [15]. Glomerular hypertension and the severity of renal damage in the FHH is further increased by UNx [3,4]. The glomerular capillary pressure (PGC) level in UNx-FHH rats was found to be higher than those reported following UNx in MW or Wistar Kyoto (WKY) rats [11,12], strains that are less susceptible to the development of renal damage than the FHH rat. However, PGC in UNx-FHH is comparable to that reported for MW rats with remnant kidneys [13]. In general, normotensive rat strains appear less susceptible than hypertensive strains. We have previously studied the normotensive WAG, in which survival time after UNx is only slightly reduced compared to the 2K WAG [9,14]. In ACI rats in the present study, hardly any renal damage develops even after a substantial elevation of systemic blood pressure. However, normal blood pressure is no guarantee for low susceptibility. The Milan normotensive rat, in contrast to the Milan hypertensive rat spontaneously develops renal damage without the development of hypertension [15].
Similar to male FHH rats, male MunichWistar-Fromter (MWF) rats spontaneously develop glomerular and systemic hypertension [16], which is also greatly worsened by UNx [17]. In spontaneously hypertensive rats (SHR), where systemic blood pressure is much higher, glomerulosclerosis and renal failure do not develop early in life [13]. When renal mass is reduced, the resistance of the afferent arteriole decreases in SHR and allows the transmission of systemic hypertension into the glomerular capillary network [13]. Subsequent proteinuria (UpV) develops, albeit much more slowly than in FHH and MWF rats following UNx [10,11]. Even a further increase in systemic pressure using L-NAME does not severely impair renal function in SHR because of adequate control of afferent arteriolar resistance [18]. Thus, it appears that adequate renal autoregulation is of major importance in protecting the glomerulus from the transmission of systemic pressure into the capillary bed. We recently observed an impaired renal autoregulation leading to glomerular hypertension in the FHH rat [19]. Moreover, isolated perfused interlobular arteries of the FHH rat showed an impaired myogenic response, which contributes to the development of renal damage in this animal model [20].
Regulation of renal vascular resistance, intraglomerular pressure, and glomerular filtration might be genetically determined and thereby predispose to renal failure if PGC is excessively high. The recent findings that genes determining renal failure and blood pressure in the FHH rat are separate and distinct from each other could be a possible explanation for the strain differences in the susceptibility to develop glomerular damage. Experiments in which rats of an F2 cross between ACI and FHH rats were studied after UNx, not only confirmed the results of an earlier study in a back-cross of both strains, but also revealed the presence of three additional loci for renal failure [5,6]. These findings underscore the complexity of the genetics to progressive renal failure and might point towards specific genetically regulated mechanisms related to changes induced by UNx.
Alterations in the glomerular permeability of proteins, allowing the development of UpV, can occur while GFR remains within normal limits in different renal diseases both in animals and humans [21]. The persistence of UpV in the presence of normal GFR can be considered an independent risk factor for the development of renal failure, since no correlations were found between UpV and GFR in the rats used in the first study after UNx and in the second study after L-NAME and ACEI treatment. The levels of final GFR in the present study showed that, compared with ACI rats, the compensatory increase in GFR after UNx in F1 rats is larger than the decrease in GFR caused by NO synthase inhibition.
Although our data strongly suggest an important role for the elevated blood pressure in the development of glomerular damage, the effects of reducing synthesis may have other effects on the kidney as well. Likewise, chronic ACE-i mediates more than reverse the hypertension. When angiotensin II (Ang II) formation is chronically inhibited with ACEI, glomerular hypertension is controlled while glomerular hyperfiltration is usually unaffected. In contrast, when Ang II activity is increased by chronic infusion, intraglomerular pressure, proteinuria, and FGS are further elevated [22]. Although the role of the reninangiotensin system (RAS) in chronic NO inhibition remains somewhat controversial, the development of hypertension in this model is thought to be renin-dependent and may result partly from increased vascular Ang II receptor expression [23]. Various reports suggest that the endothelial NO synthase system and the RAS interact as regulators of the glomerular microcirculation [24].
Another interesting finding was that, at similar doses of L-NAME, SBP increased significantly more in F1 rats than ACI rats at both 6 and 12 weeks after UNx. In our previous studies [7], higher doses of L-NAME induced similar increases in SBP both in F1 and ACI rats. Furthermore, the enhancement of blood pressure values in F1-UNx rats seems to be higher in than in F1-2K rats with a dose of L-NAME 9 mg/kg BW. These data suggest an alteration in the synthesis or in the metabolism of endothelial NO in F1 rats.
In conclusion, after UNx and chronic NO synthase inhibition, the F1(FHHxACI) rat, which is heterozygous for genes that determine the susceptibility to renal damage in the FHH rat, is not completely protected from developing hypertension-associated renal damage. Furthermore, we conclude that renal damage in hypertensive F1 rats is not a direct effect of L-NAME, but the result of the high blood pressure or another action depending on the activation of the RAS.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|