Abstract
Abstract. Chronic inhibition of nitric oxide synthase (NOS) is known to cause renal parenchymal injury with systemic hypertension. To elucidate the pathogenetic mechanism in renal damage induced by NOS inhibition, Nω-nitro-L-arginine methyl ester (L-NAME) was given orally for 12 wk in Wistar rats, and the roles of tissue renin-angiotensin system and transforming growth factor-β1 (TGF-β1) were investigated. BP and urinary protein excretion increased significantly in L-NAME rats compared with control rats, and glomerulosclerosis and interstitial fibrosis developed. In L-NAME rats, the cortical tissue levels of angiotensin-converting enzyme activity and angiotensin II were significantly higher than those in control rats. The cortical mRNA expressions of both TGF-β1 and fibronectin were significantly elevated in L-NAME rats. Immunohistochemically, increased expressions of both fibronectin and α-smooth muscle actin were also revealed in L-NAME rats. In L-NAME rats, these histologic injuries and the increased expression of TGF-β1 were equally emeliorated by either angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor antagonist, but not by hydralazine. In conclusion, the locally activated renin-angiotensin system in connection with the increased TGF-β1 expression is a major pathogenetic feature of renal injury in chronically NOS-inhibited rats.
Nitric oxide (NO) is a labile radical gas and is believed to have many important biologic actions in various tissues (1). NO is synthesized from L-arginine and is mediated by nitric oxide synthase (NOS). Many L-arginine analogues inhibit NO synthesis and are reported to cause tissue injuries (2,3). In the kidney, NO participates in the regulation of glomerular and medullary hemodynamics, namely via tubuloglomerular feedback response, renin release, and by controlling extracellular fluid volume status (4,5,6). Recent experimental studies in rats have shown that chronic administration of an L-arginine analogue, Nω-nitro-L-arginine methyl ester (L-NAME), caused systemic hypertension and renal parenchymal injury, i.e., glomerulosclerosis and interstitial fibrosis (7,8,9). In these models, it was also reported that the renin-angiotensin system (RAS) was stimulated and that the RAS inhibition with either angiotensin-converting enzyme (ACE) inhibitor (ACEI) (10,11) or angiotensin II type I receptor antagonist (AIIA) prevented both the onset of hypertension and renal damage (8,9,12).
Transforming growth factor-β1 (TGF-β1) acts as a key fibrogenic cytokine in many tissues by enhancing extracellular matrix (ECM) synthesis (13). In terms of renal damage, it was also revealed that the increases in both the production and the activation of TGF-β1 are the final common step leading to glomerulosclerosis and interstitial fibrosis (14,15,16). Recently, scientific interest has been focused on the augmentation of the local RAS in connection with TGF-β1 (17). It has been shown that the enhanced ECM synthesis due to angiotensin II (AngII) is mediated by the generation of active TGF-β1, indicating that AngII induces the conversion of the latent form of TGF-β1 to active form (18).
However, it remains unclear whether a locally activated RAS in the renal cortex contributes to the renal parenchymal damage in the experimental model of chronic NOS inhibition. In the present study, the association of the local RAS and TGF-β1 for the renal injury in chronically NOS-inhibited rats and the effects of RAS inhibition with either ACE inhibitor or AIIA were examined.
Materials and Methods
Experimental Protocol I
Male Wistar rats, 10 wk old and weighing 210 to 230 g, were used. All rats were treated according to the protocols approved by the Kyushu University Animal Care Committees at the Center Animal Care facility. Two groups of rats were studied. The L-NAME group (L-NAME rats, n = 25) received L-NAME in the drinking water (0.25 mg/ml). At this concentration, the daily intake of L-NAME was 20 to 30 mg/kg per d. (2) The control group (control rats, n = 20) received untreated drinking water. All rats were kept in individual cages and were fed regular rat chow in a special pyrogen-free facility. The actual volume of water ingested by each group of rats was measured weekly, and it was confirmed that all animals drank 20 to 30 ml/d. Every 4 wk, body weight (BW), systolic BP (SBP), and urinary protein excretion (UP) were measured. At weeks 8 and 12, the rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg, intraperitoneally) and sacrificed. At week 8, cortical tissue levels of ACE activity, AngII levels, TGF-β1, and fibronectin (FN) mRNA expression were examined. At week 12, ACE activity, AngII levels, the expression of TGF-β1 and FN mRNA, renal histology, and serum creatinine (SCr) were examined.
Blood samples were obtained in situ via the aorta and then centrifuged and stored frozen at -80°C until measurement. The kidneys were perfused with cold phosphate-buffered saline (pH 7.4) and then excised. The renal capsules were gently removed and the cortex was trimmed off with scissors. Renal cortical tissue samples for measurements of ACE activity, AngII, and for mRNA expression were frozen in liquid nitrogen and stored at -80°C until these analyses.
Experimental Protocol II
The effects of the RAS inhibitors on both renal lesions and the expression of TGF-β1 mRNA were also examined in L-NAME-treated rats. The L-NAME-treated rats were induced by the same methods as in Experimental Protocol I. Five groups were studied for 12 wk. The first group (L-NAME, n = 8) received L-NAME in its drinking water (0.25 mg/ml). The other groups studied were as follows: the second group (L-NAME + ACEI, n = 8) received L-NAME (0.25 mg/ml) and ACEI (imidapril, 0.05 mg/ml); the third group (L-NAME + AIIA, n = 8) received L-NAME (0.25 mg/ml) and a specific angiotensin II type 1 receptor antagonist (TCV-116, 0.1 mg/ml); the fourth group (L-NAME + Hyd, n = 8) received L-NAME (0.25 mg/ml) and hydralazine (0.15 mg/ml); and the fifth group (control, n = 8) received untreated drinking water. All agents were given in the drinking water. Every 4 wk, BW, SBP, and UP were measured. At week 12, renal histology, the expression of TGF-β1 mRNA, and SCr were examined.
Analytical Methods
SBP was measured in a conscious state by the tail-cuff method. UP was examined by the sulfosalicylic acid method. SCr was measured for the assessment of renal function using a Hitachi 7170 autoanalyzer (Hitachi, Tokyo, Japan).
Histologic Examination
The kidneys were fixed with Methacarn solution (19) and embedded in paraffin for a light microscopic study. Sections of 2-μm thickness were stained with Masson's trichrome. A histologic examination was done independently by two pathologists blinded to the experimental groups. To semiquantify glomerular mesangial matrix, 100 glomeruli were selected at random, and the degree of glomerular mesangial matrix expansion was determined using the method of Raij et al. (20). The percentage of each glomerulus occupied by mesangial matrix was estimated and scored as follows: 0, no lesion; 1 +, 1 to 25%; 2 +, 26 to 50%; 3 +, 51 to 75%; and 4 +, 76 to 100%. The number of glomeruli showing no lesion was set to n0; similarly, 1 + was n1, 2 + was n2, 3 + was n3, and 4 + was n4, respectively. One hundred glomeruli were examined independently and then the sclerosis index was calculated by the following formula: Glomerulosclerosis index = [(0 × n0 + 1 × n1 + 2 × n2 + 3 × n3 + 4 × n4)/100] × 100.
A representative section from the entire cortex was analyzed by a point-counting technique to obtain the relative interstitial volume (21). The relative interstitial volume of the kidney was examined with a 121-point (100 square) eyepiece micrometer. The renal interstitial injury was estimated by counting the relative interstitial volume. A minimum of five sections (605 points) was randomly selected and counted in all cases.
Immunohistochemical Examinations
The method of immunohistochemical staining has been described previously (22). Briefly, the kidney tissues were fixed in Methacarn solution. Paraffin sections were cut at 4-μm thickness. After deparaffinization and blocking of endogenous peroxidase in 0.3% H2O2 in methanol for 30 min, the sections were incubated overnight at 4°C with mouse monoclonal anti-α-smooth muscle actin (α-SMA) (Nichirei Corp., Tokyo, Japan). After extensive washings, the sections were blocked with normal rabbit serum, then incubated with affinity-purified rabbit anti-mouse IgG and avidin enzyme complex (Nichirei Corp.). Staining was visualized by incubating with diaminobenzidine (Nichirei Corp.) and counterstaining with hematoxylin. Controls were obtained by replacing the primary antibody with normal mouse IgG (Sigma Chemical Co., St. Louis, MO) at equivalent concentrations.
The distribution of FN was examined by performing the indirect immunofluorescence study (15). A frozen tissue was cryostat-sectioned at 4-μm thickness. The air-dried sections were fixed in cold acetone and were incubated with polyclonal rabbit anti-rat FN antibody (Chemicon, Temecula, CA), and then reacted with FITC-labeled goat anti-rabbit IgG (Organon Teknika Corp., West Chester, PA). As a control experiment, the tissue sections were incubated with normal rabbit sera, followed by either FITC-labeled goat anti-rabbit IgG or secondary antibody only.
RNA Extraction and Northern Blotting Analysis
The renal cortical tissue samples were obtained as described previously. The total RNA was isolated from the renal cortical tissue using Isogen (Nippon Gene, Tokyo, Japan) based on the acid-guanidinium thiocyanate-phenol-chloroform extraction method (23). Twenty micrograms of total RNA was subjected to electrophoresis in 2.2 M formaldehyde 1% agarose gel, transferred to Hybond N nylon membranes (Amersham, Arlington Heights, IL), and then fixed by baking at 80°C for 2 h.
The cDNA probes for the rat TGF-β1 (provided by Dr. T. Nakamura, Kyushu University, Fukuoka, Japan) (24) and the FN (provided by Dr. R. O. Hynes, Massachusetts Institute of Technology, Cambridge, MA) (25) were used. The cDNA probe for the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as an internal control probe, was prepared using reverse transcription (RT)-PCR, as described previously (26). The cDNA probe was labeled with 32P-dCTP by the random primer method.
After prehybridization and hybridization in Quik-Hyb (Stratagene, La Jolla, CA) according to the manufacturer's protocol at 64°C for 2 h, the membranes were washed twice for 15 min at room temperature with a 2 × SSC buffer and 0.1% sodium dodecyl sulfate (SDS) solution, and washed once for 30 min at 60°C with a 0.1 × SSC buffer and 0.1% SDS solution. Autoradiography was performed by the standard methods. The densitometric values of each mRNA were corrected against the value of GAPDH mRNA. The ratio of each mRNA/GAPDH mRNA from eight rats in same group was averaged.
Measurement of ACE Activity
ACE activities in both the renal cortical tissue and the serum were measured using a fluorometric assay, described by Cheung and Cushman (27). For determination of renal-cortical ACE activity, the cortical tissue was homogenized with 9 vol of ice-cold 10 mM Tris-HCl buffer solution (pH 7.4) containing 0.2 M sucrose and centrifuged at 500 × g at 4°C for 10 min. Fifty microliters of the supernatant was added to 400 μl of 1 mM Hippuryl-His-Leu (Sigma Chemical Co.) in 50 mM Tris-NaCl buffer (pH 7.4) and incubated at 37°C for 1 h. The reaction was stopped by adding 2 ml of methanol, and the reaction tubes were chilled in an ice-water bath, then hippuric acid was measured. Serum ACE activity was calculated as micromoles of His-Leu generated per milliliter of serum per hour, and the tissue ACE activity was calculated as micromoles of His-Leu generated per gram of tissue weight per hour.
Measurement of Tissue AngII Level
AngII from the renal cortical tissue was measured using RIA. Cortical tissue was homogenized with 9 vol of ice-cold saline containing 0.1N HCl and 5% aprotinin, and centrifuged at 12,000 × g at 4°C for 20 min. The collected supernatant was added to florisil (Sigma Chemical Co.) and then centrifuged at 1000 × g for 2 min. The supernatant was discarded and the pellet was washed 3 times with distilled water, followed by acetone containing 0.5N HCl, and petroleum ether. The extract was dried under vacuum and reconstituted in Tris buffer (pH 8.5) for RIA. The incubation mixture consisted of sample or standard and anti-AngII rabbit antisera (SRL, Tokyo, Japan). The AngII antibody used in our experiment has been shown to have a very low cross-reactivity (0.037%) with angiotensin I (28). The incubation was carried out at 4°C for 24 h and followed by the late addition of 125I-AngII and further incubated at 4°C for 8 h. To separate bound radioactivity from free ligands, anti-rabbit Ig goat antisera and polyethylene-glycol were added, incubated at 4°C for 1 h, and centrifuged at 2000 × g for 20 min. The radioactivity in the precipitate was counted in a γ-spectrometer.
Drugs Used
The drugs used in the present study were L-NAME (Sigma Chemical Co.), imidapril (Tanabe Pharmaceutical Co., Osaka, Japan), AngII type 1 receptor antagonist (TCV-116; Takeda Pharmaceutical Co., Osaka, Japan), and hydralazine (Novartis Pharmaceutical Co., Tokyo, Japan).
Statistical Analyses
Results are presented as mean ± SEM. Statistical comparisons were done using the StatView program (Abacus Concepts, Berkeley, CA). One-way ANOVA was followed by t test with Bonferroni correction when indicated. A P value < 0.05 was considered statistically significant.
Results
Experimental Protocol I
BW, SBP, UP, and Renal Function. BW, SBP, UP, and SCr are shown in Table 1. BW values were not significantly different between the groups except for BW at week 12, when it was significantly lower in the L-NAME rats (P < 0.01). The SBP in L-NAME rats rose progressively and reached a significant level at week 4 compared with control rats. The SBP kept a significantly higher level throughout the observation period of 12 wk. UP at the baseline was rather stable until week 8 in both groups, but it rapidly increased at week 12 in the L-NAME rats. The renal function estimated by SCr at week 12 is also presented in Table 1. The SCr in the L-NAME rats was significantly higher than that in control rats (P < 0.01).
Clinical parameters in Experimental Protocol Ia
Histologic Changes. The renal histologic injury was examined semiquantitatively by counting the glomerulosclerosis index and the relative volume of interstitial fibrosis on sacrifice at week 12. The representative figures of glomeruli stained with Masson's trichrome in both groups are shown in Figure 1. Table 2 depicts the semiquantitative data of the histologic changes. It was apparent that both the glomerulosclerosis index and the relative interstitial volume were significantly higher in L-NAME rats compared with control rats (P < 0.01).
Micrographs of renal tissue specimens with Masson's trichrome stain in control rats (a and b) and in Nω-nitro-L-arginine methyl ester (L-NAME) rats (c and d) at week 12, Experimental Protocol I. Panels show kidney sections from control rats with intact glomeruli (a), intact interstitium (b), sclerosing glomeruli in L-NAME rats (c), and focal tubular atrophy and dilation with interstitial fibrosis (d). Magnification: ×400 in a and c; ×50 in b and d.
Histologic analysis in Experimental Protocol Ia
α-SMA and Fibronectin Protein in the L-NAME Rats. α-SMA is a good marker of TGF-β1-induced phenotypic change of the cell (29). To determine the tissue localization of α-SMA in the kidney, an immunohistochemical staining was performed. The results are shown in Figure 2. In control rats, α-SMA was positively stained only in the small arteries, but negative in either glomeruli or interstitium. In contrast, α-SMA was markedly expressed in the sclerosing glomeruli and fibrous interstitium of the kidney in L-NAME rats.
Immunohistochemical micrographs of anti-α-smooth muscle actin (anti-α-SMA) antibody on renal sections from control rats (a and b) and L-NAME rats (c and d) at week 12, Experimental Protocol I. An avidin-biotin-diaminobenzidine detection system was used with hematoxylin counter stain. The anti-α-SMA antibody-positive cells were not detected in any glomeruli or in the interstitium of control rats. The α-SMA-positive cells presented in the glomeruli and interstitium of L-NAME rats. Magnification: ×50 in a and c; ×200 in b and d.
The immunofluorescence microscopic examination revealed that FN was basically present in the glomeruli and interstitium of control rats. However, in the sclerotic and fibrotic lesions in L-NAME rats, the FN staining was more prominent in its intensity and the area was wider, as shown in Figure 3.
Immunofluorescence micrographs with anti-fibronectin antibody on renal sections from control rats (a) and L-NAME rats (b) at week 12, Experimental Protocol I. Fibronectin was basically present in both glomeruli and interstitium of control rats. Increased fibronectin quantity was found in the sclerosing glomeruli and in the widened interstitium of L-NAME rats. Magnification: ×400 in a and b.
Renal Cortical mRNA Levels for TGF-β1 and FN. To examine the synthesis of TGF-β1 in the renal cortex, Northern blotting analysis for TGF-β1 mRNA (2.5 kb) was performed on total RNA isolated from the cortical tissues of both groups at weeks 8 and 12. At week 8, the expression of TGF-β1 mRNA transcript was detected in the kidney tissue of L-NAME rats. The level of TGF-β1 mRNA was enhanced by about threefold compared with the control rats. TGF-β1 mRNA in the cortex of L-NAME rats increased progressively at week 12. At week 8, no cortical FN transcripts could be detected in control rats. However, the FN mRNA (8.0 kb) transcript of the L-NAME rat was detected weakly. At week 12, the FN mRNA expression of L-NAME rats was increased by about twofold compared with control rats. Similar results were obtained in three different experiments. Representative results are shown in Figure 4, A and B.
(a) Northern blot analysis of fibronectin mRNA, transforming growth factor-β1 (TGF-β1) mRNA in the cortex at week 8, Experimental Protocol I. Each lane contains 20 μg of total RNA from a single sample from one animal of either control rats or L-NAME rats. The blots were rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. (b) At week 12, Experimental Protocol I. Each lane contains 20 μg of total RNA from a single sample from one animal. The same results were obtained in four different experiments. Arrows indicate the sizes of the major transcripts for fibronectin (8.0 kb), TGF-β1 (2.5 kb), and GAPDH (1.3 kb).
Renin-Angiotensin System. The levels of ACE activities and AngII levels are shown in Table 3. The cortical tissue levels of ACE activities in L-NAME rats were markedly increased after weeks 8 and 12 of treatment. Similarly, the cortical tissue levels of AngII were significantly higher in L-NAME rats compared with control rats. Compared with the control group, serum ACE activity in the L-NAME group did not change.
Renin-angiotensin system in Experimental Protocol Ia
Experimental Protocol II
BW, SBP, UP, and Renal Function. The changes in BW, SBP, UP, and SCr are shown in Table 4. BW at the baseline did not differ significantly among the groups. In L-NAME rats, the gain of BW was lowest at week 12. Coadministration of either imidapril, TCV-116, or hydralazine (Hyd) equally lowered SBP throughout the period, and the levels of SBP in these three groups did not differ from those in control rats. UP at the baseline was stable until week 8 in all groups, but it increased significantly at week 12 in the L-NAME + Hyd group (P < 0.05). SCr at sacrifice in both L-NAME and L-NAME + Hyd was significantly higher than that in the other three groups (P < 0.05), as shown in Table 4.
Clinical parameters in Experimental Protocol IIa
Histologic Changes. The semiquantitative analysis of renal injury in this experiment is shown in Table 5. Interestingly, the coadministration of either imidapril or TCV-116 completely prevented the renal injury induced by chronic L-NAME administration, compared with that in the L-NAME + Hyd group. Despite the similar antihypertensive effect, hydralazine could not ameliorate the L-NAME-induced histologic damage.
Histologic analysis in Experimental Protocol IIa
Renal Cortical mRNA Levels for TGF-β1. The expression of TGF-β1 mRNA transcript in all groups is shown in Figure 5. Similar to Experimental Protocol I, the level of TGF-β1 mRNA in the L-NAME group was enhanced compared with the control rats. However, the levels of TGF-β1 mRNA in both L-NAME + ACEI and L-NAME + AIIA were not enhanced, being similar to the control rats. In contrast, the level of TGF-β1 mRNA in the L-NAME + Hyd was not decreased, compared with the L-NAME rats.
Expression of TGF-β1 mRNA on the cortical tissue of five groups at week 12, Experimental Protocol II. The expression of TGF-β1 mRNA in both L-NAME + angiotensin-converting enzyme inhibitor (ACEI) and L-NAME + angiotensin II type I receptor antagonist (AIIA) were reduced compared with that in the L-NAME group. However, L-NAME + hydralazine (Hyd) was not.
Discussion
The present study demonstrated that the chronic NOS inhibition with L-NAME caused severe glomerulosclerosis and interstitial fibrosis in association with systemic hypertension. These changes were characterized by the increased production of both TGF-β1 and FN. The tissue ACE activity and the AngII level in the renal cortex were upregulated. These histologic injuries and the increased expression of TGF-β1 were equally ameliorated by the coadministration of specific RAS inhibitors, such as ACEI and AIIA, but not by hydralazine. Thus, it was suggested that in the chronic NOS inhibition with L-NAME, nonselective total suppression of NO synthesis stimulated RAS in the tissue level, with a resultant activation of fibrogenic processes.
Baylis et al. (7) demonstrated that the chronically NO-blocked rats by oral administration of L-NAME (L-NAME, 5 mg/dl in drinking water) for 2 mo developed systemic hypertension and renal vasoconstriction with an elevation of intraglomerular BP. They also noted that NO-blocked rats developed proteinuria and glomerular sclerotic injury. Fujihara et al. (8) evaluated the renal effects of NO blockade for 30 d in Munich-Wistar rats, giving 65 mg/dl L-NAME in the drinking water. They found that L-NAME-treated rats presented with marked hypertension, glomerulosclerosis, and renal interstitial fibrosis. In our study, L-NAME (25 mg/dl in drinking water) was given orally for 12 wk in Wistar rats. Because the SBP in L-NAME rats rose to significant levels compared with control rats, the dose of L-NAME administration was sufficient enough to cause tissue injuries. BP and UP increased significantly more in L-NAME rats than in control rats, and glomerulosclerosis and interstitial fibrosis developed.
The activation of RAS in the model of chronic administration of L-NAME was suggested by recent studies. Ribeiro et al. (9) reported that plasma renin activity was markedly elevated in rats that had received L-NAME for more than 1 mo. Administration of losartan, a specific AngII antagonist, largely attenuated the development of systemic hypertension and prevented both renal functional and morphologic deterioration in L-NAME-treated rats. Navarro et al. (10) have shown that the coadministration of ACEI with L-NAME reduced the elevation of BP and proteinuria induced by chronic NOS inhibition. Fujihara et al. (8) found similar results with AngII antagonist. However, it remains unclear what the effects of NOS inhibition on tissue RAS in the renal cortex are, and how it contributes to the renal parenchymal damage in this model. In this study, we examined both ACE activity and AngII levels in the renal cortex and found that they were increased twofold compared with control rats. Qiu et al. (30) reported variable levels of plasma renin activity during chronic L-NAME administration and suggested that the AngII dependence of renal damage is not mediated via changes in circulating AngII. The present study is the first to find that the activation of tissue RAS contributes to glomerulosclerosis and interstitial fibrosis in the chronic L-NAME-administered rat model.
To confirm the involvement of activated tissue RAS in the renal cortex in renal injuries, the effects of the RAS inhibitors on both the renal lesions and the expression of TGF-β1 mRNA were examined in L-NAME-treated rats. The glomerulosclerosis and interstitial fibrosis were markedly reduced by both ACEI and AIIA, and the degrees of amelioration were equal. The increase in the expression of TGF-β1 mRNA was also attenuated. The mechanisms by which ACEI may exert its beneficial effects on parenchymal damage in this model are supposed to be mediated by either the suppression of AngII generation or by the increase in tissue bradykinin levels, or both. Tojo et al. (11) reported that the nitrite production was significantly suppressed from the kidney slices of L-NAME-treated rats, and that ACEI restored the nitrite production. In their study, the diminished immunoreactivity for both the neuronal NOS in the macula densa and the endothelial NOS in the renal vasculature of L-NAME-treated rats recovered with ACEI. Farhy et al. (31) showed that ACEI prevents the neointimal formation after balloon injury in the rat carotid artery and that this effect of ACEI was reduced by more than 50% with the kinin receptor antagonist. They suggested that the beneficial effect of ACEI on the neointimal formation is mediated either by bradykinin per se or through the stimulation of NO synthesis. However, the renal parenchymal damage was effectively abolished by AIIA, which has no effect on bradykinin generation. In this context, the mechanism of the prevention of renal fibrosis by ACEI was caused by the inhibitory effect of AngII generation, and bradykinin might not play a major role in preventing renal tissue damage in chronic NOS inhibition. Coadministration of ACEI, AIIA, and hydralazine equally lowered BP throughout the period, and the levels of SBP in these three groups did not differ from that in control rats. Because hydralazine lowered BP, hydralazine reduced proteinuria and tended to reduce the histologic injury scores at week 12. However, both ACEI and AIIA completely prevented the proteinuria and histologic injury. Hydralazine had no effect on the prevention of renal damage contrary to ACEI and AIIA. Thus, the prevention of renal damage by both ACEI and AIIA was not mediated by changes in systemic hemodynamics by the reduction in arterial BP.
TGF-β is known to be a major regulator in the sclerotic course among many cytokines, participating in the sclerotic course in both the glomerulus and the renal interstitium (32,33,34,35). We have recently shown that TGF-β1 is involved in the glomerulosclerosis and interstitial fibrosis in adriamycin-induced nephropathy in rats (15) and in hypertensive renal injury in Dahl salt-sensitive rats (16). Thus, TGF-β1 is a key fibrogenic cytokine in kidney damage, in which excess amounts of ECM are produced. In the present study, the renal parenchymal injury correlated well with the increased expression of TGF-β mRNA in L-NAME rats, which was also accompanied by the overexpression of both FN mRNA and FN protein. The α-SMA protein was markedly expressed in the sclerosing glomeruli and fibrous interstitium. TGF-β1 is also known to induce myofibroblast modulation by regulating the expression of α-SMA (29). Thus, TGF-β1 plays a major role in renal injuries directly, as well as indirectly by inducing phenotypic modulation in renal parenchymal cells in chronically NOS-inhibited rats.
In summary, severe histologic and biochemical injuries were induced by chronic NOS inhibition with orally administered L-NAME. ACEI and AIIA were equally effective for ameliorating renal injury. In this model, the locally activated RAS in association with TGF-β1 upregulation is a major pathogenetic feature.
Acknowledgments
Acknowledgments
We thank K. Iwaki (Research Institute of Cardiac Surgery, Kyushu University) and H. Noguchi for their excellent technical assistance. We also thank Dr. S. S. Deenitchina for her comments on the manuscript.
Footnotes
This work was presented in part at the 30th Annual Meeting of the American Society of Nephrology, San Antonio, Texas, November 2-5, 1997 (J Am Soc Nephrol 8: 499A, 1997).
American Society of Nephrology
- © 2000 American Society of Nephrology
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