Short-term nitric oxide inhibition induces progressive nephropathy after regression of initial renal injury
http://www.100md.com
《美国生理学杂志》
Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of So Paulo, So Paulo, Brazil
ABSTRACT
Chronic nitric oxide (NO) inhibition and salt overload (HS) promote severe hypertension and renal injury, which regress quickly, although not completely, on treatment withdrawal. We investigated whether renal function and structure remain stable 6 mo after cessation of these treatments. Adult male Munich-Wistar rats were distributed among three groups: HS, receiving 3.1% Na diet; HS+N, receiving HS and the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME; 30 mg·kg–1·day–1 orally); and HS+N+L, receiving HS+N and the ANG II blocker losartan (L; 50 mg·kg–1·day–1 orally). In studies performed after 20 days of treatment (protocol 1), HS+N rats exhibited severe glomerular and systemic hypertension, massive albuminuria, glomerular and interstitial injury, and infiltration by macrophages and cells expressing ANG II. These abnormalities were largely prevented in the HS+N+L group. A second cohort (protocol 2) received HS+N for 20 days, followed by a conventional (0.5% Na) diet and no L-NAME treatment during the subsequent 30 days. At this time, systemic and glomerular pressure, along with parameters of renal injury and inflammation, were still higher than in HS or HS+N+L rats, although differences were much smaller than in protocol 1. Six months after 20-day L-NAME/salt overload treatment was ceased (protocol 3), severe albuminuria, hypertension, and renal injury developed in HS+N rats. Again, losartan prevented most of these changes. We conclude 1) short-term HS+N treatment triggers the autonomous development of progressive glomerulosclerosis; 2) this process may involve activation of the AT1 receptor; and 3) temporary HS+N treatment may represent a new model of slowly progressive chronic nephropathy.
N-L-nitro-arginine methyl ester; kidney glomerulus/physiopathology; type 1 angiotensin receptor; angiotensin II
THE PATHOGENESIS OF CHRONIC nephropathies involves the participation of a host of hemodynamic and inflammatory factors (4, 12, 15, 25, 29, 36), including glomerular hypertension, infiltration by macrophages and other cell types, and the release of inflammatory mediators such as cytokines, growth factors, and, especially, ANG II. Recent clinical and experimental studies have concentrated on the neutralization of the complex cascade of events leading to chronic nephropathies, seeking to detain, and even to reverse, their progression (10, 15, 24, 30).
Several experimental models have been used to address the pathogenesis of progressive nephropathies. Most of these models require complex and laborious procedures, such as surgery or genetic manipulation. A notable exception, in view of its simplicity and reproducibility, is represented by chronic nitric oxide (NO) inhibition, alone or in association with salt overload, which has now been used for over 12 years as a model of hypertension and progressive renal injury (6, 14, 31). Despite its advantages, this model has some drawbacks that limit its use. First, it deals with an artificial setting, not paralleled by usual clinical situations. Second, mortality is high, limiting the modeling of advanced chronic nephropathies. Third, the disease courses with severe and progressive hypertension from its very beginning, a feature that complicates the interpretation of the studies and, once again, fails to reflect the usual clinical experience.
One intriguing and somewhat unexpected feature of this model is that the severe hemodynamic and renal structural consequences of chronic NO inhibition/dietary salt overload regress rapidly, although not completely, on removal of treatment. Hypertension and albuminuria developing after 30 days of treatment with a NO inhibitor and a high-salt diet regress to near normal values after treatments are ceased (5, 14), although some evidence has been reported that this reversibility may be only partial (29). If regression is indeed incomplete, the development of a progressive nephropathy might be expected in the long run. However, long-term follow-up of these animals, which might provide evidence to this effect, has not been performed.
In the present study, partial recovery from hypertension and renal injury was corroborated in rats that had received NO inhibition and a high-salt diet for 20 days. Nevertheless, rats were shown to develop a slowly progressive nephropathy in the following 6 mo. In this manner, we were able to develop a new rat model of renal insufficiency, more akin to human disease, and to obtain additional insight into the mechanisms leading to renal injury in progressive nephropathies.
METHODS
Seventy-three adult male Munich-Wistar rats, obtained from a local facility at the University of So Paulo, and weighing initially 240–260 g, were used in this study. The animals were maintained at 23 ± 1°C on a 12:12-h light-dark cycle, with free access to tap water and food. All rats received a high-salt diet (HS; 3.2% Na, 22% protein, Harland Teklad). All experimental procedures were approved by the local Research Ethics Committee (CAPPesq, process no. 047/00) and developed in strict conformity with our institutional guidelines and with international standards for manipulation and care of laboratory animals.
Experimental Groups and Protocols
Rats were distributed among three groups: HS, receiving HS and no drug treatment; HS+N, receiving HS and the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 30 mg·kg–1·day–1) dissolved in the drinking water; and HS+N+L, receiving HS and L-NAME as in HS+N, as well as losartan potassium (L), 50 mg·kg–1·day–1 dissolved in the drinking water. The day when drug treatments were started was denoted day 0. The three groups of rats were studied according to three distinct experimental protocols, corresponding to three time points relative to day 0.
Protocol 1. In this protocol, six rats from each group were followed for 20 days and examined on the last day of drug treatment (day 20). At this time, 24-h albuminuria (UalbV) was determined by radial immunodiffusion, and the tail-cuff pressure (TCP) was assessed by an indirect method (39). Thereafter, rats underwent renal hemodynamic and morphological studies as described below.
Protocol 2. Here, six rats from the HS group, six from the HS+N group, and seven from the HS+N+L group were studied. On day 20, UalbV and TCP were obtained as in protocol 1. L-NAME treatment and salt overload were then ceased, and rats were maintained on a conventional (0.5% Na) diet until the end of the study. On day 50 (that is, 30 days after treatments had been ceased), UalbV and TCP were determined again, and rats were prepared for renal hemodynamic and morphological studies as described below.
Protocol 3. Eleven rats from the HS group, 14 from the HS+N group, and 11 from the HS+N+L group were included in this protocol. Rats received L-NAME and salt overload during 20 days as in protocols 1 and 2. L-NAME treatment and salt overload were then ceased, and rats received a conventional (0.5% Na) diet until the end of the study. UalbV and TCP were determined on day 20, day 50, and every 30 days thereafter until, and including, day 200 (i.e., 180 days after drug treatments had been ceased). At this time, rats were prepared for morphological studies as described below.
Renal Hemodynamic Studies
Rats studied on days 20 (protocol 1) and 50 (protocol 2) were anesthetized with Inactin (100 mg/kg ip) and prepared for whole kidney and glomerular hemodynamic studies, in which mean arterial pressure (MAP), glomerular filtration rate (GFR), renal plasma flow (RPF), and renal vascular resistance (RVR) were measured. Glomerular hydraulic pressure (PGC) was directly determined by micropuncture of superficial glomeruli. The details of the experimental procedures and analytical techniques utilized in these studies are given elsewhere (12).
Morphological Studies
At the end of each renal hemodynamic experiment (protocols 1 and 2), the kidneys were washed out with saline solution at the MAP and perfusion-fixed in situ, still at the MAP, with Duboscq-Brasil solution. The renal tissue was then prepared for morphological evaluation as described below.
Rats studied at day 200 (protocol 3) were anesthetized with pentobarbital sodium (50 mg/kg). The kidneys were then perfusion-fixed at the measured TCP, and the renal tissue was prepared for morphological analysis as described for the renal hemodynamic studies.
Histological and Histomorphometric Techniques
After fixation, the kidneys were weighed and two midcoronal sections were postfixed in buffered 4% formaldehyde. The renal tissue was then embedded in paraffin using standard sequential techniques and prepared for assessment of glomerular injury and interstitial tissue expansion, as well as for immunohistochemical analysis, as described previously (12). All morphometric evaluations were performed in a blinded manner by a single observer. The frequencies of glomerulosclerosis (GS) and glomerular ischemic collapse (COLL) were evaluated by consecutive examination of at least 300 glomeruli at x400. The percentage of the renal cortical area occupied by interstitial tissue (%INT) was assessed with a point-counting technique (20) in 25 consecutive microscopic fields, at a final magnification of x100 under a 176-point grid.
Immunohistochemical procedures were described in detail previously (12). Macrophages and ANG II-positive cells were detected by an indirect streptavidin-biotin alkaline phosphatase technique. For macrophage detection, a monoclonal mouse anti-rat ED-1 antibody (Serotec, Oxford, UK) was used. ANG II-positive cells were detected by a monoclonal rabbit anti-human ANG II antibody (Peninsula Laboratories, San Carlos, CA). The density of renal interstitial infiltration by macrophages (M) and ANG II-positive cells (ANG IIint) was evaluated in a blinded manner at x250 magnification and expressed as cells per square millimeter. The estimated expression of ANG II in afferent arterioles (ANG IIart) was given as positively stained arterioles/50 examined profiles.
Statistical Analysis
Differences among the three groups were analyzed using one-way ANOVA with pairwise posttest comparisons according to the Newman-Keuls method (37). Because UalbV exhibited a strong non-Gaussian distribution, log transformation of these data was performed before statistical analysis. The Pearson correlation coefficient was calculated to assess linear correlations. For all statistical tests, P values <0.05 were considered significant.
RESULTS
Protocol 1
Renal functional and hemodynamic parameters obtained on day 20 are given in Table 1. No statistically significant difference was observed among groups regarding body or left kidney weights. GFR was reduced by >50% in HS+N rats, whereas concomitant losartan treatment was associated with near normal GFR in HS+N+L rats. RPF followed a similar pattern, although hypoperfusion was only partially prevented in HS+N+L rats, whereas RVR exhibited a reciprocal behavior. PGC was elevated by almost 30 mmHg in the HS+N group. This severe glomerular hypertension was strongly attenuated, but not completely avoided, in HS+N+L rats, in which PGC values were 13 mmHg higher than in HS rats.
Figure 1 shows representative micrographs of the three main modalities of renal parenchymal injury found in this study. GS (Fig. 1A) consisted of the segmental deposition of periodic acid-Schiff-positive material in the mesangial region, leading to glomerular capillary occlusion and, often, to the formation of sinechiae with the parietal layer of Bowman's capsule. Glomerular collapse (Fig. 1B) was defined as a homogeneous shrinkage of the glomerular tuft, accompanied by wrinkling of the basement membrane. In all likelihood, these abnormalities reflect severe glomerular ischemia. Expansion of the interstitial matrix, usually associated with infiltration by mononuclear cells, is represented in Fig. 1C.
Parameters derived from histomorphometric and immunohistochemical observations made in protocol 1 are given in Table 2. The frequency of sclerotic glomeruli (%GS) was negligible in HS rats, increased significantly in the HS+N group, and was not significantly different from HS in the HS+N+L group. A similar pattern was verified regarding the frequency of collapsed glomeruli (%COLL). The fractional interstitial area, %INT, was markedly increased in the HS+N group but remained close to the HS value in the HS+N+L group. The intensity of renal interstitial inflammation, represented by the density of macrophage (M) infiltration at the interstitial area, increased fivefold in HS+N compared with HS rats (P < 0.05), remaining at levels similar to HS in the HS+N+L group.
Renal ANG II was distributed between two major compartments, namely, a vascular (mostly afferent arteriolar) compartment (ANG IIart; Fig. 2A) and an interstitial compartment (ANG IIint; Fig. 2B). Tubular expression of ANG II was comparatively negligible. Of note, ANG IIart was significantly reduced in HS+N compared with HS rats (Table 2), perhaps reflecting progressive expansion of the extracellular volume. ANG IIint more than doubled in the HS+N group, appearing mostly in inflamed interstitial areas, a finding consistent with the notion that ANG II participates in the pathogenesis of chronic progressive nephropathies. No difference was seen between HS and HS+N+L regarding ANG IIart or ANG IIint (Table 2).
PGC exhibited significant positive linear correlation with %COLL, %INT, interstitial M density, and ANG IIint (r = 0.47, P < 0.05). Similarly, all parameters of renal injury correlated significantly with interstitial M density. A significant positive linear correlation was also observed between ANG IIint and both %COLL and %INT.
Protocol 2
Functional and hemodynamic results obtained with protocol 2 (50 days from day 0 and 30 days after treatments were ceased) are shown in Table 3. Most abnormalities revealed by protocol 1 were again observed, although the differences relative to HS were much smaller than observed on day 20. MAP was elevated in HS+N rats and, to a lesser extent, in the HS+N+L group. Similarly, PGC was elevated in the HS+N group, albeit to a lesser extent than verified for protocol 1. Losartan treatment limited glomerular hypertension in the HS+N+L group, although PGC was slightly but significantly elevated in these animals compared with HS. Although no difference in GFR was observed among groups, RPF was significantly lower in HS+N compared with HS or HS+N+L rats. Accordingly, RVR was higher in the HS+N group compared with the other groups. No differences in body weight or left kidney weight were observed with this protocol.
Renal injury and inflammation (Table 4) were much milder at day 50 than observed with protocol 1. No significant difference regarding %GS was seen among the three groups. However, %COLL was higher in HS+N compared with HS rats, whereas no difference was seen between the HS and HS+N+L groups. Similarly, a moderate but significant increase in %INT was observed in HS+N vs. HS or HS+N+L rats. A similar pattern was observed regarding the intensity of macrophage infiltration. ANG IIart was not statistically different among the groups, although there was a trend toward lower values in the HS+N group. ANG IIint was similar between HS+N and HS+N+L rats. In both groups, ANG IIint was significantly higher than in the HS group.
As in protocol 1, significant positive linear correlation was observed between PGC and %GS, %COLL, %INT, M density, and ANG IIint (r = 0.55, P < 0.05). Figure 3 shows the correlations between PGC and either %GS, %COLL, or %INT obtained when data from protocols 1 and 2 were pooled. In addition, %GS and %INT correlated strongly with the density of M infiltration, whereas %INT correlated significantly with ANG IIint (not shown).
Protocol 3
The time course of TCP in rats followed from day 0 to day 200 (180 days after treatments ceased, protocol 3) is shown in Fig. 4. In the HS+N group, TCP rose >60 mmHg from day 0 to day 20, reaching almost 180 mmHg. After L-NAME and HS treatments were ceased, TCP fell markedly but never returned to pretreatment values, plateauing at 150 mmHg, still 40 mmHg above sham levels. In rats receiving HS+N+L treatment, TCP was elevated by 30 mmHg on day 20 and fell rapidly after treatments were ceased, returning to normal levels within 3 mo.
The temporal evolution of albuminuria (UalbV) is represented in Fig. 5. UalbV increased sharply during HS+L-NAME treatment, reaching 170 mg/24 h after 20 days. UalbV fell precipitously, albeit not to normal values, after treatment was ceased. From day 50 until day 200, albuminuria rose steadily, exceeding 50 mg/24 h at the end of the study, a value nearly 10-fold higher than in HS. In the HS+N+L group, UalbV reached a much lower peak value (27 mg/24 h), returning rapidly to normal after treatments were withdrawn.
Severe renal injury and inflammation were evident in HS+N rats at day 200, as shown in Table 5. %GS, %COLL, and %INT were all markedly elevated in the HS+N group compared with HS rats. The intensity of renal M infiltration was also increased in HS+N rats, as was ANG IIint. By contrast, the frequency of ANG II-positive arterioles was lower in the HS+N group compared with the HS group. Reflecting these structural abnormalities, Screat was significantly increased compared with HS rats. No significant differences, regarding either Screat or any parameters of inflammation or structural injury, were found between HS+N+L and HS rats, indicating that concomitant administration of losartan prevented the long-term consequences of HS+L-NAME treatment.
All parameters describing renal injury correlated strongly with the density of M infiltration. In addition, Fig. 6 shows that %GS, %COLL, and %INT each correlated significantly with ANG IIint when observations for all protocols were pooled. These correlations remained statistically significant when data for protocol 3 were analyzed separately.
DISCUSSION
As shown previously (12, 14), short-term treatment with L-NAME and HS (protocol 1) promoted severe hypertension and massive albuminuria, as well as intense renal structural injury, consisting mostly of tuft collapse, interstitial expansion/inflammation, and GS. Tuft collapse was the most conspicuous of these abnormalities, affecting as much as 17% of the glomerular profiles examined. Glomerular collapse was in all likelihood due to chronic ischemia, as suggested by its homogeneous nature, the small size of the affected glomeruli, and the wrinkling of the basement membrane. Glomerular ischemic collapse has been described in association with hypertensive nephrosclerosis (9, 19, 35) and may be partially responsible for the progressive structural damage and loss of renal function observed in hypertensive patients. In addition to tuft collapse, segmental GS was seen in a significant proportion of glomeruli after treatment with L-NAME and HS for 20 days, along with marked expansion of the interstitial area.
All three modalities of renal injury described in the HS+N group had apparently regressed 30 days after interruption of the HS+NAME treatment (protocol 2, day 50), although reversal was neither uniform nor complete, because some of the changes became quantitatively indistinguishable from those observed in the HS or HS+N+L group, while others remained clearly above normal. Despite its incompleteness, regression of injury is consistent with the concept that lesions associated with chronic nephropathies are not inexorably progressive and can be reversed once the underlying cause is removed or a suitable treatment is instituted (10, 24, 30). Regression of GS and interstitial inflammation/expansion is at least theoretically possible through cleavage of the excess extracellular matrix and apoptosis/removal of inflammatory cells (8, 24), at least in those cases in which the basic parenchymal structure has not been seriously compromised (23). On the other hand, the substantial reduction in the frequency of ischemic glomeruli at day 50 may reflect amelioration of afferent arteriolar vasoconstriction due to reduced inflammation and/or proliferation at the vascular walls. It is conceivable that part of this regression may have been only apparent, because more severely damaged glomeruli may have simply undergone disintegration and resorption, thus no longer being counted as such at day 50. Additionally, regression of the interstitial expansion may have reflected the presence of more readily cleaved material such as proteoglycans, rather than collagen. However, some concomitant findings strongly suggest that genuine regression did occur between day 20 and day 50. Albuminuria fell precipitously, indicating substantial amelioration of glomerular injury. Moreover, interstitial inflammation, represented by the density of M infiltration, was also reduced, along with lowering of PGC and a reduction in the frequency of ANG II-positive cells, suggesting that involution of renal damage occurred in association with amelioration of the underlying hemodynamic and inflammatory mechanisms.
Although discontinuation of HS+L-NAME treatment led to substantial regression of renal lesions at day 50 (protocol 2), several signs indicated that a significant degree of damage and dysfunction persisted at this time, because neither TCP nor albuminuria was normalized. The frequency of ischemic glomeruli and the interstitial dimensions also remained significantly increased above control values. In themselves, these findings did not necessarily indicate perpetuation of the renal damage induced by the HS+L-NAME treatment, because complete recovery might require longer than 30 days. However, examination of data obtained in protocol 3 shows that, from day 50 to day 200, a new, more insidious nephropathy developed, with progressive albuminuria and, at the end of the observation period, a much higher frequency of sclerotic and ischemic glomeruli, in addition to much more marked interstitial expansion, than in HS rats. Previous observations by Rodríguez-Iturbe and co-workers (29) showed that rats previously subjected to L-NAME treatment for 1 mo developed salt-sensitive hypertension associated with persistent low-grade inflammation of the renal tissue. Other studies have suggested that subjecting the kidneys to temporary stress not involving NO inhibition can result in subtle renal dysfunction and predisposition to progressive nephropathy in the long run. Anderson and co-workers (3) showed that rats subjected to short-term treatment with puromycin aminonucleoside developed nephrotic albuminuria but showed no glomerular histological abnormalities. After a few weeks, albuminuria receded, but PGC remained elevated; albuminuria then slowly returned to frankly increased levels, and rats were eventually shown to have developed substantial GS. Similarly, Rodríguez-Iturbe and co-workers showed that administration of protein overload (2) or ANG II infusion (33) for only 2 wk was followed by persistent infiltration of the renal interstitium by immune cells and by the development of salt-sensitive hypertension. The present findings extend the reach of those previous observations by indicating that the deleterious effects of short-term NO inhibition are not limited to salt-sensitive hypertension or to subtle renal inflammatory changes and that a peculiar form of slowly progressive nephropathy develops spontaneously in these animals.
Abundant evidence indicates that both intracapillary hypertension and renal inflammation are involved in the pathogenesis of renal injury in the renal ablation model (4, 11, 13). The present study amply corroborates this contention. In protocol 1, PGC was maximal in the HS+N group, the only one in which high-grade renal injury was seen. In addition, the magnitude of glomerular collapse and interstitial expansion correlated significantly with PGC. Conversely, in protocol 2, mild renal disease was associated with modest albeit significant glomerular hypertension in the HS+N group, whereas a significant linear correlation between each parameter of renal injury and PGC was observed. A similar association was observed, in all protocols, between the intensity of renal injury and the density of renal M infiltration, underlining the pathogenic importance of inflammation. Moreover, PGC correlated significantly with interstitial M density, suggesting a pathogenic interaction between hemodynamic and inflammatory factors. Although these associations are far from proving causality, several in vitro observations suggest that glomerular hypertension itself can trigger inflammation, because mechanical stretch has been shown to strongly stimulate the synthesis of a host of inflammatory mediators (1, 7, 22, 32).
In HS rats, ANG II was expressed almost exclusively in afferent arterioles, although less abundantly than in rats receiving diets with conventional salt content (15, 16), probably reflecting the expected participation of ANG II in the excretory response to dietary salt overload. This pattern was completely subverted after 20 days of HS+L-NAME treatment, with ANG II-positive cells now appearing mostly at inflamed areas of the renal interstitium, whereas arteriolar ANG II expression was significantly reduced. This finding, which corroborates previous observations by this laboratory in rats with chronic NO inhibition or nephrectomy (15–17), indicates that interstitial ANG II is involved in the renal inflammatory process associated with these models, rather than with responses to changes in extracellular volume. An attractive possibility is that at least part of this anomalous ANG II production reflects elevations in PGC, because mechanical stress can enhance the release of inflammatory molecules, including ANG II and the AT1 receptor, by cultivated mesangial cells (7, 18). The participation of locally produced ANG II in the pathogenesis of renal injury is further suggested by the finding that the density of ANG II-positive cells followed closely the intensity of albuminuria and the frequency of glomerular and interstitial injury. Collectively, these results are consistent with the view that local activation of the renin-angiotensin system is of paramount importance to the propagation and perpetuation of the numerous chronic inflammatory events that culminate with the development of progressive nephropathy (15, 16, 21, 26, 28, 34, 38).
As shown previously (14, 31), concomitant losartan treatment of rats subjected to the HS+NAME treatment limited systemic hypertension and kept renal injury/inflammation at levels indistinguishable from control. This finding suggests that the long-term structural damage observed in this study resulted from some effect of the temporary HS+NAME treatment, rather than from NO deficiency per se. Given the numerous hemodynamic and/or proinflammatory effects exerted by ANG II, it appears reasonable to suppose that ANG II mediated, at least in part, the early events that culminated in the establishment of a progressive nephropathy. However, it must be noted that the use of antihypertensive agents not directly related to the renin-angiotensin system, such as amlodipine, can also prevent renal injury in NO-deficient rats (27). Irrespective of the exact mechanism underlying its protective effect, administration of losartan in concomitancy with HS+L-NAME largely, although not completely, prevented the chronic nephropathy observed in rats that had received HS+L-NAME treatment alone. This finding strengthens the view that exposure to temporary hemodynamic and/or inflammatory strain can predispose renal tissue to progressive injury, which can be at least partially prevented by early and aggressive pharmacological intervention, as is often observed with human disease.
The characteristics of the nephropathy observed in this study indicate that temporary inhibition of NO synthesis may serve as a suitable new model of chronic progressive nephropathy, which may present several advantages over those currently employed, particularly the renal ablation model: preparation of the rats requires little labor and no surgery, renal disease develops in both kidneys, the accompanying hypertension is only mild, and mortality is low, yet renal insufficiency does develop. This new model may be particularly useful in evaluating the efficacy of therapeutic schemes aimed at retarding or detaining the progression of advanced chronic nephropathies.
In summary, the present observations indicate that renal injury consequent to short-term NO inhibition and salt overload is largely reversible on discontinuation of treatments. However, regression is incomplete, and an insidious but inexorable process of progressive nephropathy ensues, leading to extensive glomerular and interstitial injury, as well as to renal insufficiency, in the long run. Hemodynamic and inflammatory events seem essential to the development of this process, which also involves local activation of the renin-angiotensin system. Short-term inhibition of NO associated with salt overload may constitute a new model of progressive nephropathy.
GRANTS
This work was supported by Grants 95/4710–2 and 98/09569–4 from the State of So Paulo Foundation for Research Support. During these studies, R. Zatz was the recipient of Research Award 326.429/81 from the Brazilian Council of Scientific and Technologic Development.
DISCLOSURES
This study involves no potential conflict of interest whatsoever.
ACKNOWLEDGMENTS
We thank Gláucia Rutigliano Antunes and Luciana Faria de Carvalho for expert technical assistance. Preliminary results of this study were presented at the American Society of Nephrology/International Society of Nephrology World Congress of Nephrology, San Francisco, CA, October 10–17, 2001, and published in abstract form (J Am Soc Nephrol 12: 813A, 2001).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Akai Y, Homma T, Burns KD, Yasuda T, Badr KF, and Harris RC. Mechanical stretch/relaxation of cultured rat mesangial cells induces protooncogenes and cyclooxygenase. Am J Physiol Cell Physiol 267: C482–C490, 1994.
Alvarez V, Quiroz Y, Nava M, Pons H, and Rodriguez-Iturbe B. Overload proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of immune cells. Am J Physiol Renal Physiol 283: F1132–F1141, 2002.
Anderson S, Diamond JR, Karnovsky MJ, and Brenner BM. Mechanisms underlying transition from acute glomerular injury to late glomerular sclerosis in a rat model of nephrotic syndrome. J Clin Invest 82: 1757–1768, 1988.
Anderson S, Meyer TW, Rennke HG, and Brenner BM. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass. J Clin Invest 76: 612–619, 1985.
Arcos MI, Fujihara CK, Sesso A, Prado EBA, Prado MJA, de Nucci G, and Zatz R. Mechanisms of albuminuria in the chronic nitric oxide inhibition model. Am J Physiol Renal Physiol 279: F1060–F1066, 2000.
Baylis C, Mitruka B, and Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest 90: 278–281, 1992.
Becker BN, Yasuda T, Kondo S, Vaikunth S, Homma T, and Harris RC. Mechanical stretch/relaxation stimulates a cellular renin-angiotensin system in cultured rat mesangial cells. Exp Nephrol 6: 57–66, 1998.
Boffa JJ, Lu Y, Placier S, Stefanski A, Dussaule JC, and Chatziantoniou C. Regression of renal vascular and glomerular fibrosis: role of angiotensin II receptor antagonism and matrix metalloproteinases. J Am Soc Nephrol 14: 1132–1144, 2003.
Bonsib SM. Focal-segmental glomerulosclerosis. The relationship between tubular atrophy and segmental sclerosis. Am J Clin Pathol 111: 343–348, 1999.
Fioretto P, Steffes MW, Sutherland DE, Goetz FC, and Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 339: 69–75, 1998.
Floege J, Alpers CE, Burns MW, Pritzl P, Gordon K, Couser WG, and Johnson RJ. Glomerular cells, extracellular matrix accumulation, and the development of glomerulosclerosis in the remnant kidney model. Lab Invest 66: 485–497, 1992.
Fujihara CK, Malheiros DMAC, Noronha IL, de Nucci G, and Zatz R. Mycophenolate mofetil reduces renal injury in the chronic nitric oxide synthase inhibition model. Hypertension 37: 170–175, 2001.
Fujihara CK, Malheiros DMAC, Zatz R, and Noronha IL. Mycophenolate mofetil attenuates renal injury in the rat remnant kidney. Kidney Int 54: 1510–1519, 1998.
Fujihara CK, Michellazzo SM, de Nucci G, and Zatz R. Sodium excess aggravates hypertension and renal parenchymal injury in rats with chronic NO inhibition. Am J Physiol Renal Fluid Electrolyte Physiol 266: F697–F705, 1994.
Fujihara CK, Velho M, Malheiros DMAC, and Zatz R. An extremely high dose of losartan affords superior renoprotection in the remnant model. Kidney Int 67: 1913–1924, 2005.
Goncalves AR, Fujihara CK, Mattar AL, Malheiros DMAC, Noronha IL, de Nucci G, and Zatz R. Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory. Am J Physiol Renal Physiol 286: F945–F954, 2004.
Graciano ML, Cavaglieri RdC, Delle H, Dominguez WV, Casarini DE, Malheiros DMAC, and Noronha IL. Intrarenal renin-angiotensin system is upregulated in experimental model of progressive renal disease induced by chronic inhibition of nitric oxide synthesis. J Am Soc Nephrol 15: 1805–1815, 2004.
Hirakata M, Kaname S, Chung UG, Joki N, Hori Y, Noda M, Takuwa Y, Okazaki T, Fujita T, Katoh T, and Kurokawa K. Tyrosine kinase dependent expression of TGF- induced by stretch in mesangial cells. Kidney Int 51: 1028–1036, 1997.
Hughson MD, Johnson K, Young RJ, Hoy WE, and Bertram JF. Glomerular size and glomerulosclerosis: relationships to disease categories, glomerular solidification, and ischemic obsolescence. Am J Kidney Dis 39: 679–688, 2002.
Jepsen FL and Mortensen PB. Interstitial fibrosis of the renal cortex in minimal change lesion and its correlation with renal function. A quantitative study. Virchows Arch A Pathol Anat Histol 383: 265–270, 1979.
Kagami S, Border WA, Miller DE, and Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 93: 2431–2437, 1994.
Krepinsky JC, Ingram AJ, Tang D, Wu D, Liu L, and Scholey JW. Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation. J Am Soc Nephrol 14: 2790–2800, 2003.
Kriz W and LeHir M. Pathways to nephron loss starting from glomerular diseases-insights from animal models. Kidney Int 67: 404–419, 2005.
Ma LJ, Nakamura S, Aldigier JC, Rossini M, Yang H, Liang X, Nakamura I, Marcantoni C, and Fogo AB. Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1. J Am Soc Nephrol 16: 966–976, 2005.
Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, Schneider H, Ruiz-Ortega M, and Egido J. NF-B activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol Dial Transplant 19: 2505–2512, 2004.
Nataraj C, Oliverio MI, Mannon RB, Mannon PJ, Audoly LP, Amuchastegui CS, Ruiz P, Smithies O, and Coffman TM. Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. J Clin Invest 104: 1693–1701, 1999.
Navarro-Cid J, Maeso R, Rodrigo E, Munoz-Garcia R, Ruilope LM, Lahera V, and Cachofeiro V. Renal and vascular consequences of the chronic nitric oxide synthase inhibition. Effects of antihypertensive drugs. Am J Hypertens 9: 1077–1083, 1996.
Nishiyama A, Yoshizumi M, Rahman M, Kobori H, Seth DM, Miyatake A, Zhang GX, Yao L, Hitomi H, Shokoji T, Kiyomoto H, Kimura S, Tamaki T, Kohno M, and Abe Y. Effects of AT1 receptor blockade on renal injury and mitogen-activated protein activity in Dahl salt-sensitive rats. Kidney Int 65: 972–981, 2004.
Quiroz Y, Pons H, Gordon KL, Rincon J, Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, Johnson RJ, and Rodriguez-Iturbe B. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition. Am J Physiol Renal Physiol 281: F38–F47, 2001.
Remuzzi A, Gagliardini E, Donadoni C, Fassi A, Sangalli F, Lepre MS, Remuzzi G, and Benigni A. Effect of angiotensin II antagonism on the regression of kidney disease in the rat. Kidney Int 62: 885–894, 2002.
Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, and Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 20: 298–303, 1992.
Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, and Narins RG. Cyclic stretching force selectively up-regulates transforming growth factor- isoforms in cultured rat mesangial cells. Am J Pathol 148: 1915–1923, 1996.
Rodriguez-Iturbe B, Pons H, Quiroz Y, Gordon K, Rincon J, Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, and Johnson RJ. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from angiotensin II exposure. Kidney Int 59: 2222–2232, 2001.
Ruiz-Ortega M, Bustos C, Hernandez-Presa MA, Lorenzo O, Plaza JJ, and Egido J. Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-B activation and monocyte chemoattractant protein-1 synthesis. J Immunol 161: 430–439, 1998.
Takebayashi S, Kiyoshi Y, Hisano S, Uesugi N, Sasatomi Y, Meng J, and Sakata N. Benign nephrosclerosis: incidence, morphology and prognosis. Clin Nephrol 55: 349–356, 2001.
Utimura R, Fujihara CK, Mattar AL, Malheiros DMAC, Noronha IL, and Zatz R. Mycophenolate mofetil prevents the development of glomerular injury in experimental diabetes. Kidney Int 63: 209–216, 2003.
Wallenstein S, Zucker CL, and Fleiss JL. Some statistical methods useful in circulation research. Circ Res 47: 1–9, 1980.
Wu LL, Cox A, Roe CJ, Dziadek M, Cooper ME, and Gilbert RE. Transforming growth factor 1 and renal injury following subtotal nephrectomy in the rat: role of the renin-angiotensin system. Kidney Int 51: 1553–1567, 1997.
Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 40: 198–201, 1990.(Clarice K. Fujihara, Claudia R. Sena, De)
ABSTRACT
Chronic nitric oxide (NO) inhibition and salt overload (HS) promote severe hypertension and renal injury, which regress quickly, although not completely, on treatment withdrawal. We investigated whether renal function and structure remain stable 6 mo after cessation of these treatments. Adult male Munich-Wistar rats were distributed among three groups: HS, receiving 3.1% Na diet; HS+N, receiving HS and the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME; 30 mg·kg–1·day–1 orally); and HS+N+L, receiving HS+N and the ANG II blocker losartan (L; 50 mg·kg–1·day–1 orally). In studies performed after 20 days of treatment (protocol 1), HS+N rats exhibited severe glomerular and systemic hypertension, massive albuminuria, glomerular and interstitial injury, and infiltration by macrophages and cells expressing ANG II. These abnormalities were largely prevented in the HS+N+L group. A second cohort (protocol 2) received HS+N for 20 days, followed by a conventional (0.5% Na) diet and no L-NAME treatment during the subsequent 30 days. At this time, systemic and glomerular pressure, along with parameters of renal injury and inflammation, were still higher than in HS or HS+N+L rats, although differences were much smaller than in protocol 1. Six months after 20-day L-NAME/salt overload treatment was ceased (protocol 3), severe albuminuria, hypertension, and renal injury developed in HS+N rats. Again, losartan prevented most of these changes. We conclude 1) short-term HS+N treatment triggers the autonomous development of progressive glomerulosclerosis; 2) this process may involve activation of the AT1 receptor; and 3) temporary HS+N treatment may represent a new model of slowly progressive chronic nephropathy.
N-L-nitro-arginine methyl ester; kidney glomerulus/physiopathology; type 1 angiotensin receptor; angiotensin II
THE PATHOGENESIS OF CHRONIC nephropathies involves the participation of a host of hemodynamic and inflammatory factors (4, 12, 15, 25, 29, 36), including glomerular hypertension, infiltration by macrophages and other cell types, and the release of inflammatory mediators such as cytokines, growth factors, and, especially, ANG II. Recent clinical and experimental studies have concentrated on the neutralization of the complex cascade of events leading to chronic nephropathies, seeking to detain, and even to reverse, their progression (10, 15, 24, 30).
Several experimental models have been used to address the pathogenesis of progressive nephropathies. Most of these models require complex and laborious procedures, such as surgery or genetic manipulation. A notable exception, in view of its simplicity and reproducibility, is represented by chronic nitric oxide (NO) inhibition, alone or in association with salt overload, which has now been used for over 12 years as a model of hypertension and progressive renal injury (6, 14, 31). Despite its advantages, this model has some drawbacks that limit its use. First, it deals with an artificial setting, not paralleled by usual clinical situations. Second, mortality is high, limiting the modeling of advanced chronic nephropathies. Third, the disease courses with severe and progressive hypertension from its very beginning, a feature that complicates the interpretation of the studies and, once again, fails to reflect the usual clinical experience.
One intriguing and somewhat unexpected feature of this model is that the severe hemodynamic and renal structural consequences of chronic NO inhibition/dietary salt overload regress rapidly, although not completely, on removal of treatment. Hypertension and albuminuria developing after 30 days of treatment with a NO inhibitor and a high-salt diet regress to near normal values after treatments are ceased (5, 14), although some evidence has been reported that this reversibility may be only partial (29). If regression is indeed incomplete, the development of a progressive nephropathy might be expected in the long run. However, long-term follow-up of these animals, which might provide evidence to this effect, has not been performed.
In the present study, partial recovery from hypertension and renal injury was corroborated in rats that had received NO inhibition and a high-salt diet for 20 days. Nevertheless, rats were shown to develop a slowly progressive nephropathy in the following 6 mo. In this manner, we were able to develop a new rat model of renal insufficiency, more akin to human disease, and to obtain additional insight into the mechanisms leading to renal injury in progressive nephropathies.
METHODS
Seventy-three adult male Munich-Wistar rats, obtained from a local facility at the University of So Paulo, and weighing initially 240–260 g, were used in this study. The animals were maintained at 23 ± 1°C on a 12:12-h light-dark cycle, with free access to tap water and food. All rats received a high-salt diet (HS; 3.2% Na, 22% protein, Harland Teklad). All experimental procedures were approved by the local Research Ethics Committee (CAPPesq, process no. 047/00) and developed in strict conformity with our institutional guidelines and with international standards for manipulation and care of laboratory animals.
Experimental Groups and Protocols
Rats were distributed among three groups: HS, receiving HS and no drug treatment; HS+N, receiving HS and the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 30 mg·kg–1·day–1) dissolved in the drinking water; and HS+N+L, receiving HS and L-NAME as in HS+N, as well as losartan potassium (L), 50 mg·kg–1·day–1 dissolved in the drinking water. The day when drug treatments were started was denoted day 0. The three groups of rats were studied according to three distinct experimental protocols, corresponding to three time points relative to day 0.
Protocol 1. In this protocol, six rats from each group were followed for 20 days and examined on the last day of drug treatment (day 20). At this time, 24-h albuminuria (UalbV) was determined by radial immunodiffusion, and the tail-cuff pressure (TCP) was assessed by an indirect method (39). Thereafter, rats underwent renal hemodynamic and morphological studies as described below.
Protocol 2. Here, six rats from the HS group, six from the HS+N group, and seven from the HS+N+L group were studied. On day 20, UalbV and TCP were obtained as in protocol 1. L-NAME treatment and salt overload were then ceased, and rats were maintained on a conventional (0.5% Na) diet until the end of the study. On day 50 (that is, 30 days after treatments had been ceased), UalbV and TCP were determined again, and rats were prepared for renal hemodynamic and morphological studies as described below.
Protocol 3. Eleven rats from the HS group, 14 from the HS+N group, and 11 from the HS+N+L group were included in this protocol. Rats received L-NAME and salt overload during 20 days as in protocols 1 and 2. L-NAME treatment and salt overload were then ceased, and rats received a conventional (0.5% Na) diet until the end of the study. UalbV and TCP were determined on day 20, day 50, and every 30 days thereafter until, and including, day 200 (i.e., 180 days after drug treatments had been ceased). At this time, rats were prepared for morphological studies as described below.
Renal Hemodynamic Studies
Rats studied on days 20 (protocol 1) and 50 (protocol 2) were anesthetized with Inactin (100 mg/kg ip) and prepared for whole kidney and glomerular hemodynamic studies, in which mean arterial pressure (MAP), glomerular filtration rate (GFR), renal plasma flow (RPF), and renal vascular resistance (RVR) were measured. Glomerular hydraulic pressure (PGC) was directly determined by micropuncture of superficial glomeruli. The details of the experimental procedures and analytical techniques utilized in these studies are given elsewhere (12).
Morphological Studies
At the end of each renal hemodynamic experiment (protocols 1 and 2), the kidneys were washed out with saline solution at the MAP and perfusion-fixed in situ, still at the MAP, with Duboscq-Brasil solution. The renal tissue was then prepared for morphological evaluation as described below.
Rats studied at day 200 (protocol 3) were anesthetized with pentobarbital sodium (50 mg/kg). The kidneys were then perfusion-fixed at the measured TCP, and the renal tissue was prepared for morphological analysis as described for the renal hemodynamic studies.
Histological and Histomorphometric Techniques
After fixation, the kidneys were weighed and two midcoronal sections were postfixed in buffered 4% formaldehyde. The renal tissue was then embedded in paraffin using standard sequential techniques and prepared for assessment of glomerular injury and interstitial tissue expansion, as well as for immunohistochemical analysis, as described previously (12). All morphometric evaluations were performed in a blinded manner by a single observer. The frequencies of glomerulosclerosis (GS) and glomerular ischemic collapse (COLL) were evaluated by consecutive examination of at least 300 glomeruli at x400. The percentage of the renal cortical area occupied by interstitial tissue (%INT) was assessed with a point-counting technique (20) in 25 consecutive microscopic fields, at a final magnification of x100 under a 176-point grid.
Immunohistochemical procedures were described in detail previously (12). Macrophages and ANG II-positive cells were detected by an indirect streptavidin-biotin alkaline phosphatase technique. For macrophage detection, a monoclonal mouse anti-rat ED-1 antibody (Serotec, Oxford, UK) was used. ANG II-positive cells were detected by a monoclonal rabbit anti-human ANG II antibody (Peninsula Laboratories, San Carlos, CA). The density of renal interstitial infiltration by macrophages (M) and ANG II-positive cells (ANG IIint) was evaluated in a blinded manner at x250 magnification and expressed as cells per square millimeter. The estimated expression of ANG II in afferent arterioles (ANG IIart) was given as positively stained arterioles/50 examined profiles.
Statistical Analysis
Differences among the three groups were analyzed using one-way ANOVA with pairwise posttest comparisons according to the Newman-Keuls method (37). Because UalbV exhibited a strong non-Gaussian distribution, log transformation of these data was performed before statistical analysis. The Pearson correlation coefficient was calculated to assess linear correlations. For all statistical tests, P values <0.05 were considered significant.
RESULTS
Protocol 1
Renal functional and hemodynamic parameters obtained on day 20 are given in Table 1. No statistically significant difference was observed among groups regarding body or left kidney weights. GFR was reduced by >50% in HS+N rats, whereas concomitant losartan treatment was associated with near normal GFR in HS+N+L rats. RPF followed a similar pattern, although hypoperfusion was only partially prevented in HS+N+L rats, whereas RVR exhibited a reciprocal behavior. PGC was elevated by almost 30 mmHg in the HS+N group. This severe glomerular hypertension was strongly attenuated, but not completely avoided, in HS+N+L rats, in which PGC values were 13 mmHg higher than in HS rats.
Figure 1 shows representative micrographs of the three main modalities of renal parenchymal injury found in this study. GS (Fig. 1A) consisted of the segmental deposition of periodic acid-Schiff-positive material in the mesangial region, leading to glomerular capillary occlusion and, often, to the formation of sinechiae with the parietal layer of Bowman's capsule. Glomerular collapse (Fig. 1B) was defined as a homogeneous shrinkage of the glomerular tuft, accompanied by wrinkling of the basement membrane. In all likelihood, these abnormalities reflect severe glomerular ischemia. Expansion of the interstitial matrix, usually associated with infiltration by mononuclear cells, is represented in Fig. 1C.
Parameters derived from histomorphometric and immunohistochemical observations made in protocol 1 are given in Table 2. The frequency of sclerotic glomeruli (%GS) was negligible in HS rats, increased significantly in the HS+N group, and was not significantly different from HS in the HS+N+L group. A similar pattern was verified regarding the frequency of collapsed glomeruli (%COLL). The fractional interstitial area, %INT, was markedly increased in the HS+N group but remained close to the HS value in the HS+N+L group. The intensity of renal interstitial inflammation, represented by the density of macrophage (M) infiltration at the interstitial area, increased fivefold in HS+N compared with HS rats (P < 0.05), remaining at levels similar to HS in the HS+N+L group.
Renal ANG II was distributed between two major compartments, namely, a vascular (mostly afferent arteriolar) compartment (ANG IIart; Fig. 2A) and an interstitial compartment (ANG IIint; Fig. 2B). Tubular expression of ANG II was comparatively negligible. Of note, ANG IIart was significantly reduced in HS+N compared with HS rats (Table 2), perhaps reflecting progressive expansion of the extracellular volume. ANG IIint more than doubled in the HS+N group, appearing mostly in inflamed interstitial areas, a finding consistent with the notion that ANG II participates in the pathogenesis of chronic progressive nephropathies. No difference was seen between HS and HS+N+L regarding ANG IIart or ANG IIint (Table 2).
PGC exhibited significant positive linear correlation with %COLL, %INT, interstitial M density, and ANG IIint (r = 0.47, P < 0.05). Similarly, all parameters of renal injury correlated significantly with interstitial M density. A significant positive linear correlation was also observed between ANG IIint and both %COLL and %INT.
Protocol 2
Functional and hemodynamic results obtained with protocol 2 (50 days from day 0 and 30 days after treatments were ceased) are shown in Table 3. Most abnormalities revealed by protocol 1 were again observed, although the differences relative to HS were much smaller than observed on day 20. MAP was elevated in HS+N rats and, to a lesser extent, in the HS+N+L group. Similarly, PGC was elevated in the HS+N group, albeit to a lesser extent than verified for protocol 1. Losartan treatment limited glomerular hypertension in the HS+N+L group, although PGC was slightly but significantly elevated in these animals compared with HS. Although no difference in GFR was observed among groups, RPF was significantly lower in HS+N compared with HS or HS+N+L rats. Accordingly, RVR was higher in the HS+N group compared with the other groups. No differences in body weight or left kidney weight were observed with this protocol.
Renal injury and inflammation (Table 4) were much milder at day 50 than observed with protocol 1. No significant difference regarding %GS was seen among the three groups. However, %COLL was higher in HS+N compared with HS rats, whereas no difference was seen between the HS and HS+N+L groups. Similarly, a moderate but significant increase in %INT was observed in HS+N vs. HS or HS+N+L rats. A similar pattern was observed regarding the intensity of macrophage infiltration. ANG IIart was not statistically different among the groups, although there was a trend toward lower values in the HS+N group. ANG IIint was similar between HS+N and HS+N+L rats. In both groups, ANG IIint was significantly higher than in the HS group.
As in protocol 1, significant positive linear correlation was observed between PGC and %GS, %COLL, %INT, M density, and ANG IIint (r = 0.55, P < 0.05). Figure 3 shows the correlations between PGC and either %GS, %COLL, or %INT obtained when data from protocols 1 and 2 were pooled. In addition, %GS and %INT correlated strongly with the density of M infiltration, whereas %INT correlated significantly with ANG IIint (not shown).
Protocol 3
The time course of TCP in rats followed from day 0 to day 200 (180 days after treatments ceased, protocol 3) is shown in Fig. 4. In the HS+N group, TCP rose >60 mmHg from day 0 to day 20, reaching almost 180 mmHg. After L-NAME and HS treatments were ceased, TCP fell markedly but never returned to pretreatment values, plateauing at 150 mmHg, still 40 mmHg above sham levels. In rats receiving HS+N+L treatment, TCP was elevated by 30 mmHg on day 20 and fell rapidly after treatments were ceased, returning to normal levels within 3 mo.
The temporal evolution of albuminuria (UalbV) is represented in Fig. 5. UalbV increased sharply during HS+L-NAME treatment, reaching 170 mg/24 h after 20 days. UalbV fell precipitously, albeit not to normal values, after treatment was ceased. From day 50 until day 200, albuminuria rose steadily, exceeding 50 mg/24 h at the end of the study, a value nearly 10-fold higher than in HS. In the HS+N+L group, UalbV reached a much lower peak value (27 mg/24 h), returning rapidly to normal after treatments were withdrawn.
Severe renal injury and inflammation were evident in HS+N rats at day 200, as shown in Table 5. %GS, %COLL, and %INT were all markedly elevated in the HS+N group compared with HS rats. The intensity of renal M infiltration was also increased in HS+N rats, as was ANG IIint. By contrast, the frequency of ANG II-positive arterioles was lower in the HS+N group compared with the HS group. Reflecting these structural abnormalities, Screat was significantly increased compared with HS rats. No significant differences, regarding either Screat or any parameters of inflammation or structural injury, were found between HS+N+L and HS rats, indicating that concomitant administration of losartan prevented the long-term consequences of HS+L-NAME treatment.
All parameters describing renal injury correlated strongly with the density of M infiltration. In addition, Fig. 6 shows that %GS, %COLL, and %INT each correlated significantly with ANG IIint when observations for all protocols were pooled. These correlations remained statistically significant when data for protocol 3 were analyzed separately.
DISCUSSION
As shown previously (12, 14), short-term treatment with L-NAME and HS (protocol 1) promoted severe hypertension and massive albuminuria, as well as intense renal structural injury, consisting mostly of tuft collapse, interstitial expansion/inflammation, and GS. Tuft collapse was the most conspicuous of these abnormalities, affecting as much as 17% of the glomerular profiles examined. Glomerular collapse was in all likelihood due to chronic ischemia, as suggested by its homogeneous nature, the small size of the affected glomeruli, and the wrinkling of the basement membrane. Glomerular ischemic collapse has been described in association with hypertensive nephrosclerosis (9, 19, 35) and may be partially responsible for the progressive structural damage and loss of renal function observed in hypertensive patients. In addition to tuft collapse, segmental GS was seen in a significant proportion of glomeruli after treatment with L-NAME and HS for 20 days, along with marked expansion of the interstitial area.
All three modalities of renal injury described in the HS+N group had apparently regressed 30 days after interruption of the HS+NAME treatment (protocol 2, day 50), although reversal was neither uniform nor complete, because some of the changes became quantitatively indistinguishable from those observed in the HS or HS+N+L group, while others remained clearly above normal. Despite its incompleteness, regression of injury is consistent with the concept that lesions associated with chronic nephropathies are not inexorably progressive and can be reversed once the underlying cause is removed or a suitable treatment is instituted (10, 24, 30). Regression of GS and interstitial inflammation/expansion is at least theoretically possible through cleavage of the excess extracellular matrix and apoptosis/removal of inflammatory cells (8, 24), at least in those cases in which the basic parenchymal structure has not been seriously compromised (23). On the other hand, the substantial reduction in the frequency of ischemic glomeruli at day 50 may reflect amelioration of afferent arteriolar vasoconstriction due to reduced inflammation and/or proliferation at the vascular walls. It is conceivable that part of this regression may have been only apparent, because more severely damaged glomeruli may have simply undergone disintegration and resorption, thus no longer being counted as such at day 50. Additionally, regression of the interstitial expansion may have reflected the presence of more readily cleaved material such as proteoglycans, rather than collagen. However, some concomitant findings strongly suggest that genuine regression did occur between day 20 and day 50. Albuminuria fell precipitously, indicating substantial amelioration of glomerular injury. Moreover, interstitial inflammation, represented by the density of M infiltration, was also reduced, along with lowering of PGC and a reduction in the frequency of ANG II-positive cells, suggesting that involution of renal damage occurred in association with amelioration of the underlying hemodynamic and inflammatory mechanisms.
Although discontinuation of HS+L-NAME treatment led to substantial regression of renal lesions at day 50 (protocol 2), several signs indicated that a significant degree of damage and dysfunction persisted at this time, because neither TCP nor albuminuria was normalized. The frequency of ischemic glomeruli and the interstitial dimensions also remained significantly increased above control values. In themselves, these findings did not necessarily indicate perpetuation of the renal damage induced by the HS+L-NAME treatment, because complete recovery might require longer than 30 days. However, examination of data obtained in protocol 3 shows that, from day 50 to day 200, a new, more insidious nephropathy developed, with progressive albuminuria and, at the end of the observation period, a much higher frequency of sclerotic and ischemic glomeruli, in addition to much more marked interstitial expansion, than in HS rats. Previous observations by Rodríguez-Iturbe and co-workers (29) showed that rats previously subjected to L-NAME treatment for 1 mo developed salt-sensitive hypertension associated with persistent low-grade inflammation of the renal tissue. Other studies have suggested that subjecting the kidneys to temporary stress not involving NO inhibition can result in subtle renal dysfunction and predisposition to progressive nephropathy in the long run. Anderson and co-workers (3) showed that rats subjected to short-term treatment with puromycin aminonucleoside developed nephrotic albuminuria but showed no glomerular histological abnormalities. After a few weeks, albuminuria receded, but PGC remained elevated; albuminuria then slowly returned to frankly increased levels, and rats were eventually shown to have developed substantial GS. Similarly, Rodríguez-Iturbe and co-workers showed that administration of protein overload (2) or ANG II infusion (33) for only 2 wk was followed by persistent infiltration of the renal interstitium by immune cells and by the development of salt-sensitive hypertension. The present findings extend the reach of those previous observations by indicating that the deleterious effects of short-term NO inhibition are not limited to salt-sensitive hypertension or to subtle renal inflammatory changes and that a peculiar form of slowly progressive nephropathy develops spontaneously in these animals.
Abundant evidence indicates that both intracapillary hypertension and renal inflammation are involved in the pathogenesis of renal injury in the renal ablation model (4, 11, 13). The present study amply corroborates this contention. In protocol 1, PGC was maximal in the HS+N group, the only one in which high-grade renal injury was seen. In addition, the magnitude of glomerular collapse and interstitial expansion correlated significantly with PGC. Conversely, in protocol 2, mild renal disease was associated with modest albeit significant glomerular hypertension in the HS+N group, whereas a significant linear correlation between each parameter of renal injury and PGC was observed. A similar association was observed, in all protocols, between the intensity of renal injury and the density of renal M infiltration, underlining the pathogenic importance of inflammation. Moreover, PGC correlated significantly with interstitial M density, suggesting a pathogenic interaction between hemodynamic and inflammatory factors. Although these associations are far from proving causality, several in vitro observations suggest that glomerular hypertension itself can trigger inflammation, because mechanical stretch has been shown to strongly stimulate the synthesis of a host of inflammatory mediators (1, 7, 22, 32).
In HS rats, ANG II was expressed almost exclusively in afferent arterioles, although less abundantly than in rats receiving diets with conventional salt content (15, 16), probably reflecting the expected participation of ANG II in the excretory response to dietary salt overload. This pattern was completely subverted after 20 days of HS+L-NAME treatment, with ANG II-positive cells now appearing mostly at inflamed areas of the renal interstitium, whereas arteriolar ANG II expression was significantly reduced. This finding, which corroborates previous observations by this laboratory in rats with chronic NO inhibition or nephrectomy (15–17), indicates that interstitial ANG II is involved in the renal inflammatory process associated with these models, rather than with responses to changes in extracellular volume. An attractive possibility is that at least part of this anomalous ANG II production reflects elevations in PGC, because mechanical stress can enhance the release of inflammatory molecules, including ANG II and the AT1 receptor, by cultivated mesangial cells (7, 18). The participation of locally produced ANG II in the pathogenesis of renal injury is further suggested by the finding that the density of ANG II-positive cells followed closely the intensity of albuminuria and the frequency of glomerular and interstitial injury. Collectively, these results are consistent with the view that local activation of the renin-angiotensin system is of paramount importance to the propagation and perpetuation of the numerous chronic inflammatory events that culminate with the development of progressive nephropathy (15, 16, 21, 26, 28, 34, 38).
As shown previously (14, 31), concomitant losartan treatment of rats subjected to the HS+NAME treatment limited systemic hypertension and kept renal injury/inflammation at levels indistinguishable from control. This finding suggests that the long-term structural damage observed in this study resulted from some effect of the temporary HS+NAME treatment, rather than from NO deficiency per se. Given the numerous hemodynamic and/or proinflammatory effects exerted by ANG II, it appears reasonable to suppose that ANG II mediated, at least in part, the early events that culminated in the establishment of a progressive nephropathy. However, it must be noted that the use of antihypertensive agents not directly related to the renin-angiotensin system, such as amlodipine, can also prevent renal injury in NO-deficient rats (27). Irrespective of the exact mechanism underlying its protective effect, administration of losartan in concomitancy with HS+L-NAME largely, although not completely, prevented the chronic nephropathy observed in rats that had received HS+L-NAME treatment alone. This finding strengthens the view that exposure to temporary hemodynamic and/or inflammatory strain can predispose renal tissue to progressive injury, which can be at least partially prevented by early and aggressive pharmacological intervention, as is often observed with human disease.
The characteristics of the nephropathy observed in this study indicate that temporary inhibition of NO synthesis may serve as a suitable new model of chronic progressive nephropathy, which may present several advantages over those currently employed, particularly the renal ablation model: preparation of the rats requires little labor and no surgery, renal disease develops in both kidneys, the accompanying hypertension is only mild, and mortality is low, yet renal insufficiency does develop. This new model may be particularly useful in evaluating the efficacy of therapeutic schemes aimed at retarding or detaining the progression of advanced chronic nephropathies.
In summary, the present observations indicate that renal injury consequent to short-term NO inhibition and salt overload is largely reversible on discontinuation of treatments. However, regression is incomplete, and an insidious but inexorable process of progressive nephropathy ensues, leading to extensive glomerular and interstitial injury, as well as to renal insufficiency, in the long run. Hemodynamic and inflammatory events seem essential to the development of this process, which also involves local activation of the renin-angiotensin system. Short-term inhibition of NO associated with salt overload may constitute a new model of progressive nephropathy.
GRANTS
This work was supported by Grants 95/4710–2 and 98/09569–4 from the State of So Paulo Foundation for Research Support. During these studies, R. Zatz was the recipient of Research Award 326.429/81 from the Brazilian Council of Scientific and Technologic Development.
DISCLOSURES
This study involves no potential conflict of interest whatsoever.
ACKNOWLEDGMENTS
We thank Gláucia Rutigliano Antunes and Luciana Faria de Carvalho for expert technical assistance. Preliminary results of this study were presented at the American Society of Nephrology/International Society of Nephrology World Congress of Nephrology, San Francisco, CA, October 10–17, 2001, and published in abstract form (J Am Soc Nephrol 12: 813A, 2001).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Akai Y, Homma T, Burns KD, Yasuda T, Badr KF, and Harris RC. Mechanical stretch/relaxation of cultured rat mesangial cells induces protooncogenes and cyclooxygenase. Am J Physiol Cell Physiol 267: C482–C490, 1994.
Alvarez V, Quiroz Y, Nava M, Pons H, and Rodriguez-Iturbe B. Overload proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of immune cells. Am J Physiol Renal Physiol 283: F1132–F1141, 2002.
Anderson S, Diamond JR, Karnovsky MJ, and Brenner BM. Mechanisms underlying transition from acute glomerular injury to late glomerular sclerosis in a rat model of nephrotic syndrome. J Clin Invest 82: 1757–1768, 1988.
Anderson S, Meyer TW, Rennke HG, and Brenner BM. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass. J Clin Invest 76: 612–619, 1985.
Arcos MI, Fujihara CK, Sesso A, Prado EBA, Prado MJA, de Nucci G, and Zatz R. Mechanisms of albuminuria in the chronic nitric oxide inhibition model. Am J Physiol Renal Physiol 279: F1060–F1066, 2000.
Baylis C, Mitruka B, and Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest 90: 278–281, 1992.
Becker BN, Yasuda T, Kondo S, Vaikunth S, Homma T, and Harris RC. Mechanical stretch/relaxation stimulates a cellular renin-angiotensin system in cultured rat mesangial cells. Exp Nephrol 6: 57–66, 1998.
Boffa JJ, Lu Y, Placier S, Stefanski A, Dussaule JC, and Chatziantoniou C. Regression of renal vascular and glomerular fibrosis: role of angiotensin II receptor antagonism and matrix metalloproteinases. J Am Soc Nephrol 14: 1132–1144, 2003.
Bonsib SM. Focal-segmental glomerulosclerosis. The relationship between tubular atrophy and segmental sclerosis. Am J Clin Pathol 111: 343–348, 1999.
Fioretto P, Steffes MW, Sutherland DE, Goetz FC, and Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 339: 69–75, 1998.
Floege J, Alpers CE, Burns MW, Pritzl P, Gordon K, Couser WG, and Johnson RJ. Glomerular cells, extracellular matrix accumulation, and the development of glomerulosclerosis in the remnant kidney model. Lab Invest 66: 485–497, 1992.
Fujihara CK, Malheiros DMAC, Noronha IL, de Nucci G, and Zatz R. Mycophenolate mofetil reduces renal injury in the chronic nitric oxide synthase inhibition model. Hypertension 37: 170–175, 2001.
Fujihara CK, Malheiros DMAC, Zatz R, and Noronha IL. Mycophenolate mofetil attenuates renal injury in the rat remnant kidney. Kidney Int 54: 1510–1519, 1998.
Fujihara CK, Michellazzo SM, de Nucci G, and Zatz R. Sodium excess aggravates hypertension and renal parenchymal injury in rats with chronic NO inhibition. Am J Physiol Renal Fluid Electrolyte Physiol 266: F697–F705, 1994.
Fujihara CK, Velho M, Malheiros DMAC, and Zatz R. An extremely high dose of losartan affords superior renoprotection in the remnant model. Kidney Int 67: 1913–1924, 2005.
Goncalves AR, Fujihara CK, Mattar AL, Malheiros DMAC, Noronha IL, de Nucci G, and Zatz R. Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory. Am J Physiol Renal Physiol 286: F945–F954, 2004.
Graciano ML, Cavaglieri RdC, Delle H, Dominguez WV, Casarini DE, Malheiros DMAC, and Noronha IL. Intrarenal renin-angiotensin system is upregulated in experimental model of progressive renal disease induced by chronic inhibition of nitric oxide synthesis. J Am Soc Nephrol 15: 1805–1815, 2004.
Hirakata M, Kaname S, Chung UG, Joki N, Hori Y, Noda M, Takuwa Y, Okazaki T, Fujita T, Katoh T, and Kurokawa K. Tyrosine kinase dependent expression of TGF- induced by stretch in mesangial cells. Kidney Int 51: 1028–1036, 1997.
Hughson MD, Johnson K, Young RJ, Hoy WE, and Bertram JF. Glomerular size and glomerulosclerosis: relationships to disease categories, glomerular solidification, and ischemic obsolescence. Am J Kidney Dis 39: 679–688, 2002.
Jepsen FL and Mortensen PB. Interstitial fibrosis of the renal cortex in minimal change lesion and its correlation with renal function. A quantitative study. Virchows Arch A Pathol Anat Histol 383: 265–270, 1979.
Kagami S, Border WA, Miller DE, and Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 93: 2431–2437, 1994.
Krepinsky JC, Ingram AJ, Tang D, Wu D, Liu L, and Scholey JW. Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation. J Am Soc Nephrol 14: 2790–2800, 2003.
Kriz W and LeHir M. Pathways to nephron loss starting from glomerular diseases-insights from animal models. Kidney Int 67: 404–419, 2005.
Ma LJ, Nakamura S, Aldigier JC, Rossini M, Yang H, Liang X, Nakamura I, Marcantoni C, and Fogo AB. Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1. J Am Soc Nephrol 16: 966–976, 2005.
Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, Schneider H, Ruiz-Ortega M, and Egido J. NF-B activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol Dial Transplant 19: 2505–2512, 2004.
Nataraj C, Oliverio MI, Mannon RB, Mannon PJ, Audoly LP, Amuchastegui CS, Ruiz P, Smithies O, and Coffman TM. Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. J Clin Invest 104: 1693–1701, 1999.
Navarro-Cid J, Maeso R, Rodrigo E, Munoz-Garcia R, Ruilope LM, Lahera V, and Cachofeiro V. Renal and vascular consequences of the chronic nitric oxide synthase inhibition. Effects of antihypertensive drugs. Am J Hypertens 9: 1077–1083, 1996.
Nishiyama A, Yoshizumi M, Rahman M, Kobori H, Seth DM, Miyatake A, Zhang GX, Yao L, Hitomi H, Shokoji T, Kiyomoto H, Kimura S, Tamaki T, Kohno M, and Abe Y. Effects of AT1 receptor blockade on renal injury and mitogen-activated protein activity in Dahl salt-sensitive rats. Kidney Int 65: 972–981, 2004.
Quiroz Y, Pons H, Gordon KL, Rincon J, Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, Johnson RJ, and Rodriguez-Iturbe B. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition. Am J Physiol Renal Physiol 281: F38–F47, 2001.
Remuzzi A, Gagliardini E, Donadoni C, Fassi A, Sangalli F, Lepre MS, Remuzzi G, and Benigni A. Effect of angiotensin II antagonism on the regression of kidney disease in the rat. Kidney Int 62: 885–894, 2002.
Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, and Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 20: 298–303, 1992.
Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, and Narins RG. Cyclic stretching force selectively up-regulates transforming growth factor- isoforms in cultured rat mesangial cells. Am J Pathol 148: 1915–1923, 1996.
Rodriguez-Iturbe B, Pons H, Quiroz Y, Gordon K, Rincon J, Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, and Johnson RJ. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from angiotensin II exposure. Kidney Int 59: 2222–2232, 2001.
Ruiz-Ortega M, Bustos C, Hernandez-Presa MA, Lorenzo O, Plaza JJ, and Egido J. Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-B activation and monocyte chemoattractant protein-1 synthesis. J Immunol 161: 430–439, 1998.
Takebayashi S, Kiyoshi Y, Hisano S, Uesugi N, Sasatomi Y, Meng J, and Sakata N. Benign nephrosclerosis: incidence, morphology and prognosis. Clin Nephrol 55: 349–356, 2001.
Utimura R, Fujihara CK, Mattar AL, Malheiros DMAC, Noronha IL, and Zatz R. Mycophenolate mofetil prevents the development of glomerular injury in experimental diabetes. Kidney Int 63: 209–216, 2003.
Wallenstein S, Zucker CL, and Fleiss JL. Some statistical methods useful in circulation research. Circ Res 47: 1–9, 1980.
Wu LL, Cox A, Roe CJ, Dziadek M, Cooper ME, and Gilbert RE. Transforming growth factor 1 and renal injury following subtotal nephrectomy in the rat: role of the renin-angiotensin system. Kidney Int 51: 1553–1567, 1997.
Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 40: 198–201, 1990.(Clarice K. Fujihara, Claudia R. Sena, De)