Effects of Angiotensin II Subtype 1 Receptor Blockade by Losartan on Tubulointerstitial Lesions Caused by Hyperoxaluria

0022-5347/02/1684-1550/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 168, 1550 –1555, October 2002 Printed in U.S.A.

DOI: 10.1097/01.ju .0000023289.21352.70

EFFECTS OF ANGIOTENSIN II SUBTYPE 1 RECEPTOR BLOCKADE BY LOSARTAN ON TUBULOINTERSTITIAL LESIONS CAUSED BY HYPEROXALURIA ´ N FERDER, INE ´ S STELLA, ELENA M. V. JORGE EDUARDO TOBLLI, LEO MARGARITA ANGEROSA AND FELIPE INSERRA

DE

CAVANAGH,

From the Laboratory of Experimental Medicine, Hospital Alema´n and Instituto de Investigaciones Cardiolo´gicas (ININCA), Buenos Aires, Argentina

ABSTRACT

Purpose: Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. Materials and Methods: We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1— control, group 2— hyperoxaluria, group 3— hyperoxaluria plus losartan and group 4 —losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-␣-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-␤1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. Results: Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, ␣-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-␤1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p ⬍0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p ⬍0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p ⬍0.04). Conclusions: We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors. KEY WORDS: kidney; rats, Sprague-Dawley; losartan; fibrosis; hyperoxaluria

Oxalate, a common constituent of kidney stones, is a metabolic end product normally excreted by the kidney. After jejunoileal bypass for obesity or excess ascorbic acid ingestion a hyperoxaluric state, including primary (hereditary) oxalosis or secondary hyperoxaluria due to gastrointestinal malabsorption, can lead to tubulointerstitial damage. In addition, when these pathological circumstances are prolonged, the risk of chronic tubulointerstitial disease is increased.1 Convincing evidence indicates that oxalate and calcium oxalate crystals are harmful to renal epithelial cells in vivo as well as in cell cultures.2 Renal tubular cells have been shown to express various cytokines, chemokines and other mediators of inflammatory response, including angiotensin II.3, 4 Local angiotensin II seems to participate by inducing or amplifying the inflammatory response in the majority of glomerular as well as tubulointerstitial diseases independent of etiology. Moreover, monocyte chemoattractant protein-1, which is involved in the inflammatory process, is stimulated by angiotensin II and over Accepted for publication April 5, 2002.

expressed after contact with calcium oxalate crystal or hyperoxaluria in epithelial tubular cells.5, 6 Crystal endocytosis and subsequent tubular epithelial cell responses promote an increase in reactive oxygen species (ROS) production. ROS interacts with interstitial cells, leading to an inflammatory reaction and eventually tissue injury.7 Since fibrosis is widely considered the end point of uncontrolled extracellular matrix production, any therapeutic intervention in opposition to this process should be largely useful. A number of studies support the idea that the renin-angiotensin system has an important role in the development of tubulointerstitial fibrosis in several models of chronic tubulointerstitial injury and angiotensin II type 1 receptor stimulation could trigger the pro-fibrotic mechanism.8 Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 receptor blockers have been reported to protect against interstitial damage in different tubulointerstitial lesion models regardless of their effect on blood pressure.9, 10 In previous studies we have noted that renal-angiotensin

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EFFECTS OF ANGIOTENSIN RECEPTOR BLOCKADE ON TUBULOINTERSTITIAL LESIONS

system interaction by ACE inhibition produces a substantial benefit by controlling tubulointerstitial damage in rats subjected to ethylene glycol induced hyperoxaluria.11, 12 Angiotensin II elicits cellular responses through its binding to 2 specific receptors, namely the subtypes angiotensin II types 1 and 2 receptors.13 Most known actions of angiotensin II are mediated by angiotensin II type 1 receptor, such as vasoconstriction, inflammatory process promotion and extracellular matrix deposition.14, 15 ACE inhibition produces 2 simultaneous actions, including a decrease in angiotensin II levels and, therefore, less angiotensin II type 1 receptor binding and activation, as well as an increase in bradykinin accumulation. To our knowledge the mechanism attributable to control tubulointerstitial lesions caused by hyperoxaluria in this model remains undetermined. We evaluated whether angiotensin II type 1 receptor blockade by losartan is the main mechanism responsible for controlling or ameliorating tubulointerstitial renal lesions in hyperoxaluric rats. MATERIALS AND METHODS

Two-month-old male Sprague-Dawley rats initially weighing 250 to 280 gm. were housed in metabolic cages and divided into 4 groups of 8 each, namely control group 1, ethylene glycol group 2, ethylene glycol plus losartan group 3 and losartan group 4. All animals had access to tap water and free access to standard rat chow. For 4 weeks 1% ethylene glycol as a precursor for oxalates was administered to groups 2 and 3 continuously in drinking water. Losartan (40 mg./kg. body weight) was administered daily in groups 3 and 4 by gavage. We collected 24-hour urine samples for biochemical determination at baseline and after 4 weeks, when blood samples were also obtained for serum and blood determinations. The kidneys were harvested for histological and biochemical studies. At baseline and at the end of the experiment systolic blood pressure was measured by tail cuff plethysmography, as previously described.11, 12 Biochemical procedures. Oxalate and citrate were determined by enzymatic method (Sigma Chemical Co., St. Louis, Missouri). Calcium and magnesium were determined by standard methods using atomic absorption. Creatinine clearance was calculated using the standard formula. Urinary albumin concentration was measured by the radial immunodiffusion method. Aliquots of 24-hour urine were centrifuged at 2,000 rpm for 5 minutes to quantify crystalluria. Calcium oxalate crystals were identified using bright field phase contrast and an adapted polarized light microscope, quantified in 10 microscopic fields per sample and examined at 400⫻ magnification. Crystalluria was graded as 0 —no crystals, 1— less than 10, 2—10 to 25, 3—26 to 50 and 4 — greater than 50 crystals per field. To estimate urinary calcium oxalate supersaturation the ion activity product of calcium oxalate was determined according to the Tisselius formula.16 Kidney processing and examination. Kidneys were perfused with saline solution through the abdominal aorta until it was free of blood. Decapsulated kidneys were cut longitudinally and a portion was used to determine the products of lipid peroxidation and total glutathione content. The remaining kidney tissue was fixed in phosphate buffered 10% formaldehyde (pH 7.2) and embedded in paraffin. Sections (3 ␮.) were cut and stained with hematoxylin and eosin, periodic acid-Schiff reagent and Masson’s trichrome. Immunolabeling and optical microscopy. Renal ␣-smoothmuscle actin was quantified using anti-mouse ␣-smooth muscle actin monoclonal antibody (Sigma Chemical Co.). To identify collagen type III we used monoclonal antibody anticollagen type III (Biogen, San Roma´ n, California). Tubular and interstitial transforming growth factor-␤1 (TGF-␤1) was quantified using a polyclonal anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, California). Monocytes and macrophages were identified using ED1 mouse anti-rat

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monoclonal antibody, diluted 1:300 in phosphate buffered saline (pH 7.2 to 7.6). Morphological analysis. Morphological analysis was assessed in 10 microscopic fields per section by ⫻100 magnification with the observer blinded to the animal group. The data were averaged. Lesion scores related to tubular atrophy, unspecific inflammatory cell infiltrate, positive ED1 cells (monocytes/macrophages), crystal deposits, interstitial fibrosis, amount of ␣-smooth muscle actin, collagen type III and TGF-␤1 in renal tubulointerstitial cells. Sections were graded according to the scale, 0 —absent, 1—mild (involving 25% or less of each microscopic field), 2—moderate (greater than 26% to 50% or less), 3—severe (greater than 51% to 75% or less) and 4 —very severe (greater than 76%). When comparing various groups, the same kidney areas were analyzed. Determination of lipid peroxides (thiobarbituric acid reactive substances). The kidneys were homogenized with 4 volumes of 120 mM. KCl-30 mM. potassium phosphate, pH 7.4, and centrifuged at 600 ⫻ gravity for 10 minutes. Aliquots of the supernatant containing 0.5 mg. protein were added with 100 ␮l. butylhydroxytoluene (4% weight per volume in ethanol) before thiobarbituric acid reactive substances were determined by spectrofluorescence. Results are expressed in nM. thiobarbituric acid reactive substances or malondialdehyde equivalents) per mg. protein. Malondialdehyde standards were prepared from 1,1,3,3-tetramethoxypropane. Protein content was determined according to standard methods using bovine serum albumin as the standard. Determination of total glutathione. Renal tissue homogenates were prepared with 4 volumes of 0.33 M. HClO4 and centrifuged at 5,000 ⫻ gravity for 10 minutes. The supernatant was neutralized with 1.75 M K3PO4 and an aliquot was obtained for total glutathione measurement using the 5,5¨-dithiobis (2nitrobenzoic acid) spectrophotometric assay. Results are expressed in ␮M. glutathione equivalents per gm. wet tissue. Statistical method. Values are expressed as the mean plus or minus standard error of mean. Statistical analyses were performed using absolute values and processed using GraphPad Prism, version 2.0 (GraphPad Software, San Diego, California). For parameters with Gaussian distribution comparisons among groups were made using analysis of variance. For parameters such as histological data with nonGaussian distribution comparisons were performed using the Kruskal-Wallis (nonparametric analysis of variance) and Dunn multiple comparison tests. To explore relationships among multiple variables we used the multiple regression test. Linear correlation of the Pearson coefficient or the Spearman rank correlation was used when appropriate with p ⬍0.05 considered significant. RESULTS

There were no significant differences in baseline systolic blood pressure, urinary pH, urinary oxalate, calcium, magnesium, citrate or urinary albumin excretion among the groups. Average daily water consumption throughout the 4 weeks in groups 1 to 4 was 27.5 ⫾ 1, 27.6 ⫾ 0.8, 28.3 ⫾ 0.8 and 27.8 ⫾ 0.7 ml., respectively (p not significant). Rats in groups 2 and 3 received a daily average of 276.2 ⫾ 8.5 and 283.5 ⫾ 8.4 ␮l. ethylene glycol, respectively (p not significant). At the end of the experiment a marked elevation in urinary oxalate was observed in rats in groups 2 and 3 (table 1). Nevertheless, while the losartan treated animals in group 2 showed a high level of crystalluria, group 3 rats treated with losartan showed a significantly lower amount of calcium oxalate crystals in the urine (table 1). No significant changes in systolic blood pressure, urinary pH or citrate were observed in any group at the end of 4 weeks (table 1). Group 2 showed a significant reduction in urinary calcium excretion compared with normal control rats. On the other hand, group 3 showed enhanced urinary calcium and magnesium excretion compared with group 2. Levels were even higher than in

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EFFECTS OF ANGIOTENSIN RECEPTOR BLOCKADE ON TUBULOINTERSTITIAL LESIONS TABLE 1 Mean Parameters ⫾ SEM at 4 Wks.

Rat body Wt. (gm.) Systolic blood pressure (mm. Hg) Urine pH Urine oxalate (␮g./gm. body wt./day) Urine calcium (␮g./gm. body wt./day) Urine magnesium (␮g./gm. body wt./day) Urine citrate (␮g./gm. body wt./day) Crystalluria score Ion activity product Urinary albumin excretion (mg./day) Creatinine clearance (ml./min.) Eight rats per group.

Group 1

Group 2

Group 3

Group 4

337.5 ⫾ 4.3 120.6 ⫾ 0.8 6.43 ⫾ 0.04 1.67 ⫾ 0.06 2.87 ⫾ 0.13 5.76 ⫾ 0.21 26.4 ⫾ 1.1 0.12 ⫾ 0.12 0.16 ⫾ 0.01 0.3 ⫾ 0.13 1.38 ⫾ 0.01

322.3 ⫾ 5.3 122.2 ⫾ 1.07 6.47 ⫾ 0.04 12.9 ⫾ 1.13 1.84 ⫾ 0.13 6.09 ⫾ 0.19 25.9 ⫾ 1.2 2.87 ⫾ 0.29 1.04 ⫾ 0.12 28.3 ⫾ 2.4 0.93 ⫾ 0.02

319.7 ⫾ 5.3 119.7 ⫾ 0.5 6.48 ⫾ 0.05 13.5 ⫾ 1.06 3.50 ⫾ 0.12 7.73 ⫾ 0.20 28.4 ⫾ 1.3 0.62 ⫾ 0.18 1.20 ⫾ 0.08 4.2 ⫾ 1.4 1.18 ⫾ 0.02

321.5 ⫾ 6.3 119.2 ⫾ 0.5 6.51 ⫾ 0.05 1.7 ⫾ 0.09 3.55 ⫾ 0.18 7.51 ⫾ 0.23 27.3 ⫾ 1.2 0.12 ⫾ 0.12 0.16 ⫾ 0.01 0.2 ⫾ 0.1 1.4 ⫾ 0.01

normal controls (table 1). Regarding calcium oxalate urine supersaturation, notably there was no significant difference in groups 2 and 3 rats related to ion activity product, whereas these 2 groups showed a significant difference compared with control group 1 and control plus losartan group 4. Rats in group 2, which received ethylene glycol, showed an increase in urinary albumin excretion and lower creatinine clearance compared with the other groups (p ⬍0.01, table 1). In contrast, groups 3 had a significant reduction in urinary albumin excretion relative to group 2 (table 1). Light microscopy showed that animals in group 2 had a diffuse amount of oxalate crystals into the tubular lumina as well as in the tubular cells and interstitium, mainly in the cortex but also in the medulla. In addition, inflammatory cell infiltrates mostly corresponding to monocytes and macrophages (ED1 positive cells), tubular atrophy and interstitial fibrosis in the cortex and medulla were also present in the former group. These morphological parameters were quantified by their respective scores (table 2, figs. 1, A and 2, A). These findings were reduced in group 3 (table 2, figs. 1, B and 2, B). A significant increase in ␣-smooth muscle actin in peritubular and tubular cells (p ⬍0.01) as well as an increase in collagen type III deposition and TGF-␤1 in the interstitium and tubular cells was detected by immunostaining in rats in group 2 (table 2, figs. 3, A, 4, A and 5, A). On the other hand, group 3 rats showed small amounts of ␣-smooth muscle actin, collagen type III and TGF-␤1 (p ⬍0.01, table 2, figs. 3, B, 4, B and 5, B). Briefly, all morphological scores were notably reduced in group 3 compared with group 2, almost all to control levels, but they were not completely normalized despite rising statistical significance (table 2). Group 2 showed lower total glutathione concentration in kidney homogenates (p ⬍0.01) and a higher level of thiobarbituric acid reactive substances compared with group 3 (table 2). Furthermore, it is remarkable that the animals that received losartan (groups 3 and 4)

p Value Not significant Not significant Not significant ⬍0.01 Groups 2 ⫹ 3 vs. 1 ⫹ 4 ⬍0.01 Group 2 vs. 3 ⬍0.01 Group 2 vs. 3 Not significant p ⬍0.01 Group 2 vs. all groups ⬍0.01 Group 1 vs. 1 ⫹ 4 ⬍0.05 Group 2 vs. 3 ⬍0.01 Group 2 vs. all groups

had a total glutathione concentration that was even higher than in control group 1 and these results achieved statistical significance (table 2). Multiple regression analysis showed that thiobarbituric acid reactive substances correlate positively with interstitial fibrosis (t ratio ⫽ 4.867, p ⫽ 0.039, r ⫽ 0.9272, p ⬍0.01), indicating that lipid oxidation is likely to contribute to the development of interstitial fibrosis in this model. DISCUSSION

In the current study we found significant protection by losartan on the tubulointerstitial lesions in hyperoxaluria rats. Despite a similar urinary oxalate concentration a relevant point was that ethylene glycol plus losartan treated animals (group 3) showed different tubular damage and tubulointerstitial fibrosis scores, creatinine clearance, urinary albumin excretion, total glutathione and thiobarbituric acid reactive substance levels in kidney homogenates from those in ethylene glycol treated animals (group 2). This protective effect could be summarized as 3 relevant findings, namely a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors led concurrently to preservation of the tubular epithelial cell and renal interstitium integrity In the current study few crystal deposits were observed in rats hyperoxaluric treated rats, although urinary oxalate excretion and urinary calcium oxalate supersaturation were similar to those in the untreated hyperoxaluria group. This fact, which we have previously reported in the same model using ACE inhibition,11, 12 suggests that interaction with renal-angiotensin system provokes some favorable effect on preventing crystal deposition not only in tubular lumina, but also in the renal interstitium. Crystallization of calcium oxalate depends on the respective level of oxalate and calcium in urine as well as on other factors, such as local pH (proxi-

TABLE 2. Morphological, immunohistochemical and oxidative stress parameters at 4 weeks Mean Group 1 ⫾ SEM Score:* Tubular atrophy 0.06 ⫾ 0.06 Unspecific inflammatory cell infiltrate 0.12 ⫾ 0.12 ED1 (monocytes/macrophages) 0.06 ⫾ 0.06 Crystal deposits 0 Interstitial fibrosis 0.06 ⫾ 0.06 Smooth muscle actin 0.06 ⫾ 0.06 Collagen type III 0.12 ⫾ 0.08 0.06 ⫾ 0.06 Tubulointerstitial TGF-␤1 Oxidative stress parameters: Total glutathione (nmol./gm. wet wt.)† 273.4 ⫾ 7 Tiobarbituric acid reactive substances (nmol. malondialde0.41 ⫾ 0.02 hyde/mg. protein) Eight rats per group. * Group 2 versus all groups p ⬍0.01, group 3 versus group 1 not significant. † Group 2 versus all groups and groups 3 and 4 versus 1 p ⬍0.01. ‡ Versus group 1 p ⬍0.01.

Mean Group 2 ⫾ SEM

Mean Group 3 ⫾ SEM

Mean Group 4 ⫾ SEM

2 ⫾ 0.2 1.87 ⫾ 0.29 1.62 ⫾ 0.26 1.37 ⫾ 0.26 1.68 ⫾ 0.28 2.68 ⫾ 0.29 2.62 ⫾ 0.15 2.43 ⫾ 0.14

0.12 ⫾ 0.08 0.12 ⫾ 0.08 0.12 ⫾ 0.08 0.06 ⫾ 0.06 0.12 ⫾ 0.12 0.31 ⫾ 0.09 0.68 ⫾ 0.23 0.5 ⫾ 0.18

0.06 ⫾ 0.06 0.12 ⫾ 0.08 0.06 ⫾ 0.06 0 0.06 ⫾ 0.06 0.06 ⫾ 0.06 0.12 ⫾ 0.08 0.06 ⫾ 0.06

205.1 ⫾ 7.2 0.66 ⫾ 0.02‡

306.3 ⫾ 5.4 0.39 ⫾ 0.01

360.1 ⫾ 9 0.33 ⫾ 0.01

p Value

EFFECTS OF ANGIOTENSIN RECEPTOR BLOCKADE ON TUBULOINTERSTITIAL LESIONS

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FIG. 1. Renal tissue. A, in group 2 rat diffuse tubulointerstitial lesions are characterized by tubular atrophy and dilatation (small arrow), interstitial cellular infiltrates and interstitial fibrosis (large arrow). Note oxalate crystals (asterisk) into tubular lumina. B, group 3 rat without tubulointerstitial lesions or crystals. Masson’s trichrome, reduced from ⫻400.

FIG. 2. Renal tissue. A, in group 2 rat arrows indicate positive cells, corresponding to monocyte/macrophage infiltrates. Note oxalate crystals (asterisk) into tubular lumina. B, in group 3 rat note few positive cells. ED1 immunostaining, reduced from ⫻400.

FIG. 3. Renal tissue. A, in group 2 arrowheads indicate positive staining in peritubular interstitium and myofibroblasts. Note oxalate crystals (asterisk) into tubular lumina. B, in group 3 rat arrow indicates positive staining in vessel walls only. ␣-Smooth muscle actin immunostaining, reduced from ⫻400.

mal tubule) and the presence or absence of inhibitors of crystal formation such as urinary citrate concentration or other substances, including osteopontin, nephrocalcin, Tamm-Horsfall glycoprotein, renal lithostathine and glycosaminoglycans.17 A possible hypothesis for understanding why the animals from group 3 (ethylene glycol plus losartan) almost did not show crystals within tubular lumina is based on a reduction in the well-known action of angiotensin II on Na⫹/H⫹ antiporter activity in proximal tubule.18 This effect could modify intraluminal pH at proximal tubule, interfering with the calcium oxalate crystallization mechanism without any other substantial change in final urinary pH, as in the current experiment. SO42⫺ and HCO3- have been shown to be

inhibitors of calcium oxalate crystallization.19 Since tubular epithelial oxalate transport occurs mainly at the early segments of the nephron, any change with respect to local HCO3concentration could contribute to inhibit calcium oxalate crystallization. Furthermore, no different urinary citrate excretion was noted among the groups. Consequently the widely accepted effect of citrate regarding growth, aggregation and nucleation of calcium oxalate crystals is unlikely to be implicated in the protective mechanism of losartan. Enhanced urinary calcium and magnesium excretion has been reported using losartan.20 Since calcium and especially magnesium were increased in the urine of hyperoxaluria rats treated with losartan, the favorable effect of magnesium

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EFFECTS OF ANGIOTENSIN RECEPTOR BLOCKADE ON TUBULOINTERSTITIAL LESIONS

FIG. 4. Renal tissue. A, in group 2 rat note diffuse positive staining (brown areas) in interstitium. B, in group 3 rat note scant staining in perivascular areas only. Collagen type III immunostaining, reduced from ⫻100.

FIG. 5. Renal tissue. A, in group 2 rat arrowheads indicate large area of positive staining in tubular and peritubular cells. Note oxalate crystals (asterisk) into tubular lumina. B, in group 3 rat arrow indicates small area of positive staining. TGF-␤1 immunostaining, reduced from ⫻400.

regarding its influence on intratubular calcium oxalate formation and crystaluria21 could contribute to prevent tubulointerstitial lesions in these animals. A decrease in urinary calcium concentration has also been reported in type I primary hyperoxaluria and in experimental models using ethylene glycol.22, 23 This finding was associated with increased calcium content in dried kidney tissue and it correlated with the degree of renal damage and calcium oxalate crystal deposits. In the current study, while group 2 showed a significant reduction in urinary calcium compared with normal control rats, urinary calcium excretion in hyperoxaluric animals treated with losartan (group 3) was almost duplicated. This fact could explain part of the favorable effect by which losartan reduces the amount of calcium oxalate deposits in tubular cells as well as in the interstitium. Another important factor that could participate in the prevention of crystal deposits may be the significant reduction in the tubular cell lesions, as demonstrated by the tubular atrophy score and scant intratubular detritus production in the hyperoxaluric rats receiving losartan (table 2). Detritus and injured tubular cell membranes act as a trigger of growth, aggregation and nucleation of oxalate crystals, especially in a high urinary oxalate concentration environment.24 Preserved tubular epithelial cell integrity especially at the earlier nephron segments, which are most susceptible to calcium oxalate damage,25 may contribute to impair in crystal formation. Although other mechanisms could probably be participating in the protection against crystal deposits, we assume that angiotensin II type 1 receptor is the main agent in this process based on the evidence that angiotensin II type 1 receptor blockade and ACE inhibition have been noted to produce the same effect.11, 12 Proteinuria has been considered one of various main causes

in the pathogenic mechanisms of tubulointerstitial damage.26 In rats with intense proteinuria ACE and angiotensinogen are up-regulated and angiotensin II produces its deleterious action on the interstitium through the activation of angiotensin II type 1 receptor.27 In agreement with this finding in the current study we noted a correlation of urinary albumin excretion with the tubulointerstitial fibrosis score in untreated hyperoxaluria rats. In contrast, animals with hyperoxaluria that received losartan showed lower urinary albumin excretion without this relationship. The urinary albumin excretion reduction may participate as a protective effect by interfering with cytokine dependent damage mechanisms, as in other models of proteinuria.26 Moreover, it is well known that the monocyte/ macrophage infiltrate has relevant participation as a source of TGF-␤, and other cytokine and chemokine production as well as being a potent source of ROS, which are important factors implicated in the mechanisms of damage to renal tubulointerstitial tissue. Interestingly in the current study losartan treated animals showed lower TGF-␤1 immunostaining than those not treated. Also, these results agreed with similar data in other experiments.27, 28 It is recognized that TGF-␤1 has a central role in the renal fibrotic process and it is postulated to be a key mediator of renal fibrotic disease.29 Since a strong interaction of angiotensin II and TGF-␤1 has been demonstrated in several models, we speculate that a protective effects of losartan in our model may have been mediated by a decrease in TGF-␤1.30 Unfortunately to our knowledge the primary stimulus that starts the pathway for tubular and interstitial lesions by hyperoxaluria is unclear. Beyond its capacity to generate crystals hyperoxaluria has been shown to damage renal epithelial cells. A high concentration of oxalate into the epithelial cell is toxic since it increases free radical production and lipid peroxidation.31, 32 Furthermore, hypoxia produced by

EFFECTS OF ANGIOTENSIN RECEPTOR BLOCKADE ON TUBULOINTERSTITIAL LESIONS

injury and loss of peritubular capillaries due to interstitial damage and accumulation of extracellular matrix in the interstitium can contribute to ROS overproduction, especially in high metabolic cellular activity situations such as oxalate tubular overload. An additional source of ROS may be inflammatory cell infiltrates, which are also present in this environment. Nicotinamide adenine dinucleotide oxidation and oxalate deposition in the kidney correlate negatively with mitochondrial glutathione depletion,33 suggesting that mitochondrial dysfunction results from total glutathione depletion. In the current study hyperoxaluric animals untreated with losartan showed remarkably lower total glutathione concentration in kidney tissue and an increased level of thiobarbituric acid reactive substances, a marker of lipid peroxidation. Moreover, lipid peroxidation was the major variable involved in tubulointerstitial fibrosis on multiple regression analysis. These abnormalities were dramatically reduced by losartan treatment. Interestingly all animals that received losartan showed an increase in total glutathione to a level significantly higher than in control rats. Increased antioxidant defenses by interacting against the renalangiotensin system have previously been observed in studies of our group not only in normal rodents, but also in patients with chronic renal failure.34, 35 In conclusion, renalangiotensin system interaction with losartan produces a beneficial effect against tubulointerstitial lesions caused by hyperoxaluria, suggesting that the principal mechanism of action should be mediated by angiotensin II type 1 receptors. Ana Uceda and Andrea Biscochea assisted with laboratory experiments and Jaquelina Mastantuono reviewed manuscript style. REFERENCES

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