Inflammation is probably not a prerequisite for renal interstitial fibrosis in normoglycemic obese rats

Stéphanie Lavaud1, Bruno Poirier1, Chantal Mandet1, Marie-France Bélair1, Théano Irinopoulou1, Didier Heudes1, Raymond Bazin2, Jean Bariéty1, Isaac Myara1,3, and Jacques Chevalier1

1 Institut National de la Santé et de la Recherche Médicale Unité 430, Broussais Hospital, 75014 Paris; Claude Bernard Association, 2 Institut National de la Santé et de la Recherche Médicale Unité 465, Institut des Cordeliers, 75005 Paris; and 3 Laboratory of Applied Biochemistry, Faculty of Pharmaceutical and Biological Sciences, 92296 Châtenay-Malabry, France


    ABSTRACT
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INTRODUCTION
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We examined the role of inflammation in the development of renal interstitial fibrosis in Zucker obese rats, which rapidly present kidney lesions in the absence of hypertension and hyperglycemia. Type I and III collagens were quantified using a polarized light and computer-assisted image analyzer. The expression of mRNA encoding matrix components, adhesion molecules, chemokines, and growth factors was followed by RT-PCR. The presence of synthesized proteins as well as lymphocytes and macrophages was determined by immunohistochemistry. Interstitial fibrosis developed in two phases. The first phase occurred as early as 3 mo and resulted from a neosynthesis of type III collagen and fibronectin and a reduction of extracellular matrix catabolism, in parallel with an overexpression of transforming growth factor-beta 1 and in the absence of any lymphocyte or macrophage infiltration. After 6 mo, interstitial fibrosis worsened with a large accumulation of type I collagen, concomitantly with a large macrophage infiltration. Thus inflammation cannot explain the onset of interstitial fibrosis that developed in young, insulinoresistant, normoglycemic, obese Zucker rats but aggravated this process afterward.

Zucker rat; hyperlipidemia; hyperinsulinemia; collagen; fibronectin; tissue inhibitor of metalloproteinase-1; transforming growth factor-beta 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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CHRONIC DISEASES of the kidney have an intrinsic tendency to progress to parenchymal scars and loss of renal function, requiring chronic dialysis or transplantation. A large consensus associates an inflammatory process with renal glomerular and interstitial fibrosis, an irreversible accumulation of extracellular matrix (ECM) components. This paradigm was drawn from several animal models such as renal ablation, uninephrectomized hypercholesterolemic rats, puromycin aminonucleoside-induced nephrosis (PAN), protein-overload proteinuria, experimental autoimmune tubulointerstitial nephritis, spontaneous murine lupus nephritis, aging rats, streptozotocin-induced diabetes, and from humans showing anti-glomerular basement membrane (GBM) antibody disease, cyclosporine nephrotoxicity, various forms of proliferative and nonproliferative glomerulonephritis, or diabetic nephropathy. Conversely, maneuvers that reduce the macrophage infiltration also reduce the gravity of the lesions. Consequently, macrophages have been proposed as propagating initial renal injury to glomerulosclerosis and/or tubulointerstitial fibrosis in a way similar to that observed in atherosclerosis (8).

Numerous studies have been devoted to the mechanisms of glomerulosclerosis, but the role of inflammation in the pathogenesis of kidney interstitial fibrosis that occurs in patients or animals suffering from metabolic disorders such as diabetes mellitus, obesity, and/or hyperlipidemia remains relatively unknown as most of the data concerning the genesis of tubulointerstitial lesions come from experimental models of PAN (9) and partial nephrectomy (11, 13). Moreover, the cross-talk between resident kidney cells, invasive inflammatory cells, and the high levels of metabolic factors such as plasma cholesterol, triglycerides, glucose, and/or insulin is still unclear. Each of these metabolic factors may induce, on its own, an activation of the target cells, as suggested by in vitro experiments (1, 21, 40). In this respect, the Zucker obese (ZO) rat, a useful model of genetic obesity, is of special interest to specifically address the pathogenesis of renal interstitial fibrosis without interference of hypertension and high plasma glucose concentrations and subsequent glycation complication (Amadori products, advanced glycation end products) often encountered in other animal models. Indeed, ZO rats show abnormal glucose tolerance and peripheral insulin resistance similar to that patients with type II non-insulin-dependent diabetes mellitus but still control their glycemia at a normal level at the cost of severe hyperinsulinemia (27). ZO rats express, at weaning, pronounced hyperlipidemia that rapidly worsens with age. Whereas lean (ZL) littermate rats, used as internal controls, have normal levels of serum lipids, glucose, and insulin and normal renal structure and function, ZO rats develop, at 3 mo onward, renal functional impairment, tubular alterations, interstitial fibrosis, glomerulosclerosis, and focal and segmental glomerular hyalinosis that aggravate with age (23). These lesions occur in the absence of hypertension (23) or renal hemodynamic modification (31). Using automated image analysis, RT-PCR, and immunocytochemistry, we examined in this study the time course of interstitial fibrosis in the ZO rat kidney, with a peculiar interest in the specific contribution of inflammation to the onset and to the aggravation of this fibrosis process.


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Animals. Male Zucker lean (Fa/fa, ZL) and obese (fa/fa, ZO) rats (Institut National de la Santé et de la Recherche Médicale Unité 465 husbandry, Dr. Raymond Bazin, Paris, France) were identified and selected at 4 wk of age by visual examination of inguinal fat deposits. They were raised under standard husbandry conditions, fed regular laboratory chow ad libitum (M20 diet; Extralabo, Provins, France), and had free access to water until death at 5 wk (1 mo) and 3, 6, 9, and 12 mo of age. For determination of biological parameters, fasting animals were housed individually in metabolic cages with free access to water. Twenty-four-hour urine samples were collected, and a blood sample was obtained by orbital sinus puncture in tubes containing heparin. At the time of death, animals were anesthetized with pentobarbital sodium (0.1 ml/100 g body wt ip), and the kidneys were removed and weighed. For subsequent histological and immunohistochemical analysis of the kidneys, transverse sections were immediately made at the hilus and were either frozen and stored in liquid nitrogen or fixed in alcoholic Bouin's solution or in paraformaldehyde before embedding in paraffin. Animal care complied with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1989, authorization 00577, 1989, Paris, France).

Biological parameters. Conscious systemic blood pressure was measured by a tail-cuff system (Ugo Basile Apelex, Varese, Italia). Blood samples were centrifuged at 2,500 rpm for 10 min at 4°C, and aliquots of plasma were frozen and stored at -20°C. Triglycerides and total cholesterol were determined by the enzymatic and colorimetric GPO-PAP and CHOD-PAP detection kits, respectively (Boehringer, Mannheim, Germany). Glycemia and plasma and urine creatinine concentrations were measured on a Synchron CX7 Beckman analyzer (Beckman, Fullerton, CA). Plasma insulin was measured by RIA (CIS, Gif sur Yvette, France) with a rat insulin standard (Novo, Copenhagen, Denmark). Proteinuria was determined using the Coomassie Protein Assay Reagent (Pierce, Rockford, IL) with BSA as standard.

Kidney structure. For light microscopy, transverse kidney sections fixed in alcoholic Bouin's solution and embedded in paraffin were cut (4-µm thick) and stained with Masson's trichrome or sirius red F3BA (0.1% in saturated aqueous picric acid) according to routine histological techniques. Tubulointerstitial injury was defined as tubular dilation, degenerating tubular cells, and cellular debris in the lumen, proteinaceous tubular casts, interstitial fibrosis, and interstitial inflammatory cell infiltrates. It was graded on Masson's trichrome-stained sections on a scale of 0-4 (0 = normal; 0.5 = small focal areas of damage; 1 = involvement of <10% of the cortex; 2 = involvement of 10-25% of the cortex; 3 = involvement of 25-75% of the cortex; 4 = extensive damage involving >75% of the cortex) to form a semiquantitative index of tubulointerstitial damage (16).

Morphometric measurements. Collagen density was estimated by computer-assisted morphometry on transverse kidney sections processed with sirius red staining. Using a cross-polarizing filter, collagens appear as green (mainly type III collagen) and yellow-red structures (mainly type I collagen; see Ref. 20). Treatment and analysis of the images were performed using the public domain National Institutes of Health (NIH) Image 1.62b7 program. A sequence of mathematical and morphological operations, corresponding to a personal program written in Pascal-like language, allowed the identification and quantification of all structures of interest. Parameters such as the surface area, the perimeter of all collagenic structures, and a reference surface area were measured. Because of the large variability in the surface area of the medullary tubules and interstitium from one section to the other, a comparative morphometric study of the medullary domain has little significance, and solely the kidney cortex was analyzed. Interstitial areas were considered, to the exclusion of glomeruli and vascular zones. Both green and yellow-red structures were measured together with no discrimination between the two figures. The computer program ensured that the same analysis sequence was used for each field. Survey of the mean and variance of the data defined the number of fields needed to obtain a convergent estimate of fibrosis density, determined to be 30. To sample the whole surface of the cortex, one field in four was quantified. A preliminary assay carried out on a random sample of 600 images distributed over the six groups of animals studied (1-, 6-, and 12-mo-old ZL and ZO rats) showed a bimodal distribution of collagens in two types of structures: collagen accumulation in small foci between 5 and 300 µm2 and accumulations in large areas over 300 µm2. It also showed that small foci increased regularly between 1, 6, and 12 mo, whereas large foci accumulated only in old ZO rat kidney. Therefore, we have not performed the analysis at intermediate ages of 3 and 9 mo, since it would not have provided significant complementary information. Structures smaller than 5 µm2 corresponded to a background signal. Small foci and large areas of collagens have been distinguished in the measurement procedure. Two parameters were determined for each size of collagen structures: the "surface density" as collagen object surface area divided by the reference surface area and the "number density" as collagen object number divided by the reference surface area.

Immunohistochemical studies. The lineage of interstitial mononuclear cells was assessed by incubating frozen kidney sections from rats aged 1, 3, 6, or 9 mo with a mouse monoclonal antibody specific for a monocyte/macrophage cytoplasmic marker (ED1 antibody, Serotec, Oxford, UK) diluted 1:1,000 in 0.05 M Tris-buffered saline, pH 7.4, containing 0.1% BSA (Sigma, St. Louis, MO) for 60 min at room temperature. Because interstitial fibrosis and tubular lesions were particularly well developed in the kidneys of the 12-mo-old obese rats, rendering the estimation of mononuclear cell densities difficult and imprecise, this age group was not included in the immunohistochemical study. Because the density of macrophages was very low in young, 3-mo-old ZL and ZO rats, we then searched for another cell type at this early age in frozen kidney sections of lean and obese rats using a mouse monoclonal antibody directed against T cell receptors (1:100, 30 min, clone RT3; Serotec). Next, the sections were washed in Tris-buffered saline and incubated with rabbit anti-mouse immunoglobulin antibody (Dako, Carpinteria, CA) and APAAP complexes (alkaline phosphatase-anti-alkaline phosphatase) (diluted 1:75; Dako). The enzyme was revealed with a freshly prepared Fast Red Substrate System (Dako) containing 0.33 mg/ml levamisole (Sigma) to reduce the staining background. Sections were counterstained with hematoxylin. The number of positive cells in squares of 1.105 mm2 distributed over the whole kidney was counted, with a minimum of 13 squares surveyed per kidney section as defined by convergent analysis. All other immunostainings were performed on formaldehyde-fixed, deparaffinized kidney sections from 3- and 9-mo-old ZL and ZO rats. Anti-type I (1:50) and anti-type III (1:20) collagen antibodies (Biogenesis, Pool, UK), anti-monocyte-chemoattractant protein-1 (MCP-1) antibody (1:30; Serotec), and anti-transforming growth factor (TGF)-beta 1 antibody (1:100, clone TB21; Serotec) were used after a 2-h enzymatic digestion of the section at 37°C with 1 mg/ml pepsin (Sigma) in 0.5 M acetic acid. For fibronectin detection (1:50, rabbit anti-human fibronectin cross-reacting with rat fibronectin; Dako), the enzymatic digestion was made using 0.1 M pronase (Sigma) for 10 min at 37°C. Anti-type IV collagen (1:300, clone CIV22; Neomarkers, Union City, CA) and anti-alpha -smooth muscle actin (alpha -SMA, 1:300, clone 1A4; Neomarkers) antibodies were used directly, without prior enzymatic digestion. Sections were incubated for 30 min at room temperature with the primary antibody diluted in PBS, pH 7.2, rinsed in PBS, and subsequently incubated for 30 min at room temperature with a biotinylated secondary antibody (anti-rabbit or anti-mouse antibody; Biosys, Burlingame, CA) diluted 1:200 in PBS. These secondary antibodies were revealed using a fluorescence probe (Fluorolink Cy 2; Amersham Life Science, Little Chalfond, UK) for 30 min. Immunolabeled sections were observed under a Leica TCS SP confocal microscope (Leica, Heidelberg, Germany).

Total RNA extraction. For mRNA expression analysis, a second series of lean and obese rats (1, 3, 6, 9, and 14 mo old) was killed. Rats were anesthetized with pentobarbital sodium, and the left kidney was perfused at 4°C, first with 135 mM NaCl, 1 mM Na2HPO4, 1.2 mM Na2SO4, 1.2 mM MgSO4, 5 mM KCl, 2 mM CaCl2, 5.5 mM glucose, and 5 mM HEPES, pH 7.4 (solution I), and then with 3 ml of solution I containing 1 mg/ml type I collagenase (300 U/mg; Sigma) and 1 mg/ml BSA (Sigma). A piece of renal cortex was removed from the left kidney and rapidly frozen, grounded, and homogenized in 4 M guanidium isothiocyanate. Total mRNAs were extracted with TRIzol (1 ml/100 mg of tissue; Life Technologies, Grand Island, NY). To ensure sufficient material for the subsequent PCR analysis, in all experimental groups, four different pools of mRNAs extracted from three rats each were used. For each pool, PCR was run in duplicate.

RT-PCR. mRNAs were reverse transcribed into cDNA with oligo(dT) and Moloney murine leukemia virus RT (GIBCO-BRL, Gaithersburg, MD). For each experiment, stock solutions of the different cDNAs were adjusted by diluting with bidistilled water to a final concentration in such a way as to yield identical levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All PCRs were carried out on 2 µl of these adjusted cDNA solutions added to 23 µl of a mixture containing 0.2 mM dNTP, PCR buffer (10 mM Tris · HCl, pH 8.3, 50 mM KCl, 0.001% gelatin), DMSO, MgCl2 (see concentrations in Table 1), and 2.5 units AmpliTaq polymerase (Perkin-Elmer, Norwalk, CT). The cDNA were amplified with the primers listed in Table 1. All sequences were found in the GenBank database (National Center for Biotechnology Information, NIH, Bethesda, MD). Each sample was incubated in a DNA thermal cycler (Perkin-Elmer) at 54-58°C for 30-40 cycles, depending on the primer (Table 1). The PCR fragments were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining (Eurobio, Les Ulis, France). Polaroid photographs of ethidium bromide-stained gels were digitized in 512-512 pixel gray scale images. The amount of nucleic acid, determined by densitometric analysis of the dots, was proportional to the logarithm of the optic density. Analysis was performed using the public domain NIH Image 1.62b7 program. The intensities of the cDNA bands for each protein were normalized to the GAPDH band intensities.

                              
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Table 1.   Primer list and characteristics of polymerization

Statistical analysis. Results are expressed as means ± SE. Statistical analysis (Statview 5.0 software; Abaccus Concepts, Berkeley, CA) was carried out using two-way ANOVA with age and genotype as factors. Statistical significance was achieved at P < 0.05. In the case of interaction between the factors, one-factor ANOVA was used at one level of the other factor. The Spearman rank correlation coefficient was calculated between macrophage density and different biological parameters.


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Biological parameters. As shown in Table 2, mean systemic tail-cuff blood pressure remained at a normal range whatever the age and the genotype of the animals. Plasma levels of cholesterol and triglycerides were higher in ZO than in ZL rats as early as the first month and worsened afterward in ZO rats. Plasma glucose concentration did not change with age and remained at identical normal values in both ZL and ZO groups. Insulinemia, higher in the obese than in the lean group at 1 mo onward, plateaued at 3-6 mo and declined thereafter. No difference was observed between the two groups by 12 mo. Marked proteinuria developed in ZO rats after 3 mo of age and worsened between 6 and 9 mo to exceed 20- to 30-fold the ZL values at 9 and 12 mo. Plasma creatinine concentration increased with age in parallel in both groups of animals up to 6 mo but rose markedly thereafter in ZO rats. The creatinine clearance rate was lower in ZO than in ZL rats (ZO vs. ZL rats, in ml/min: 3 mo, 1.20 ± 0.11, n = 11 vs. 1.65 ± 0.18, n = 11; 9 mo, 1.37 ± 0.15, n = 12 vs. 2.31 ± 0.23, n = 13; group effect, P = 0.002; age effect, P < 0.0001; interaction, P = 0.084).

                              
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Table 2.   Physiological parameters

Histology, automated image analysis. The first histological signs of interstitial fibrosis and tubular alterations were detected as early as 3 mo in ZO rat kidneys (Fig. 1). These alterations rapidly widened and worsened to affect large areas of the kidney cortex (Figs. 1 and 2). As shown in Fig. 3, collagen fibers appear as green (type III collagen) and yellow-red structures (type I collagen) under polarized light. Green structures were found in small foci, randomly dispersed in a field. Their surface (Fig. 3E) and number (data not shown) densities increased regularly with age in both groups, with the increment being more in ZO than in ZL rats. By contrast, yellow-red structures accumulated in large figures (>= 300 µm2) and almost never in small foci. Large figures, insignificant at 1 mo, appeared scattered at 6 mo onward in each measured field. With age, their surface (Fig. 3F) and number (not shown) densities increased in parallel, slowly in ZL but dramatically in ZO rats, to overcome, at 12 mo, fivefold the lean value.


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Fig. 1.   Semiquantitative index of tubulointerstitial damage. Kidney lesions, graded on a scale of 0-4 (see METHODS), occurred as early as 3 mo in Zucker obese (ZO) rats and extensively increased with age (n = 6 rats/group except in 12-mo-old obese group where n = 5 rats). *P < 0.05, ZO vs. Zucker lean (ZL) values at a given age.



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Fig. 2.   Histological aspect of 9-mo-old lean (A) and obese (B) rat kidneys. Focal and segmental glomerular hyalinosis as well as tubular and interstitial lesions were commonly seen in obese rats but not in lean littermates. Mononuclear cells invaded areas of interstitial fibrosis (*) in close vicinity of sclerosed glomeruli (arrows) and damaged tubules, which present microcystic formations without (T) or with (C) tubular casts. Bar = 50 µm.



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Fig. 3.   Computer-assisted image analysis of kidney cortex sections of ZL (A and C) and ZO (B and D) rats at 3 (A and B) and 12 (C and D) mo of age. With the use of cross-polarizing filter and sirius red staining, collagens appear as green deposits (mainly type III collagen) found in small foci (arrows) and yellow-red structures (mainly type I collagen) found in large figures and little in small foci. Small foci surface density was higher in ZO than in ZL rats at all ages (E). Large figures dramatically extended after 6 mo in ZO rats (D and F; n = 6 rats/group except in 12-mo-old obese group where n = 5 rats). Bar = 50 µm. When age-group interaction was not significant, ZO vs. ZL values differed at all ages (P < 0.05). *P < 0.05, ZO vs. ZL values at a given age.

Immunohistochemistry. Type I collagen immunolabeling was occasionally and equally seen in ZL rats (Fig. 4A) and in young ZO rats (data not shown), limited to the periglomerular and periarterial interstitium. By contrast, in old ZO rats, a large accumulation of collagen I was found in areas of interstitial fibrosis (Fig. 4B). Type III collagen (Fig. 4, C and D) was evenly and similarly detected in arterial and arteriolar walls, whatever the age and the genotype of the animals. In young ZO rats, it was also scattered in the periarteriolar and peritubular interstitium, with the pattern increasing with age (Fig. 4D). Type IV collagen (data not shown) was limited to the glomerular matrixes and to the tubular basement membranes. It increased with age, more in ZO than in ZL rats. In young rats, fibronectin was mainly seen in arterial and arteriolar walls. Like collagen III, a few labeled dots were found scattered in the interstitium of young ZO rats (data not shown). In old rats (Fig. 4, E and F), fine and sparse fibronectin labeling was evidenced in ZL rats, whereas extensive labeling was detected in areas of interstitial fibrosis in ZO littermates. In young rats, a sparse TGF-beta 1 immunostaining (Fig. 5, A and B) was confined to perivascular and periglomerular areas. It increased with age in both groups of animals (Fig. 5, C and D) and was extensively found superimposed to large areas of interstitial fibrosis in 9-mo-old ZO rats. TGF-beta 1 immunostaining was never observed in glomeruli or in tubular epithelial cells. Intense immunostaining of alpha -SMA protein (Fig. 5, E and F) was constantly and similarly observed in the arterial and arteriolar walls of both young and old ZL and ZO rat kidneys. In old ZO rats, additional staining was seen in areas of interstitial fibrosis (Fig. 5F). MCP-1 was never detected whatever the age and the genotype of the animals (data not shown). In ZL rats and young ZO rats, a small, similar amount of monocytes/macrophages (2-3 ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cells/mm2 interstitial surface area) was occasionally seen scattered in the kidney cortex. In ZO rats, ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cell density markedly increased after 6 mo to overcome fivefold the ZL value at 9 mo (Fig. 6). ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cell values correlated well with proteinuria, cholesterolemia, and triglyceridemia (r comprised between 0.625 and 0.835, P < 0.0001), whereas a strong correlation was found between the histological tubulointerstitial injury index and ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cells, proteinuria, cholesterolemia, and triglyceridemia (r > 0.880 for those parameters, P < 0.0001). At 3 mo, the density of lymphocytes in the interstitial area was similar in both groups of animals (ZO vs. ZL rats: 120.5 ± 21.8 vs. 126.7 ± 11.2 cells/mm2, P = 0.81, n = 5 rats/group).


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Fig. 4.   Immunolabeling of extracellular matrix components in 9-mo-old ZL (A, C, and E) and ZO (B, D, and F) rats. Type I collagen (A and B) was extensively found in areas of fibrosis in ZO rats (B). Small labeled spots of type III collagen (C and D) were seen in vessel and arterial walls of both ZL and ZO rats (arrows) and in areas of fibrosis in ZO rats (D). Fibronectin (E and F) also contributed to the fibrosis process in ZO rats (F). Bar = 100 µm.



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Fig. 5.   Immunolabeling of transforming growth factor (TGF)-beta 1 in 3- and 9-mo-old ZL (A and C) and ZO (B and D) rats and of alpha -smooth muscle actin in 9-mo-old ZL (E) and ZO (F) rats. TGF-beta 1, restricted to perivascular and periglomerular interstitial areas, was detected as early as 3 mo in ZL (A) and ZO (B) rats. It was abundantly found in areas of fibrosis in old ZO rats (D). alpha -smooth muscle actin (SMA) was constantly observed in the arterial and arteriolar walls of both ZL (E) and ZO (F) rats (arrows), whereas additional staining was seen in areas of fibrosis (F, double arrow). Bar = 100 µm.



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Fig. 6.   Immunohistochemical detection of monocytes/macrophages (ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cells). ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cell density markedly increased in ZO rats after 6 mo (n = 6 rats/group except in 12-mo-old obese group where n = 5 rats). *P < 0.05, ZO vs. ZL values at a given age (age-group interaction).

RT-PCR. As shown in Fig. 7, mRNAs of the proteins involved in the ECM turnover [type I and III collagens, fibronectin, and tissue inhibitor of metalloproteinase (TIMP)-1] were generally overexpressed in both lean and obese groups at 1 mo compared with the 3-mo expression, reflecting the growing process of young pups. The expression of alpha 2-collagen I mRNA remained at a low level and was roughly similar in lean and obese rat kidney cortex until 6 mo, when it increased in ZO rats to over eight- and fourfold the ZL value at 9 and 14 mo, respectively. mRNA for alpha 1-collagen III was overexpressed in ZO rats compared with ZL rats at 3 mo and onward. After 6 mo, the value increased modestly in ZL rats but rose in ZO rats to over fourfold the ZL value at 14 mo. alpha 1-Collagen IV mRNA was steadily and roughly equally expressed in both groups of animals (data not shown). After 1 mo, the expression of fibronectin mRNA increased in ZO rat kidney between 3 and 9 mo and rose significantly afterward, whereas fibronectin mRNA was almost undetectable in ZL animals before 9 mo. mRNA encoding TIMP-1 was overexpressed in ZO rats compared with ZL littermates as early as 3 mo of age. With age, it regularly increased in the ZO group but remained steadily expressed in ZL rats. By contrast, no difference in the expression of mRNA coding for TIMP-2 was noted between ZL and ZO rats, despite its age-dependent increase in both groups (data not shown). alpha -SMA mRNA remained steadily expressed, at a similar level, in both ZL and ZO rats. Although no difference in the expression of platelet-derived growth factor A (PDGF-A) mRNA was noticed between ZL and ZO rats, whatever the age of the animals (not shown), renal TGF-beta 1 mRNA was overexpressed in ZO rat cortex at the third month and onward. Osteopontin (Opn) mRNA increased in ZO rat kidney cortex at 3 mo and onward compared with lean littermates. RANTES (regulated on activation, normal T cell expressed and secreted) mRNA remained at the same range of expression at all ages, and no difference was seen between ZL and ZO rats. No enhanced expression of intercellular and vascular cell adhesion molecule-1 (ICAM-1 and VCAM-1, respectively) mRNA was noticed in ZO rats compared with ZL littermates, and mRNA coding for MCP-1 was never detected whatever the age and the genotype of the animals (data not shown).


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Fig. 7.   Synthesis of mRNAs encoding alpha 2- and alpha 1-chains of type I and III collagens (Coll), fibronectin, tissue inhibitor of metalloproteinase (TIMP)-1, alpha -SMA, TGF-beta 1, osteopontin, and RANTES in ZL and (regulated on activation, normal T cell expressed and secreted) ZO rats. In all PCR experiments, the intensities of the cDNA bands for each protein were normalized to the glyceraldehyde-3-phosphate dehydrogenase band intensities. See text for additional information.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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To establish the time course of the renal interstitial fibrosis process in the ZO rat model, we followed at regular time points, in a long-course study, the expression of mRNA coding for proteins involved in ECM turnover. Protein accumulation was evaluated by immunolabeling of kidney sections from 3-mo-old rats, when the first lesions became visible, and 9-mo-old rats, when lesions are well established (Fig. 1 and Refs. 23 and 32). Interstitial fibrosis developed early in young obese rats: the synthesis of type III collagen and fibronectin was amplified in 3-mo-old ZO rat kidney compared with age-matched ZL littermates, as shown by corresponding mRNA overexpression and protein accumulation in scattered interstitial dots. Concomitantly, the overexpression of TIMP-1 mRNA probably led to a reduction in ECM catabolism. These changes were associated with an increased expression of TGF-beta 1 and occurred in the absence of any infiltration of macrophages and lymphocytes in the interstitium. In a second phase, numerous macrophages invaded the interstitium between 6 and 9 mo, in parallel with an overexpression of type I collagen mRNA and accumulation of the protein in large areas of interstitial fibrosis and a worsening of tubulointerstitial damages and proteinuria.

The technique of collagen fiber density evaluation by computer-assisted morphometry and sirius red polarization microscopy has been validated by several authors (30 and references therein). The increased expression of collagen III mRNA fits well with the accumulation of the protein, shown by morphometry as green small foci whose surface area density increased with age, more in ZO than in ZL rat kidneys, and by immunolabeling of the interstitium. This increased expression reflects the regular thickening process of the kidney cortex arterial walls that occurs with age. It also mirrors the development and progression of focal interstitital fibrosis in ZO rats. Although fibronectin also participated in the fibrosis process, another ECM component, type IV collagen, was confined to glomeruli and tubular basement membranes. When measured on microdissected, isolated glomeruli, the expression of alpha 1-collagen IV mRNA increased with age (23), but it remained at a steady level between ZL and ZO rats when measured on the whole cortex. This is probably due to the high ratio of tubular basement membranes over glomerular ECM, which masks the increment of glomerular alpha 1-collagen IV mRNA. By contrast, collagen I mRNA and protein accumulation did not differ between the two experimental groups at 3 mo.

Besides de novo synthesis of ECM components, reduction of their catabolism should occur in young, 3-mo-old ZO rats. The observation of an increased expression of mRNA coding for TIMP-1, an enzyme that specifically inhibits 72-kDa metalloproteinase, and data showing an upregulation of TIMP-1 in several experimental models of renal fibrosis (12, 15, 19, 41) converge to make TIMP-1 an important and common mediator of interstitial fibrosis. By contrast, the expression of mRNA for TIMP-2, an inhibitor of 92-kDa metalloproteinase, remained similar in ZO and ZL rats at all ages. This result differs from the observations we have made on isolated glomeruli of the same animals, in which both TIMP-1 and TIMP-2 mRNAs were overexpressed (23). This indicates that the turnover of ECM components in the glomerulus and in the interstitium diverges. The source of TIMP-1 remains unknown. It could come from the glomerulus (23) and/or be synthesized by arterial or arteriolar endothelial cells as suggested by the presence of TIMP-1 mRNA in endothelial cells from the heart (6) or from areas of head and neck carcinomas (33). Further in situ hybridization experiments are necessary to precisely define the cellular source of this metalloproteinase inhibitor.

In response to one or several stimuli, TGF-beta 1, but not PDGF-A, is produced in ZO rat kidney cortex as shown by RT-PCR and immunohistochemistry experiments. TGF-beta 1 mRNA was overexpressed as early as 3 mo and increased afterward. TGF-beta 1 protein was restricted to the interstitial area but was never observed in glomeruli or in tubular epithelial cells. It accumulated with age in ZO rats in areas of fibrosis. The lack of labeling in tubular cells remains puzzling compared with the increase of TGF-beta 1 seen in tubular epithelial cells in nephrotic syndrome and diabetic nephropathy (43), in streptozotocin-induced diabetic rats (17), or in aging rats (37) and questions the source of circulating TGF-beta 1 seen in ZO rats. One possibility could be an autocrine production of this cytokine by interstitial fibroblasts on activation. Indeed, besides an intense immunolabeling of alpha -SMA in vessel and arteriolar walls, an additional staining was observed in areas of interstitial fibrosis in old ZO rats, indicating the presence of myofibroblasts in the interstitium. However, the expression of alpha -SMA mRNA remained at a steady level in both groups of animals at all ages. This is probably due to the high ratio of alpha -SMA in vessel walls over areas of fibrosis, which masks the fibrotic increment of alpha -SMA mRNA. The scatter of alpha -SMA immunolabeling that we have observed may also suggest that activation of fibroblasts was limited to cytokine and/or ECM synthesis rather than to cell proliferation and general transformation into myofibroblasts. Further experiments are necessary to clarify these points.

The onset of interstitial fibrosis at 3 mo appeared independent of any inflammatory cell accumulation in ZO rat kidneys. The first sign of macrophage/ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cell infiltration in the interstitium occurred at 6 mo, long after the onset of interstitial fibrosis. If macrophages do not trigger the fibrosis onset, they most likely worsen the fibrosis process once initiated, as already suggested in ZO rats (24, 25) and in other experimental models (reviewed in Ref. 10). Yet, an activation of a few resident monocytes/macrophages in ZO, but not in ZL rat kidney, at an early age cannot be completely excluded. However, the density of such resident cells was low (2-3 cells/mm2 interstitial surface area). Moreover, the lack of expression of several major components generally found during inflammation strengthens this immunohistochemical evidence of a nonoccurrence of inflammatory cells in the first steps of interstitial fibrosis in young, 3-mo-old ZO rats. mRNAs coding for either RANTES, a potent inflammatory chemokine expressed in several glomerulopathies and in experimental models of protein overload (46), or ICAM-1 and VCAM-1, two specific adhesion molecules generally involved in the trapping of circulating inflammatory cells (5), remained similarly expressed in the two groups of animals. More surprinsingly, MCP-1, a C-C chemokine for monocytes that has been detected in a variety of experimental and human glomerulonephritis (36) and in diabetic patients (35) and animals (38), was not seen in this ZO rat model. We failed to detect any mRNA coding for MCP-1 in lean or obese Zucker rats while, with the same primer and conditions of polymerization, MCP-1 mRNA was detected in our laboratory in streptozotocin-induced diabetic rats (38). Immunohistochemical detection of MCP-1 was also negative, regardless of the experimental group and the age of the animals. Recently, Ihm (18) has proposed that MCP-1 production and secretion are stimulated by high glucose concentrations and may have a role in the pathogenesis of diabetic nephropathy in its early phase. Treatment of streptozotocin-induced diabetic rats with insulin also dropped the MCP-1 mRNA expression to the nondiabetic, control rat values, in parallel with a drop in hyperglycemia (38). These observations might explain the absence of MCP-1 expression in our normoglycemic, obese Zucker rats. Conversely, in a recent paper, Coimbra et al. (7) have shown an influx of monocytes/macrophages (ED<UP><SUB>1</SUB><SUP>+</SUP></UP> cells) in the cortical and medullary interstitium of ZO rats that peaked at 14 wk (3 mo) and decreased thereafter. However, the strain of Zucker rats that has been used by these authors developed a mild hyperglycemia at 14 wk and onward. Altogether, these observations suggest that glucose must have a peculiar role in the recruitment of inflammatory cells that accompanies the renal fibrosis process in several circumstances such as diabetic nephropathy. The invasion of macrophages in the renal interstitium that we have observed from 6 mo onward might be the result of the overexpression of Opn mRNA and release of the protein in ZO rat kidney. Opn is a highly acidic, secreted glycoprotein that was originally isolated as a bone matrix molecule. It has chemotactic effects, and recent studies have shown an association between mononuclear cell infiltrates and the expression of Opn (reviewed in Ref. 45). The stimulus for Opn mRNA expression in ZO rats still remains unknown. It could be a direct consequence of the tubular injury that developed at 3 mo and onward. Unless insulin and other growth factors have a direct effect on tubular and interstitial cells, tubular alterations might probably result from the alteration of the glomerular filtration barrier and of the ensuing leak of proteins (29, 42). Indeed, the first step of podocytosis, in which glomerular podocytes are filled with numerous albumin vesicles, occurs very early in our rat model, long before definite proteinuria is measured (2, 27). Next, proximal tubular cells become rapidly clogged with lysosomes of endocytosed proteins and release into the intercellular medium inflammatory molecules and cellular debris that, in turn, induce interstitial inflammation and fibrosis (14, 22, 28).

Instead of inflammation, hyperinsulinemia and hyperlipidemia are good candidates to trigger the renal fibrotic process in young, 3-mo-old ZO rats. In vitro experiments suggest that insulin activates glomerular mesangial cells (1). However, to date, no clear data can strengthen this hypothesis in vivo (27). On the other hand, low (LDL)- and very-low-density lipoproteins (VLDL) likely play a key role in the genesis of interstitial fibrosis in obese Zucker rats. Hyperlipidemia is present as early as weaning and worsens at an early age in ZO rats, and native LDL/VLDL are able to activate target cells presenting apo B/E receptors (4, 26, 44). We have recently demonstrated that an oxidative stress occurs in ZO rat kidney as early as 3 mo (32). Such an oxidative stress could trigger, on its own, the fibrosis process, as recently shown by Scheuer et al. (39) in hyperlipidemia-induced renal damage. Reactive oxygen species are also known to stimulate target cells (34) and to alter the glomerular filtration barrier, leading to the leak of proteins in glomerular ultrafiltrate (3) and the ensuing activation of tubular cells.

In conclusion, this study shows that interstitial fibrosis first occurred in 3-mo-old ZO rat kidney, probably independent of any lymphocyte/macrophage recruitment. The high level of LDL/VLDL that exists in young ZO pups and the occurrence of an oxidative stress that develops at an early age (32) are conceivably two key factors of the fibrotic process. Once activated, interstitial fibroblasts and/or tubular cells might synthesize ECM components, either directly or through the stimulus of cytokines (21). They might also synthesize chemokines, such as Opn, which induces an inflammatory process and provides an amplifier loop in the fibrotic process with an additional release of TGF-beta 1 and an overexpression of type I and III collagens.


    ACKNOWLEDGEMENTS

We acknowledge the skillful technical assistance of Michel Paing in photography. We also thank Drs. Jean-Paul Duong Van Huyen, Srinivas Kaveri, and Antonino Nicoletti (Institut National de la Santé et de la Recherche Médicale Unité 430) for helpful discussions during the preparation of the manuscript.


    FOOTNOTES

Part of this work was presented in abstract form at the American Society of Nephrology Annual Meeting in San Antonio, TX, in November 1997.

Address for reprint requests and other correspondence: J. Chevalier, Immunopathologie Rénale, INSERM U 430, Hôpital Broussais, 96 Rue Didot, 75674 Paris Cedex 14, France (E-mail: jacques.chevalier{at}brs.ap-hop-paris.fr).

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.

Received 1 May 2000; accepted in final form 18 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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