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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-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-1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-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-
-smooth muscle actin (
-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.
|
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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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.
|
|
|
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-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-
1 immunostaining was never observed in glomeruli or in tubular epithelial cells. Intense immunostaining of
-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
|
|
|
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 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
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.
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).
-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-
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-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
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
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-1, but not
PDGF-A, is produced in ZO rat kidney cortex as shown by RT-PCR and immunohistochemistry experiments. TGF-
1 mRNA was
overexpressed as early as 3 mo and increased afterward.
TGF-
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-
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-
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
-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
-SMA mRNA remained at a steady level in both groups of animals at
all ages. This is probably due to the high ratio of
-SMA in vessel
walls over areas of fibrosis, which masks the fibrotic increment of
-SMA mRNA. The scatter of
-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
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-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abrass, CK,
Spicer D,
and
Raugi GJ.
Induction of nodular sclerosis by insulin in rat mesangial cells in vitro: studies of collagen.
Kidney Int
47:
25-37,
1995[ISI][Medline].
2.
Bariéty, J,
Chevalier J,
Michel O,
Nochy D,
and
Callard P.
La biopsie rénale dans la hyalinose segmentaire et focale des glomérules.
In: La Biopsie Rénale, edited by Droz D,
and Lantz B.. Paris, France: Les Editions INSERM, 1996, p. 109-134.
3.
Binder, CJ,
Weiher H,
Exner M,
and
Kerjaschki D.
Glomerular overproduction of oxygen radicals in Mpv17 gene-inactivated mice causes podocyte foot process flattening and proteinuria: a model of steroid-resistant nephrosis sensitive to radical scavenger therapy.
Am J Pathol
154:
1067-1075,
1999
4.
Björkerud, S,
and
Björkerud B.
Growth-stimulating effect of lipoproteins on human arterial smooth-muscle cells and lung fibroblasts is due to apo b-containing lipoproteins, type LDL and VLDL, and requires LDL receptors.
Biochim Biophys Acta Mol Cell Res
1268:
237-247,
1995[ISI][Medline].
5.
Brady, HR.
Leukocyte adhesion molecules and kidney diseases.
Kidney Int
45:
1285-1300,
1994[ISI][Medline].
6.
Chua, CC,
Hamdy RC,
and
Chua BHL
Angiotensin II induces TIMP-1 production in rat heart endothelial cells.
Biochim Biophys Acta Mol Cell Res
1311:
175-180,
1996[ISI][Medline].
7.
Coimbra, TM,
Janssen U,
Grone HJ,
Ostendorf T,
Kunter U,
Schmidt H,
Brabant G,
and
Floege J.
Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes.
Kidney Int
57:
167-182,
2000[ISI][Medline].
8.
Diamond, JR.
Analogous pathobiologic mechanisms in glomerulosclerosis and atherosclerosis.
Kidney Int
39:
S29-S34,
1991[ISI].
9.
Drukker, A,
and
Eddy AA.
Failure of antioxidant therapy to attenuate interstitial disease in rats with reversible nephrotic syndrome.
J Am Soc Nephrol
9:
243-251,
1998[Abstract].
10.
Eddy, AA.
Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions.
J Am Soc Nephrol
5:
1273-1287,
1994[Abstract].
11.
Eddy, AA.
Interstitial inflammation and fibrosis in rats with diet-induced hypercholesterolemia.
Kidney Int
50:
1139-1149,
1996[ISI][Medline].
12.
Eddy, AA,
Giachelli CM,
McCulloch L,
and
Liu E.
Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria.
Kidney Int
47:
1546-1557,
1995[ISI][Medline].
13.
Eddy, AA,
Liu E,
and
McCulloch L.
Interstitial fibrosis in hypercholesterolemic rats: role of oxidation, matrix synthesis, and proteolytic cascades.
Kidney Int
53:
1182-1189,
1998[ISI][Medline].
14.
Eddy, AA,
McCulloch L,
Liu E,
and
Adams I.
A relationship between proteinuria and acute tubulointerstitial disease in rats with experimental nephrotic syndrome.
Am J Pathol
138:
1111-1123,
1991[Abstract].
15.
Engelmyer, E,
Van Goor H,
Edwards DR,
and
Diamond JR.
Differential mRNA expression of renal cortical tissue inhibitor of metalloproteinase-1, -2, and -3 in experimental hydronephrosis.
J Am Soc Nephrol
5:
1675-1683,
1995[Abstract].
16.
Floege, J,
Hackmann B,
Kliem V,
Kriz W,
Alpers CE,
Johnson RJ,
Kuhn KW,
Koch KM,
and
Brunkhorst R.
Age-related glomerulosclerosis and interstitial fibrosis in Milan normotensive rats: a podocyte disease.
Kidney Int
51:
230-243,
1997[ISI][Medline].
17.
Gilbert, RE,
Cox A,
Wu LL,
Allen TJ,
Hulthen UL,
Jerums G,
and
Cooper ME.
Expression of transforming growth factor-b1 and type IV collagen in the renal tubulointerstitium in experimental diabetes. Effects of ACE inhibition.
Diabetes
47:
414-422,
1998[Abstract].
18.
Ihm, CG.
Monocyte chemotactic peptide-1 in diabetic nephropathy.
Kidney Int
52:
S20-S22,
1997.
19.
Jones, CL,
Bush S,
Post M,
McCulloch L,
Liu E,
and
Eddy AA.
Pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis.
Kidney Int
40:
1020-1031,
1991[ISI][Medline].
20.
Junqueira, LCU,
Cossermelli W,
and
Brentani R.
Differential staining of collagens type I, II and III by Sirius red polarization microscopy.
Arch Histol Japn
41:
267-274,
1978[Medline].
21.
Keane, WF,
Madias NE,
Harrington JT,
King A,
Pereira B,
Singh A,
Lafayette R,
Neuringer J,
and
Natov SN.
Lipids and the kidney.
Kidney Int
46:
910-920,
1994[ISI][Medline].
22.
Kees-Folts, D,
Sadow JL,
and
Schreiner GF.
Tubular catabolism of albumin is associated with the release of an inflammatory lipid.
Kidney Int
45:
1697-1709,
1994[ISI][Medline].
23.
Lavaud, S,
Michel O,
Sassy-Prigent C,
Heudes D,
Bazin R,
Bariéty J,
and
Chevalier J.
Early influx of glomerular macrophages precedes glomerulosclerosis in the obese Zucker rat model.
J Am Soc Nephrol
7:
2604-2615,
1996[Abstract].
24.
Magil, AB.
Tubulointerstitial lesions in young Zucker rats.
Am J Kidney Dis
25:
478-485,
1995[ISI][Medline].
25.
Matsuda, S,
Arai T,
Iwata K,
Oka M,
and
Nagase M.
A high-fat diet aggravates tubulointerstitial but not glomerular lesions in obese Zucker rats.
Kidney Int
56:
S150-S152,
1999[ISI].
26.
Mazière, C,
Auclair M,
Djavaheri-Mergny M,
Packer L,
and
Mazière JC.
Oxidized low density lipoprotein induces activation of the transcription factor NFkB in fibroblasts, endothelial and smooth muscle cells.
Biochem Mol Biol Int
39:
1201-1207,
1996[ISI][Medline].
27.
Michel, O,
Heudes D,
Lamarre I,
Masurier C,
Lavau M,
Bariéty J,
and
Chevalier J.
Reduction of insulin and triglycerides delays glomerulosclerosis in obese Zucker rats.
Kidney Int
52:
1532-1542,
1997[ISI][Medline].
28.
Nath, KA.
Tubulointerstitial changes as a major determinant in the progression of renal damage.
Am J Kidney Dis
20:
1-17,
1992[ISI][Medline].
29.
Nath, KA.
The tubulointerstitium in progressive renal disease.
Kidney Int
54:
992-994,
1998[ISI][Medline].
30.
Nicoletti, A,
Heudes D,
Hinglais N,
Appay MD,
Philippe M,
Sassy-Prigent C,
Bariéty J,
and
Michel JB.
Left ventricular fibrosis in renovascular hypertensive rats. Effects of losartan and spironolactone.
Hypertension
26:
101-111,
1995
31.
O'Donnell, MP,
Kasiske BL,
Cleary MP,
and
Keane WF.
Effects of genetic obesity on renal structure and function in the Zucker rat. II. Micropuncture studies.
J Lab Clin Med
106:
605-610,
1985[ISI][Medline].
32.
Poirier, B,
Lannaud-Bournoville M,
Conti M,
Bazin R,
Michel O,
Bariéty J,
Chevalier J,
and
Myara I.
Oxidative stress occurs in absence of hyperglycaemia and inflammation in the onset of kidney lesions in normotensive obese rats.
Nephrol Dial Transplant
15:
467-476,
2000
33.
Polette, M,
Clavel C,
Birembaut P,
and
Declerck YA.
Localization by insitu hybridization of messenger RNAs encoding stromelysin-3 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 in human head and neck carcinomas.
Pathol Res Pract
189:
1052-1057,
1993[ISI][Medline].
34.
Poli, G,
and
Parola M.
Oxidative damage and fibrogenesis.
Free Radic Biol Med
22:
287-305,
1997[ISI][Medline].
35.
Rovin, BH,
Doe N,
and
Tan LC.
Monocyte chemoatractant protein-1 levels in patients with glomerular disease.
Am J Kidney Dis
27:
640-646,
1996[ISI][Medline].
36.
Rovin, BH,
Rumancik M,
Tan L,
and
Dickerson J.
Glomerular expression of monocyte chemoattractant protein-1 in experimental and human glomerulonephritis.
Lab Invest
71:
536-542,
1994[ISI][Medline].
37.
Ruiz-Torres, MP,
Bosch RJ,
O'Valle F,
del Moral RG,
Ramirez C,
Masseroli M,
Perez-Caballero C,
Iglesias MC,
Rodriguez-Puyol M,
and
Rodriguez-Puyol D.
Age-related increase in expression of TGF-b1 in the rat kidney. Relationship to morphologic changes.
J Am Soc Nephrol
9:
782-791,
1998[Abstract].
38.
Sassy-Prigent, C,
Heudes D,
Mandet C,
Bélair MF,
Michel O,
Perdereau B,
Bariéty J,
and
Bruneval P.
Early glomerular macrophage recruitment in streptozotocin-induced diabetic rats.
Diabetes
49:
466-475,
2000[Abstract].
39.
Scheuer, H,
Gwinner W,
Hohbach J,
Grone EF,
Brandes RP,
Malle E,
Olbricht CJ,
Walli AK,
and
Grone HJ.
Oxidant stress in hyperlipidemia-induced renal damage.
Am J Physiol Renal Physiol
278:
F63-F74,
2000
40.
Striker, GE,
and
Striker LJ.
Recent advances in diabetic nephropathy: how big a culprit is glucose?
Diabetes Metab
22:
407-414,
1996[ISI][Medline].
41.
Tang, WW,
Feng LL,
Xia YY,
and
Wilson CB.
Extracellular matrix accumulation in immune-mediated tubulointerstitial injury.
Kidney Int
45:
1077-1084,
1994[ISI][Medline].
42.
Van Goor, H,
Ding GH,
Kees-Folts D,
Grond J,
Schreiner GF,
and
Diamond JR.
Macrophages and renal disease.
Lab Invest
71:
456-464,
1994[ISI][Medline].
43.
Wang, SN,
and
Hirschberg R.
Tubular epithelial cell activation and interstitial fibrosis. The role of glomerular ultrafiltration of growth factors in the nephrotic syndrome and diabetic nephropathy.
Nephrol Dial Transplant
14:
2072-2074,
1999
44.
Wheeler, DC,
and
Chana RS.
Interactions between lipoproteins, glomerular cells and matrix.
Miner Electrolyte Metab
19:
149-164,
1993[ISI][Medline].
45.
Wüthrich, RP.
The complex role of osteopontin in renal disease.
Nephrol Dial Transplant
13:
2448-2450,
1998
46.
Zoja, C,
Donadelli R,
Colleoni S,
Figliuzzi M,
Bonazzola S,
Morigi M,
and
Remuzzi G.
Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kB activation.
Kidney Int
53:
1608-1615,
1998[ISI][Medline].