Transforming growth factor-ß1 in the kidney and urine of patients with glomerular disease and proteinuria

Dimitrios S. Goumenos1,, Sotiris Tsakas1, Abdel Meguid El Nahas2, Sotiria Alexandri1, Simon Oldroyd2, Pantelitsa Kalliakmani1 and John G. Vlachojannis1

1 Department of Internal Medicine—Nephrology, University Hospital, Patras, Greece and 2 Sheffield Kidney Institute, Northern General Hospital, Sheffield, UK



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Transforming growth factor-ß1 (TGF-ß1) is the major fibrogenic growth factor implicated in the pathogenesis of renal scarring. Proteinuria is a poor prognostic feature for various types of glomerular disease and its toxic action may be related to the activation of tubular epithelial cells towards increased production of cytokines and chemoattractant peptides. In this work we studied the site of synthesis and expression profile of TGF-ß1 in the renal tissue of patients with heavy proteinuria and examined the relation of this expression with the urinary excretion of TGF-ß1.

Methods. Twenty-five patients with heavy proteinuria (8.4±3.0 g/24 h) were included in the study. All patients underwent a diagnostic kidney biopsy and were commenced on immunosuppressive therapy with corticosteroids and cyclosporin. The sites of synthesis and expression profile of TGF-ß1 mRNA and protein in the kidney were examined by in situ hybridization and immunohistochemistry. Urinary and plasma TGF-ß1 levels were determined by ELISA before the initiation of treatment and 6 months later and compared with those of normal subjects and of patients with IgA nephropathy and normal urinary protein excretion.

Results. The site of synthesis and expression of TGF-ß1 in the renal tissue of patients with heavy proteinuria was mainly localized within the cytoplasm of tubular epithelial cells. Interstitial expression was also present but glomerular TGF-ß1 expression was found only in patients with mesangial proliferation. Urinary TGF-ß1 excretion was significantly higher in nephrotic patients compared with normal subjects and with patients with IgA nephropathy and normal urinary protein excretion (783±280 vs 310±140 and 375±90 ng/24 h, respectively; P<0.01). In patients with remission of proteinuria after immunosuppressive therapy, urinary TGF-ß1 excretion was significantly reduced (from 749±290 to 495±130 ng/24 h; P<0.01), while in patients with persistent nephrotic syndrome, it remained elevated.

Conclusions. The localization of TGF-ß1 mRNA and protein within tubular epithelial cells, along with its increased urinary excretion in patients with nephrotic syndrome, suggest the activation of these cells by filtered protein towards increased TGF-ß1 production.

Keywords: glomerular injury; proteinuria; transforming growth factor-ß1



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß) represents a group of 25-kDa proteins that are actively involved in the development and differentiation of various tissues [1]. TGF-ß was originally isolated from platelets but it has also been found in cell cultures of monocytes/macrophages and mesangial cells [2]. Three isoforms of TGF-ß have been identified in mammalian species: TGF-ß1, TGF-ß2 and TGF-ß3. TGF-ß1, the most important isoform in humans, is secreted from cells in the form of a high-molecular-weight latent complex [3]. Cleavage of this larger precursor molecule is necessary for TGF-ß1 activation [2,3]. TGF-ß1 targets specific receptors in various cell types and often exerts its biological effects through autocrine and paracrine pathways [2,3]. It is implicated in the accumulation of extracellular matrix (ECM) via increased synthesis and decreased degradation of its components and via upregulation of integrins on cell surface, thus facilitating the deposition of matrix [4].

TGF-ß1 is the main modulator of the healing process that follows injury of various tissues [4]. Normally, TGF-ß1 release ceases by feedback mechanisms when the healing process has been completed [3,4]. However, if TGF-ß1 release is not switched off, ECM components are accumulated and tissue fibrosis occurs [4]. Increased TGF-ß1 synthesis in the renal tissue followed by accumulation of collagen and development of glomerulosclerosis has been documented in experimentally-induced glomerular disease [5,6], while the administration of specific antiserum against TGF-ß1 results in amelioration of glomerular injury [7]. Although the exact role of TGF-ß1 in the progression of human renal disease has not been established, accumulating evidence suggests that TGF-ß1 is actively involved in the development of renal scarring [8,9].

Proteinuria is a hallmark of many types of glomerular disease and represents an independent risk factor for poor outcome [10]. Its toxic action is attributed to the release of various mediators by tubular epithelial cells following the uptake of filtered protein [10,11]. Exposure of proximal tubular epithelial cells in culture to albumin, immunoglobulins or transferrin is accompanied by enhanced synthesis of vasoactive and chemoattractant peptides, such as endothelin-1 and monocyte chemoattractant protein-1 (MCP-1), that are mainly polarized towards the basolateral compartment [12,13]. This might be crucial for the tubulointerstitial inflammatory response and structural injury observed in proteinuric nephropathies [11].

The purpose of this study was to investigate TGF-ß1 mRNA and peptide localization in the renal tissue of patients with proteinuric nephropathies and to examine any potential relationship of TGF-ß1 renal expression with its excretion in the urine along with the degree of proteinuria and the severity of histopathological involvement.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Twenty-five patients (14 males and 11 females), 51±16 years old, with severe proteinuria (8.4±3.0 g/24 h) and normal renal function (creatinine clearance 91±10 ml/min), were studied. The histopathological diagnoses were membranous nephropathy (n=11), focal segmental glomerulosclerosis (FSGS) (n=5), minimal changes disease (n=3), mesangiocapillary glomerulonephritis (n=3) and diffuse lupus nephritis (n=3). None of the patients had been treated by either angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists for hypertension or proteinuria. All patients were commenced on immunosuppressive therapy with prednisolone (0.5–1 mg/kg/day) and cyclosporin A (2–3 mg/kg/day). The dose of prednisolone was gradually reduced to 5–10 mg on alternate days at the end of a 6 month follow-up period while the dose of cyclosporin A was regulated according to the blood levels (80–100 ng/ml).

The localization of TGF-ß1 mRNA and TGF-ß1 protein were studied by in situ hybridization and immunohistochemistry, respectively, in kidney sections from the biopsies of nephrotic patients. Renal tissue sections with normal renal parenchyma, from 10 patients who underwent nephrectomy for renal cell carcinoma, were used as controls.

Protein excretion and TGF-ß1 levels were measured at the day of renal biopsy and before the initiation of immunosuppressive therapy, in 24 h urine collection. A second measurement of urinary TGF-ß1 levels was performed after 6 months of treatment. Eight patients with IgA nephropathy and normal renal function without proteinuria (urinary protein <0.3 g/24 h) and 14 healthy subjects were used as control groups for the comparison of urinary TGF-ß1 excretion and plasma levels.

Urinary protein was measured in 24 h urine collection at least once every month of follow-up. Complete remission of the nephrotic syndrome was considered in cases with proteinuria <0.3 g/24 h, while partial remission when proteinuria was between 0.3 and 3 g/24 h. No remission was considered in cases with persistent proteinuria above 3 g/24 h.

Conventional histopathology
Renal tissues were taken with a Tru-cut 14G biopsy needle and fixed in 10% neutral formalin, embedded in paraffin and examined in multiple sections (4 µm each). The histological study included haematoxylin and eosin staining, periodic acid–Schiff (PAS), Masson trichrome and reticulin–silver methenamine staining. The severity of glomerular disease was estimated by light microscopy. Samples containing less than 10 glomeruli were excluded. The severity of glomerulosclerosis was expressed as the percentage of sclerosed glomeruli. The severity of interstitial fibrosis and inflammation as well as tubular atrophy was estimated from Masson trichrome stained sections by morphometric analysis based on point counting as previously described [14,15].

In situ hybridization
A cDNA sequence specific for murine TGF-ß1 in pBluesribe (+) was kindly provided by Dr R.J. Akhurst (Glasgow University, UK). The TGF-ß1 insert was placed in the sense orientation relative to the T3 promoter. Antisense and sense RNA riboprobes were generated following digestion with the restriction enzymes (Appligenen) EcoRI and HindIII, respectively [16]. A 600 bp fragment of the RGR5 cDNA sequence [17] specific for rat pro {alpha}1 (III) collagen was kindly provided by Dr V. Wilson (Guy's and St Thomas' Medical School, UK). The BamHI/EcoRI fragment was inserted into pGem 3Z+ in the sense orientation relative to the T7 promoter. Antisense and sense RNA riboprobes were generated following digestion with the restriction enzymes EcoRI and BamHI, respectively. Riboprobes were labelled with digoxigenin rUTP (Boehringer Mannheim, UK) using Riboprobe Gemini II in vitro Transcription System (Promega, UK) (21 µM rUTP, 11 µM DIG-r-UTP). Following labelling the RNA probe was cleaned using nick extraction. The resulting RNA pellet was resuspended at a concentration of 100 ng/µl. Five-micrometre sections of formalin fixed paraffin-embedded renal biopsy material were placed onto 3-amino-propyltriethoxysilane (APES)-coated microscope slides. Sections were rehydrated, treated with 5 µM levamisole—an inhibitor of endogenous alkaline phosphatase—and 0.2 N hydrochloric acid for 20 min and then digested with 5 µg/ml proteinase K (Sigma) for 30 min at 37°C. The sections were subsequently postfixed in 1% paraformaldehyde, washed and dehydrated. Digoxigenin (DIG)-labelled riboprobes were denatured for 10 min at 65°C and added to the hybridization solution containing 50% deionized formamide, 5 mM EDTA, 10 mM NaH2PO4, 0.2 mg/ml herring sperm DNA (Promega), 0.1 mg/ml yeast tRNA (Sigma), 2x SSC, 1x Denhardt's solution and 10% dextran sulphate. Hybridization was performed with 10 ng/ml of DIG-labelled riboprobes added to each slide and incubated for 18 h at 50°C in a humid chamber. The slides were washed sequentially with 5x SSC and 2x SSC containing 50% formamide at 50°C and then treated with 20 µg/ml RNase A (Sigma) for 30 min at 37°C. Further washing was continued once with 2x SSC and once with 0.2x SSC at 37°C. Slides were washed with Tris-buffered saline (TBS; 0.1 M Tris–HCl pH 7.5 and 0.15 M NaCl) and incubated with TBS containing 10% normal sheep serum and 1.5% blocking reagent (Boehringer Mannheim, UK), for 60 min at room temperature (RT). The sections were then incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (Boehringer Mannheim) diluted 1:500 (1.5 U/ml) in TBS containing 1% normal sheep serum, for 60 min at RT. The sections were finally washed in TBS and then in alkaline buffer (0.1 M Tris–HCl pH 9.5, 0.1 M MgCl2). The colour substrate solution consisting of nitroblue-tetrazolium-chloride/5-bromo-4-chloro-3-indoylphosphate (NBT/BCIP) ready-to-use tablets (1 tablet to 10 ml DEPC-treated water) was added to each section. The colour reaction was developed in the dark at 37°C, stopped with distilled water and mounted under aqueous mounting medium (60% glycerol).

Immunohistochemistry
TGF-ß1 was detected immunohistochemically on kidney biopsy sections. Briefly, formalin-fixed paraffin-embedded kidney sections (4 µm) were cut and mounted on gelatinized slides. Dewaxed, hydrated sections were processed in a microwave oven for 3 cycles (5 min each at 450 W) in citrate buffer pH 6.0. After cooling, sections were quenched with H2O2 3% in 100% methanol for 20 min to inhibit endogenous peroxidase activity. Samples were then treated with Protein Blocking Agent to reduce the non-specific binding of antibodies. Slides were washed in phosphate buffered saline (PBS) and incubated with a polyclonal anti-TGF-ß1 antibody (1:50) (Santa Cruz, USA) for 1 h at 37°C, in a humid atmosphere. Following antibody treatment, slides were washed in PBS and incubated for 10 min with a polyvalent biotinylated secondary antibody (Kwik kit, IMMUNONTM, USA). Slides were then washed in PBS, incubated with streptavidin–peroxidase reagent for 10 min and washed again with PBS. Visualization of the antigen–antibody complex was achieved by incubating slides in diamine-benzidine (DAB) chromogen solution for 10–20 min, until a satisfactory colour development. Finally, slides were washed in water, counterstained with haematoxylin for 2 min and mounted with resin mounting medium. All steps, except that of the primary antibody, were performed at RT. Control sections were incubated with non-immune rabbit antiserum or processed after the omission of the primary antibody.

Morphometric analysis
The extent of TGF-ß1 immunostaining as well as the severity of interstitial fibrosis, inflammation and tubular atrophy were estimated stereologically [15]. Transverse sections of the cortical zone of each kidney section were selected and viewed under a light microscope at magnification x200. A square lattice of 100 points with a total surface area of 0.016 mm2 was superimposed onto the tissue. Data were collected from a series of randomly selected adjacent fields (10–15) extending throughout the biopsy core. Points falling on stained tissue were recorded and their percentage out of the total number of measured points was calculated.

Determination of urinary and plasma TGF-ß1 levels
The concentration of TGF-ß1 was measured by enzyme immunoassay, according to Honkanen et al. [18]. Microtitre plates (Costar, USA) were coated with 0.1 µg/well mouse monoclonal anti-TGF-ßs antibodies (Genzyme Co., USA) in 0.05 M Na2CO3 buffer pH 9.0, by incubating overnight at 4°C. The wells were washed with PBS, 0.05% Tween-20. One-hundred microlitres of standard dilutions (R&D Systems, UK) and undiluted samples were acid-activated (1 N HCl, for 2 h at RT) and neutralized (1.2 N NaOH/0.5 M HEPES). One-hundred microlitres of neutralized samples were incubated in the wells overnight at 4°C. The TGF-ß1 bound onto the wells was then detected with a rabbit polyclonal anti-TGF-ß1 antibody (R&D Systems) labelled with horseradish peroxidase (200 µl, 1.5 h at RT). Peroxidase activity was determined using tetramethylbenzidine (TMB) as a substrate (R&D Systems). The intra-assay and inter-assay coefficients of variation (CV) were 7.4 and 6.3%, respectively. The recovery of TGF-ß1 standards (50 and 100 pg/ml) ranged from 83 to 112%. The TGF-ß1 urinary excretion was calculated as ng/24 h and plasma TGF-ß1 concentration was calculated as ng/ml.

Statistical analysis
The results were expressed as means±SD. Differences of TGF-ß1 excretion in urine between the patient's group and the group of healthy subjects were determined by comparison of their mean values, using Student's t-test for unpaired data. Differences of TGF-ß1 excretion in urine between first and second measurement in proteinuric patients treated by immunosuppressive drugs were determined using Student's t-test for paired data. Linear regression analysis was used to correlate the severity of immunostaining for TGF-ß1 with histological markers of renal damage. A P-value of <0.05 was considered to be significant.



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
General observations
All patients were treated with a combination of prednisolone and cyclosporin A. Twenty-one patients showed remission of the nephrotic syndrome (14 complete and seven partial), while four patients had persistent nephrotic syndrome after 6 months of treatment (Table 1Go). The mean time to remission was 4.0±0.5 months. Relapses of the nephrotic syndrome were observed in six out of the 21 patients (30%) following the reduction of prednisolone dose. The diagnoses of these patients were membranous nephropathy in three, minimal changes in two and FSGS in one, and all patients responded to increase dose of prednisolone. The renal function was stable in all patients and none of them developed arterial hypertension or other adverse reactions related to the administration of cyclosporin and corticosteroids.


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Table 1.  Remission rate of nephrotic syndrome in patients with various types of glomerular disease

 

In situ hybridization
TGF-ß1 mRNA was detected by in situ hybridization in renal biopsies from patients with glomerular disease and heavy proteinuria. The TGF-ß1 mRNA was mainly localized within proximal and distal tubular epithelial cells (Figure 1aGo) and, to a lesser extent, in the renal interstitium. Sparse TGF-ß1 mRNA expression was detected in the glomeruli of two patients with severe mesangial proliferation. Sections stained with a sense riboprobe for TGF-ß1 were negative (Figure 1bGo). Sections from the control group were also negative for TGF-ß1 mRNA.



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Fig. 1.  (a) In situ hybridization histochemistry (digoxigenin) showing mRNA for TGF-ß1 (red colour) in the tubules of a kidney section with diffuse lupus nephropathy (x400). (b) In situ hybridization with a sense riboprobe for TGF-ß1 showing no staining in the kidney of a patient with membranous nephropathy (x400).

 

Immunohistochemistry
Renal tissue sections were stained immunohistochemically for TGF-ß1 and displayed TGF-ß1 protein expression within the cytoplasm of proximal, distal and collecting tubular epithelial cells of proteinuric patients (Figure 2aGo). Interstitial expression was less prominent and not present in all patients, whereas glomerular TGF-ß1 expression was found only in the renal tissue from patients with mesangial proliferation. Sections from the control group were negative for TGF-ß1 protein (Figure 2bGo). Intense TGF-ß1 immunostaining in the tubulointerstitial area was observed in seven patients with membranous nephropathy (63%), in three with FSGS (60%), in one with minimal changes disease (33%), in two with mesangiocapillary glomerulonephritis (66%) and in two with diffused lupus nephritis (66%). The rest of the patients showed a weak expression of TGF-ß1 in the same area. However, it is of note that the severity of TGF-ß1 immunostaining in the tubulointerstitial area was positively correlated with the degree of interstitial inflammation (r=0.510, P<0.05), fibrosis (r=0.454, P<0.05) and tubular atrophy (r=0.475, P<0.05).



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Fig. 2.  (a) Immunohistochemistry for TGF-ß1 protein expression (brown colour) within the tubular epithelial cells of a kidney with membranous nephropathy (x200). (b) Immunohistochemistry for TGF-ß1 protein in a control kidney section, showing no TGF-ß1 expression (x200).

 

Urinary TGF-ß1 excretion and plasma TGF-ß1 concentration
TGF-ß1 was determined in 24 h urine collections. The excreted amount of TGF-ß1 in the urine of patients with proteinuria was significantly higher compared with that of patients with IgA nephropathy and normal urinary protein excretion and with that of healthy subjects (783±280 vs 375±90 and 310±140 ng/24 h, respectively; P<0.01). No difference was observed between the amount of excreted TGF-ß1 in the urine and the type of glomerular disease. However, a significant positive correlation was observed between the urinary TGF-ß1 levels and the amount of protein excretion (r=0.618, P<0.01). Twenty-one patients went into complete or partial remission of the nephrotic syndrome (urinary protein from 8.4±6.0 to 0.2±0.1 and 2.1±1.2 g/24 h, respectively; P<0.01). Four patients remained nephrotic after 6 months of immunosuppressive therapy (urinary protein from 12.5±1.7 to 9.9±3.2 g/24 h; P=NS). The amount of TGF-ß1 excreted in the urine significantly decreased (from 749±290 to 495±130 ng/24 h; P<0.01) in patients with remission of proteinuria (Figure 3Go). However, it remained elevated, without significant difference between the two measurements (962±400 vs 1012±370 ng/24 h; P=NS), in patients with persistent nephrotic syndrome. The plasma concentration of TGF-ß1 showed no difference between patients and healthy subjects (31±18 vs 28±15 ng/ml) as well as between patients with and without remission of proteinuria after immunosuppressive therapy (32±16 vs 30±9 ng/ml; P=NS).



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Fig. 3.  Urinary TGF-ß1 levels before and after 6 months of immunosuppressive therapy in patients with complete and partial remission of nephrotic syndrome (n=21).

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study extends other observations for the involvement of TGF-ß1 in the progression of human glomerular disease [8,9]. Increased production and expression of TGF-ß1 has been described in various types of glomerular disease and it is implicated in the development of renal scarring by facilitating the accumulation of ECM components [4,5].

In the present work, the distribution of TGF-ß1 mRNA and protein in the kidney as well as TGF-ß1 excretion in the urine of patients with glomerular disease and heavy proteinuria were examined. In situ hybridization and immunohistochemistry using specific antibodies revealed that TGF-ß1 mRNA and protein are localized within the cytoplasm of tubular epithelial cells. The extent of TGF-ß1 protein expression differs not only among patients with various types of proteinuric nephropathies, but also among patients with the same type of glomerular disease. However, the extent of TGF-ß1 expression is related to the degree of tubulointerstitial injury. Urinary TGF-ß1 excretion is increased in patients with proteinuria due to glomerular dysfunction, compared with healthy subjects and patients with glomerular disease without proteinuria. In addition, remission of proteinuria with immunosuppressive treatment is followed by decreased urinary TGF-ß1 excretion. It is unlikely that the changes observed in urinary TGF-ß1 excretion are related to the immunosuppressive therapy, as the latter was administered at comparable doses to patients who went into remission and those who did not. Furthermore, cyclosporin has been reported to increase, rather than decrease, renal TGF-ß1 levels [19]. Finally, we cannot attribute the changes in urinary TGF-ß1 excretion to reduction in its filtration in response to cyclosporin-induced decrease in glomerular filtration rate, as there is no reason to believe that the haemodynamic effect of cyclosporin would differ in patients who underwent remission compared with the others. Our findings of increased urinary TGF-ß1 excretion in nephrotic patients are in agreement with those of Honkanen et al. [18], who reported elevated urinary TGF-ß1 levels in patients with membranous nephropathy compared with patients with other types of glomerular disease without heavy proteinuria [18].

It is well known that heavy proteinuria represents an independent risk factor for poor outcome in most types of glomerular disease and it is related to the severity of tubulointerstitial injury [10,11]. The deleterious effect of proteins is attributed to the release of toxic agents from lysosomes that follow the uptake of a large amount of filtered protein from the tubular epithelial cells by endocytosis [10,11]. The stimulation of tubular epithelial cells triggers a cascade of biological reactions that culminate in the transcriptional activation of genes coding for vasoactive and chemoattractant peptides, such as ET-1 and MCP-1 [11,12]. The increased release of such peptides may play a role in the accumulation of monocytes in the renal interstitium and the stimulation of fibroblasts/myofibroblasts in the same area leading to interstitial inflammation and fibrosis [9,20].

TGF-ß1 is a pluripotent growth factor [2]. It is one of the most fibrogenic growth factors implicated in the pathogenesis of experimental and clinical renal fibrosis [5,9]. The involvement of TGF-ß1 in the development of renal scarring is related to increased synthesis and decreased degradation of ECM components, as well as to the activation of myofibroblasts in the tubulointerstitial area [2,21]. It is also involved in the evolution of tubular apoptosis that is related to progressive loss of intrinsic renal cells and their replacement by fibrotic tissue [22]. Myofibroblast invasion and the apoptotic process are both involved in the development of renal scarring. TGF-ß1 bears the capacity to activate interstitial fibroblasts, induce apoptosis and mediate the differentiation of tubular epithelial cells into myofibroblasts [22,23]. Our observations of increased expression of TGF-ß1 within tubular epithelial cells in patients with glomerular disease and heavy proteinuria and the correlation of this expression to the severity of interstitial fibrosis and tubular atrophy are in concert with these data. Furthermore, out of the three patients with minimal changes disease and mild tubulointerstitial injury, the extent of TGF-ß1 expression was intense only in one. However, urinary TGF-ß1 excretion was elevated in all patients. Whether the observed discrepancy between increased urinary excretion and weak renal expression of TGF-ß1 is due to the small number of patients or to the minimal changes disease per se needs further investigation.

Increased TGF-ß1 levels in the urine of nephrotic patients have been reported previously [18]. The elevated urinary excretion of TGF-ß1 along with its increased synthesis and expression within tubular epithelial cells observed in this study are suggestive of upregulation of renal TGF-ß1 by the filtered protein. The latter is further supported by the fact that a significant decrease of TGF-ß1 urinary excretion was observed in nephrotic patients who showed remission of proteinuria with immunosuppressive treatment. The TGF-ß1 produced by activated tubular epithelial and interstitial cells is probably excreted in the urine but is also accumulated in the renal interstitium and is involved in the activation of cells synthesizing ECM components leading to interstitial fibrosis and scarring.

Although the exact pathways involved in the deleterious effect of proteinuria in the progression of glomerular disease remain elusive, a considerable knowledge has grown from in vitro studies of exposure of tubular epithelial cells to protein. Our findings in nephrotic patients are in agreement with these studies showing activation of tubular epithelial cells and increased production of cytokines and growth factors that lead to the development of tubulointerstitial inflammation and scarring. Further research is required to delineate the pathways involved in the toxic effect of proteinuria and to exploit the TGF-ß1 signalling system effectively in new therapeutic approaches to renal scarring.



   Notes
 
Correspondence and offprint requests to: Dr D. Goumenos, Department of Internal Medicine—Nephrology, University Hospital of Patras, Patras 26500, Greece. Email: dgoumenos{at}med.upatras.gr Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
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Received for publication: 1. 4.02
Accepted in revised form: 22. 7.02