Increased expression of TGF-β1 but not of its receptors contributes to human obstructive nephropathy

Kidney International, Vol. 56 (1999), pp. 2137–2146

Increased expression of TGF-b1 but not of its receptors contributes to human obstructive nephropathy HIROYUKI KANETO, HARUO OHTANI, ATSUSHI FUKUZAKI, SHIGETO ISHIDOYA, ATSUSHI TAKEDA, YUKIHIKO OGATA, HIROSHI NAGURA, and SEIICHI ORIKASA Department of Urology and Pathology, Tohoku University School of Medicine, Sendai, Japan

Increased expression of TGF-b1 but not of its receptors contributes to human obstructive nephropathy. Background. Previous studies have revealed an increased expression of transforming growth factor-b1 (TGF-b1) and deposition of extracellular matrix in the kidney of animals with ureteral obstruction. However, these relationships have not been elucidated in the hydronephrotic kidney of humans. Methods. We analyzed the tissue expression of extracellular matrix proteins, TGF-b1, and its receptors in the human kidney with ureteral obstruction by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Obstructed kidneys (OBKs) were obtained from patients with ureteral tumors. A kidney specimen from patients with a renal tumor was used as control (CNKs). Results. The interstitial volume was significantly increased in OBKs in comparison with CNKs. OBKs showed increased deposition of collagen types I and IV and fibronectin in the renal interstitium. RT-PCR revealed overexpression of collagen a1(IV) mRNA and fibronectin mRNA in OBKs. OBKs showed a significantly increased mRNA expression of TGF-b1 in comparison with CNKs. The immunoreactivity for TGF-b1 increased markedly in the interstitium of OBKs. There was a significant correlation between the TGF-b1 mRNA level and the interstitial volume. However, there was no significant difference between OBKs and CNKs in the relative mRNA level nor in immunoreactivity for TGF-b receptors. Conclusions. These data suggest that TGF-b1 may contribute to the interstitial fibrosis found in the human kidney with ureteral obstruction, mainly because of an increase in the expression of this cytokine without significant changes to its receptors.

Animal experimental models have elucidated the mechanisms of tubulointerstitial injury in the kidney with ureteral obstruction. Transforming growth factor-b1 (TGF-b1) is a major cytokine involved in this injury [1–5]. These authors first demonstrated that TGF-b1 mRNA levels are up-regulated in the renal cortex of the Key words: tubulointerstitial disease, cytokine, unilateral ureteral obstruction, fibrosis, renal injury, hydronephrosis. Received for publication May 12, 1999 and in revised form July 20, 1999 Accepted for publication August 3, 1999

 1999 by the International Society of Nephrology

obstructed kidney (OBK) at three days after unilateral ureteral obstruction (UUO), and that the level continued to be elevated until the 14th day of UUO [1]. Other investigators showed an increase in TGF-b1 mRNA as early as 12 hours of UUO [3]. Thus, an increase in TGF-b1 contributes to tubulointerstitial fibrosis in both acute and chronic phases of obstructive nephropathy. Palmer et al demonstrated that the urinary TGF-b1 concentration in the renal pelvis of children with congenital hydronephrosis was significantly higher than that in the bladder urine of the same group or that in the control group [6]. Thus, it is conceivable that in humans, TGF-b1 contributes to renal fibrosis in the setting of acute or chronic ureteral obstruction by similar mechanisms to those described in the experimental animal models. Although several receptors for TGF-b have been identified, an interaction between type I and type II receptors has an important role in signal transduction through the serine/threonine kinase domain [7, 8]. However, there are few reports describing the expression of TGF-b receptors in renal injury. Tamaki et al found mRNA upregulation of both TGF-b and type II receptors in animals with adriamycin-induced nephropathy, a model of glomerulosclerosis and interstitial fibrosis [9]. In this model, up-regulation of both the ligand (TGF-b1) and its receptor may have a role in interstitial fibrosis. Recently, Sutaria et al demonstrated up-regulation of TGF-b type I and type II receptors in both obstructed and contralateral kidneys of rats with UUO [10]. This study was therefore designed to analyze the expression of extracellular matrix (ECM) proteins and TGF-b1 and to investigate whether receptors for TGF-b have a role in obstructive nephropathy in the human kidney with ureteral obstruction. METHODS Materials and tissue preparation The obstructed kidneys (OBKs) were obtained from nine patients who underwent nephroureterectomy be-

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cause of transitional cell carcinoma of the ureter (5 males and 4 females, mean age 68.4 years, range 50 to 81 years). Ultrasonography and/or intravenous urography revealed mild to severe degrees of hydronephrosis. However, we could not confirm the onset of ureteral obstruction because all patients had shown hydronephrosis at the time of their visit to the hospital. The control kidneys (CNKs), which did not show hydronephrosis, were obtained from eight patients who underwent nephrectomy because of renal cell carcinoma (6 males and 2 females, mean age 52.8 years, range 27 to 69 years). We obtained tissue from morphologically normal areas of these CNKs. For histological studies, a surgical specimen of each kidney was fixed in 4% paraformaldehyde (PFA) for two to four hours at 48C, washed in phosphate-buffered saline (PBS), immersed in 10% and 20% sucrose/PBS each for four hours, embedded in OCT compound, and frozen in liquid nitrogen. PFA-fixed, paraffin-embedded sections were also prepared for conventional histological examination. For RNA extraction, the renal cortex was dissected, immediately frozen, and stored at 2708C until use. Morphometric analysis of interstitial volume A standard point-counting method was used to quantitate the interstitial volume on the stained sections, as described previously [11, 12]. We used a grid containing 36 sampling points engraved on an eyepiece and counted the points falling on the renal interstitium, including tubular basement membrane, directly in the microscopic field under 3200 magnification in consecutive nonoverlapping fields (over 50 fields). We determined the percentage of the total points/36 3 the number of counted fields as the volume fraction (Vv). Immunohistochemical study Paraffin sections (3 mm in thickness) were deparaffinized and used for immunostaining of fibronectin and collagen types I and IV. The deparaffinized sections were treated in 0.05 m Tris-HCl, pH 7.6, and digested with 0.1% trypsin in 0.1% CaCl2/0.05 m Tris-HCl, pH 7.6, for 30 minutes at room temperature. Frozen sections (4 mm in thickness) were used for immunostaining of latencyassociated peptide (LAP) for TGF-b1, and TGF-b type I (TbRI) and type II (TbRII) receptors. The sections were washed in PBS and immersed in 10% sheep or donkey normal serum to block nonspecific binding sites for 30 minutes at room temperature and were then incubated in the primary antibody overnight at 48C. The sections were immersed in 0.03% H2O2 in methanol to block endogenous peroxidase activity and were then incubated in the secondary antibody (1:100 dilution) for one hour at room temperature. The sections were then washed in PBS, followed by incubation with 0.005% H2O2/0.03% diaminobenzidine (DAB) in 0.05 m Tris-HCl, pH 7.6, and were counterstained with 4% methylgreen.

The primary antibodies used are listed in Table 1. TGF-b1 antibody (Ab96), TGF-b receptor type I antibody (VPN), and TGF-b receptor type II antibody (DRL) recognize LAPs of TGF-b1 and intracellular domains of TbRI and TbRII, respectively. The properties of these antibodies have been described previously [13–15]. The second antibodies were peroxidase-conjugated sheep antimouse IgG, F(ab9)2 and donkey antirabbit IgG, F(ab9)2 (Amersham, Buckinghamshire, UK). Rabbit immunoglobulin or mouse IgG1 (Dako, Tokyo, Japan) was used as a negative control. Characterization and quantitation of mRNA by RT-PCR Total RNA was extracted from the renal cortex by RNAzol B (Biotecx Laboratories, Houston, TX, USA) and dissolved in TE buffer [10 mm Tris, 1 mm ethylenediaminetetraacetic acid (EDTA)]. cDNA was synthesized from 8 mg of each total RNA using oligo (dT) 15 primer. The mixture of RNA and oligo dT was incubated for 10 minutes at 658C and chilled on ice. The reverse transcriptase (RT) buffer, 5 mm MgCl2, 20 U of RNase inhibitor, 1 mm dNTP and 15 U of AMV RT (Promega, Madison, WI, USA) were added, incubated at 428C for 60 minutes, heated to 958C for three minutes, and chilled on ice. Three microliters of each cDNA were used for polymerase chain reaction (PCR). To each cDNA was applied 47 ml of reaction mixture containing PCR buffer (50 mm KCl, 10 mm Tris-HCl, and 1.5 mm MgCl2), 200 mm of each dNTP, and 50 pmol of oligonucleotide primers for TGF-b1, TbRI, TbRII, and collagen a1(IV), 25 pmol of fibronectin primer, or 20 pmol of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer. The mixture was first incubated in a thermal cycler at 728C, and then 1.25 U of Taq polymerase (Pharmacia Biotech, Milwaukee, WI, USA) was added to each tube. PCR was carried out with a Perkin Elmer Cetus DNA Thermal Cycler using the following programs (optimal cycles were developed previously for each primer): 948C (1 min), 608C (1 min), and 728C (1 min) for TGF-b1 (30 cycles), collagen a1(IV) (26 cycles), fibronectin (28 cycles), and GAPDH (26 cycles); 948C (1 min), 658C (1 min), and 728C (1 min) for TbRI (34 cycles) and TbRII (32 cycles). The primers were designed according to the human cDNA sequences for TGF-b1, TbRI, TbRII, collagen a1(IV), fibronectin, and GAPDH (Table 2). Fibronectin primers were designed to flank the EDA region of the human cDNA. The resultant sizes of PCR products were 496 bp and 226 bp of EDA positive (EDA1) and EDA negative (EDA2) variants, respectively. PCR products were sequenced using a dGTP BigDye Terminator Cycle Sequencing kit and an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA) to confirm the identity of PCR product as target cDNA. After amplification, 15 ml of each PCR

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Kaneto et al: TGF-b1 in human obstructive nephropathy Table 1. Antibodies used in the present study Antibody Fibronectin Collagen type I Collagen type IV TGF-b1 (Ab96) TGF-b receptor type I (VPN) TGF-b receptor type II (DRL) a

Clone

Final dilution

Source

Polyclonal Polyclonal CIV22 Polyclonal Polyclonal Polyclonal

1:1000 1:200 1:100 1:200 1:50 1:10

Dako, Tokyo, Japan Chemicon, Temecula, USA Dako, Glostrup, Denmark The Cancer Institutea The Cancer Institutea The Cancer Institutea

These antibodies were kindly provided by Dr. K. Miyazono, Department of Biochemistry, The Cancer Institute, Tokyo, Japan

Table 2. Primer sequences Gene TGF-b1 TbRI TbRII Collagen a1(IV) Fibronectin GAPDH

Primers (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense) (sense)

59-T TGCAGTGTGT TAT C CGTGC TGT C 59-CAGAAATACAGCAACAAT T C C TGG 59-GACAGTGCGGT TGTGGCAGAT 59-TGT T T C TGC CAC C T C TGTAC 59-T C T CAAAC TGC T C TGAAGTGT 59-AC C T C CAT C TGTGAGAAGC C 59-GGGTGTGT TAGT TACGCAAAT 59-GC TGTGGAT CGGC TAC T C T T 59-T T C T T T CAT TGGT C CGGT C T T 59-GCAGC C CACAGTGGAGTATGT 59-T C CAC CAC C C TGT TGC TGTA 59-T C C TGCAC CAC CAAC TGC T T

product were electrophoresed through 2.0% agarose gel with ethidium bromide (0.5 mg/ml). The gel was photographed with Polaroid type 665 negative film over ultraviolet light. The negative film was developed, and the bands on the film were scanned by densitometry (Molecular Analyst software; Bio-Rad, Richmond, CA, USA). The ratio of optical density of each target mRNA against that of GAPDH mRNA was determined as a relative mRNA level. Statistical analysis Data are shown as mean 6 sd and were analyzed by unpaired Student’s t-test. The relationship between TGF-b1 mRNA levels and the interstitial volume was examined by linear regression analysis. RESULTS Morphology of renal cortex Figure 1A shows the renal cortex of a CNK (40-yearold male) stained with periodic acid-Schiff. The peritubular space was narrow, and there were few interstitial cells. In contrast to this, the OBK (70-year-old male) showed an increased interstitial space with deposition of ECM and a marked atrophy of cortical tubules (Fig. 1B). Interstitial volume of renal cortex The range of interstitial volume was 13.1 to 19.1% in CNKs and 14 to 68% in OBKs. Eight of nine OBKs had over 20%, and five of these had over 40% interstitial

Size

Reference

187 bp

[16]

354 bp

[14]

598 bp

[17]

434 bp

[18]

496 bp 226 bp 527 bp

[19] [20]

volume. The interstitial volume of OBKs was significantly greater than that of CNKs (40 6 19.4% vs. 16 6 2.3%, P , 0.01). There was a correlation between the degree of hydronephrosis assessed by ultrasonography and the interstitial volume in the OBK group. However, there was no significant correlation between the interstitial volume and the ages of the patients in either the OBK or CNK groups. Immunostaining for ECM proteins Figure 2 demonstrates the immunohistochemical appearance of ECM proteins. Fibronectin was weakly stained and confined to the peritubular space in the renal cortex of CNKs (Fig. 2A). OBKs showed a remarkable increase in fibronectin in the widened interstitial space, but not in the tubular basement membrane (Fig. 2B). Collagen type I (as with fibronectin) was increased in the interstitium of OBKs (Fig. 2D) in comparison with that of CNKs (Fig. 2C). Collagen type IV was confined to tubular basement membranes and peritubular capillaries and was not found in the interstitial space in CNKs (Fig. 2E). In contrast, OBKs showed an increased deposition of collagen type IV in both the interstitium and the tubular basement membrane (Fig. 2F). mRNA expression for collagen a1(IV) and fibronectin We examined gene expression of collagen type IV as a membranous ECM and that of fibronectin as an interstitial ECM. The representative PCR products of collagen a1(IV), fibronectin, and GAPDH are shown in

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Fig. 1. Morphology of the renal cortex of control kidney (CNK; 40-year-old male; a) and obstructed kidney (OBK; 70-year-old male; b; PAS staining; original magnification 3200).

Figure 3. The GAPDH level was approximately the same in every case. OBKs showed higher collagen a1(IV) mRNA levels than CNKs. Both EDA1 and EDA2 variants of fibronectin mRNA were increased in the OBKs as compared with CNKs, although the increase in expression was slight in two cases of OBKs. The ratio of EDA1 to EDA2 mRNA level was not different between CNKs and OBKs. Immunostaining for TGF-b1, TbRI, and TbRII Ab96 recognizes TGF-b1 LAP either with or without mature TGF-b1 and LAP bound to latent TGF-b–binding protein [13]. Figure 4 a and b demonstrate the immunostaining of TGF-b in the renal cortex. Immunoreactivity for TGF-b was observed in the interstitial space in both CNKs and OBKs. In CNKs, TGF-b was seen around the peritubular capillaries, but not in the renal tubular cells or in glomeruli (Fig. 4a). OBKs showed intense immunoreactivity for TGF-b in the increased interstitial space, especially around interstitial cells, but not in renal tubules (Fig. 4b). OBKs with 14% interstitial volume showed the same immunoreactivity for TGF-b as CNKs. There was no detectable difference in the pattern of TGF-b1 distribution among OBKs. VPN staining produced positive signals for TbRI in collecting ducts and distal tubules and showed no quantitative differences between CNK (Fig. 4c) and OBK (Fig. 4d). DRL staining for TbRII exhibited the same pattern of distribution as TbRI. In CNKs, TbRII was seen in collecting ducts and distal tubules, whereas in some specimens, TbRII was present weakly beneath the basement membrane of proximal tubules (Fig. 4e). In OBKs, the pattern was approximately the same as in CNKs (Fig. 4f). No immunoreactivity for TbRI or TbRII was observed in interstitial cells or glomeruli either in CNKs or OBKs. The localization of TbRI- and TbRII-positive cells was

not consistent with the region of increased deposition of ECM. mRNA expression for TGF-b1, TbRI, and TbRII We first amplified cDNA synthesized with or without RT from the total RNA of CNK to confirm the specificity of PCR primers. The PCR product of the predicted size was obtained by using cDNA synthesized with RT, whereas no bands were seen in the PCR product from cDNA synthesized without RT. Figure 5 demonstrates representative PCR products of TGF-b1, TbRI, TbRII, and GAPDH obtained from OBKs (2 cases) and CNKs (2 cases). GAPDH mRNA levels were not different among these four cases. The amount of TGF-b1 PCR product was slightly greater in OBKs than in CNKs. OBKs showed approximately the same mRNA levels of TbRI and TbRII as CNKs. We evaluated the relative mRNA levels by calculating the ratio of TGF-b1, TbRI, or TbRII mRNA against GAPDH mRNA (Fig. 6). The relative amount of TGF-b1 mRNA in OBKs was significantly greater than that in CNKs (0.93 6 0.138 vs. 0.69 6 0.186, P , 0.05). However, the mRNA levels of TbRI and TbRII were not significantly different between OBKs and CNKs. Correlation between interstitial volume and TGF-b1 mRNA level of obstructed kidneys Based on the assumption that the interstitial volume of the renal cortex in OBKs may reflect the degree of fibrogenesis in the interstitium, we analyzed the relationship between interstitial volume (Vv) and the ratio of OBK TGF-b1 mRNA (Fig. 7), revealing a positive correlation (r 5 0.799, P , 0.01, Pearson). This positive correlation was confirmed with the addition of data from CNKs (data not shown).

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Fig. 2. Immunostaining for extracellular matrix (ECM) proteins in the renal cortex of CNK (a, c, and e) and OBK (b, d, and f ). (a and b) Fibronectin. (c and d) Collagen type I. (e and f) Collagen type IV. Original magnification 3400.

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Fig. 3. Representative polymerase chain reaction (PCR) products of collagen a1(IV), fibronectin, and GAPDH in CNKs (1–4) and OBKs (5–8). Fibronectin shows two mRNA variants of EDA1 (496 bp) and EDA2 (226 bp). M, DNA size marker.

DISCUSSION We have demonstrated that the renal cortex of OBKs shows variable degrees of increase in the relative volume of the cortical interstitium from values in the normal range up to 68%, whereas the interstitial volume of normal CNKs was 13 to 19%. The variance of the increase of the interstitial volume of OBKs may be due to the degree and duration of ureteral obstruction because there was a correlation between the degree of hydronephrosis assessed by ultrasonography and the interstitial volume. In the experimental model of UUO, it has been shown that interstitial volume increases in OBKs of rats as early as three days after complete ureteral ligation [12] and that in the chronic phase, the interstitial volume in OBKs increased by 40% from a value of 2.8% in the kidney of normal rabbits [4]. This immunohistochemical study revealed that the expansion of the cortical interstitium of OBKs was partly due to the increased deposition of both interstitial and membranous ECM. Fibronectin and collagen type I were confined to the cortical interstitium in both CNKs and OBKs, whereas collagen type IV increased in both the interstitium and the basement membrane of OBKs. mRNA expression for collagen type IV and fibronectin was up-regulated in OBKs as compared with CNKs. Fibronectin mRNA precursor is alternatively spliced to yield several isoforms [21]. It has been shown that TGF-b preferentially up-regulates the EDA-containing isoform (both mRNA and protein) in cultured human fibroblasts [22, 23]. Viedt, Burger, and Hansch demonstrated that human tubular epithelial cells exhibit a preferential increase in EDA1 mRNA after stimulation by TGF-b [24, 25]. In the experimental UUO model, OBKs overexpressed EDIIIA (the rat equivalent to human EDA)positive mRNA but not the ED-IIIA-negative variant [1]. However, in this study, both EDA1 and EDA2 variants increased in OBKs as compared with CNKs. The ratio of EDA1 to EDA2 mRNA level was not different between CNKs and OBKs. Thus, it may be that

TGF-b up-regulates both fibronectin isoforms or that EDA2 mRNA is regulated by other cytokines in the kidneys of humans with obstructive nephropathy. The mechanisms underlying the progression of tubulointerstitial injuries have been studied by many investigators. TGF-b is the major cytokine promoting the deposition of ECM during fibrogenesis in renal injury [reviewed in 26]. However, there have been few reports concerning TGF-b in the onset of kidney disease in humans [27, 28]. We found an increase in the expression of TGF-b1 mRNA and protein in human kidneys with ureteral obstruction. A positive correlation between TGF-b1 mRNA expression and the interstitial volume suggests that sustained TGF-b1 up-regulation results in severe renal fibrosis in OBKs in humans. The expansion of the interstitial space was due mainly to the accumulation of ECM proteins in the interstitium. Thus, these data suggest that TGF-b1 contributes to interstitial fibrosis in the chronic phase of human obstructive nephropathy. There is still controversy concerning the cellular sources of TGF-b1 in tubulointerstitial fibrosis during UUO. Macrophages are potential sources of TGF-b1 in OBKs. It has been shown that monocytes/macrophages infiltrate the renal interstitium of OBKs as early as four hours of UUO [29]. Diamond et al demonstrated that intracellular TGF-b1 was positive in the monocytes/macrophages that had infiltrated the renal interstitium of OBKs, and that there was a significant correlation between the number of infiltrating mononuclear cells and the TGF-b1 mRNA level [3]. Other investigators found immunoreactivity for TGF-b1 in interstitial mononuclear cells in the OBKs 24 hours after the commencement of UUO [5]. They also detected TGF-b1 in the juxtaglomerular apparatus, peritubular capillaries, and tubular cells. Yamamoto et al demonstrated that in an anti-mesangial model of serum nephritis, TGF-b–positive interstitial mononuclear cells exhibit immunoreactivity for fibroblastic/myofibroblastic cells but not for monocytes/macrophages [30]. They also showed an increase in TGF-b transcripts

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Fig. 4. Immunostaining for TGF-b1 (a and b), TbRI (c and d ) and TbRII (e and f ) in the renal cortex of CNK (a, c, and e) and OBK (b, d, and f; original magnification 3400).

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Fig. 5. Representative PCR products of TGF-b1, TbRI, and TbRII in OBKs (1, 2) and CNKs (3, 4). M, DNA size marker.

Fig. 6. Relative amounts of TGF-b1, TbRI, and TbRII mRNA in OBKs and CNKs. The vertical axis expresses mRNA as a ratio of GAPDH mRNA (N 5 8 for CNKs and N 5 9 for OBKs). #P , 0.05 vs. CNK.

Fig. 7. Correlation between the relative amount of TGF-b1 mRNA and the interstitial volume (Vv) of OBKs. There is a significant correlation.

in both glomeruli and the renal interstitium of the affected kidney. Renal tubular cells may be responsible for production of TGF-b1 because an up-regulation of TGF-b1 mRNA was observed in renal cortical tubules rather than glomerular cells of OBKs [1]. Tamaki et

al reported that TGF-b1 was positive both in tubular epithelial cells and in interstitial cells in adriamycin nephropathy [9]. They suggested that only a part of the population of infiltrating macrophages is the source of TGF-b1 because the number of ED-1–positive cells exceeded that of TGF-b1–positive cells. Thus, both resident renal tubular cells and interstitial cells, including infiltrating mononuclear cells, may be responsible for TGF-b production in experimental model. Transforming growth factor-b is synthesized in a precursor form and is cleaved to active TGF-b by a LAP. Niemir et al found overexpression of LAP but a low amount of active TGF-b1 in the interstitium of human IgA glomerulonephritic kidneys and suggested that conversion to active TGF-b from latent TGF-b was limited in this model [27]. In this investigation, we used an antibody that recognizes LAP with or without active TGF-b1. LAP was positive mainly in the interstitium, but not in the interstitial mononuclear cells or in renal tubular cells, in both CNKs and OBKs. There was no difference in the pattern of TGF-b1 distribution among OBKs. Because latent TGF-b is produced in response to up-regulation of TGF-b transcription in the OBK, it is likely that latent TGF-b is produced and promptly

Kaneto et al: TGF-b1 in human obstructive nephropathy

secreted into the interstitium, where it is subsequently converted to the active form [31]. Wright et al previously showed an up-regulation of TGF-b in OBKs but not in the contralateral kidneys of rats after 3 and 14 days of UUO [32]. The same group demonstrated an increase in mRNA and protein expressions for TbRI and TbRII in both obstructed and contralateral kidneys [10]. They suggested that TGF-b has a possible role in fibrosis of OBKs and in hypertrophy of the contralateral kidneys through the up-regulation of TGF-b receptor. Horvath et al showed intense immunoreactivity for TbRI and TbRII in the proximal and distal tubules in normal human kidney, whereas in the allograft rejection, the immunoreactivity decreased in these cells [33]. We showed that TbRI and TbRII are localized in the collecting ducts and distal tubules but not in the interstitial cells or glomeruli, in both CNKs and OBKs. However, we found no significant quantitative changes in the protein and mRNA levels of these receptors in OBKs. Because the localization of these cells was not necessarily consistent with the region of increased deposition of ECM, TGF-b receptors expressed in these renal tubular cells may not contribute to the interstitial fibrosis. It is possible that in the chronic phase of renal fibrosis, the immunoreactivity for receptors is too low to be detected by immunohistochemistry. The difference in expression of ligand and receptors reported by different investigators may be due to the immunoreactivity of the antibodies used or the different types of inflammation encountered. We conclude that TGF-b1 is involved in the interstitial deposition of ECM proteins in human hydronephrotic kidneys caused by ureteral obstruction, via up-regulation of TGF-b1 expression, without affecting the expression of TGF-b receptors. ACKNOWLEDGMENTS We thank Kohei Miyazono, M.D., and Michiyasu Kato, M.D., Department of Biochemistry, The Cancer Institute, Tokyo, Japan, for providing the antibodies for TGF-b and its receptors. Reprint requests to Hiroyuki Kaneto, M.D., Department of Urology, Tohoku University School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. E-mail: [email protected]

REFERENCES 1. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-b1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 44:313–321, 1993 2. Ishidoya S, Morrissey J, McCracken R, Klahr S: Angiotensin II receptor antagonist ameliorates renal tubulointerstitial fibrosis caused by unilateral ureteral obstruction. Kidney Int 47:1285–1294, 1995 3. Diamond JR, Kees-Folts D, Ding G, Frye JE, Restrepo NC: Macrophages, monocyte chemoattractant peptide-1, and TGF-b1 in experimental hydronephrosis. Am J Physiol 266:F926–F933, 1994

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4. Sharma AK, Mauer SM, Kim Y, Michael AF: Interstitial fibrosis in obstructive nephropathy. Kidney Int 44:774–788, 1993 5. Pimentel JL, Sundell CL, Wang S, Kopp JB, Montero A, Martinez-Maldonado M: Role of angiotensin II in the expression and regulation of transforming growth factor-b in obstructive nephropathy. Kidney Int 48:1233–1246, 1995 6. Palmer LS, Maizels M, Kaplan WE, Firlit CF, Cheng EY: Urine levels of transforming growth factor-b1 in children with ureteropelvic junction obstruction. Urology 50:769–773, 1997 7. Okadome T, Yamashita H, Franzen P, Moren A, Heldin CH, Miyazono K: Distinct roles of the intracellular domains of transforming growth factor-b type I and type II receptors in signal transduction. J Biol Chem 269:30753–30756, 1994 8. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J: Mechanism of activation of the TGF-b receptor. Nature 370:341–347, 1994 9. Tamaki K, Okuda S, Ando T, Iwamoto T, Nakayama N, Fujishima M: TGF-b1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy. Kidney Int 45:525–536, 1994 10. Sutaria PM, Ohebshalom M, McCaffrey TA, Vaughan ED Jr, Felsen D: Transforming growth factor-b receptor types I and II are expressed in renal tubules and are increased after chronic unilateral ureteral obstruction. Life Sci 62:1965–1972, 1998 11. Moller JC, Skriver E, Olsen S, Maunsbach AB: Ultrastructural analysis of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis). Virchows Arch 402:209– 237, 1984 12. Kaneto H, Morrissey J, MacCracken R, Reyes AA, Klahr S: Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed kidney of the rat. Kidney Int 45:1637–1647, 1994 13. Olofsson A, Miyazono K, Kanzaki T, Colosetti P, Engstrom U, Heldin CH: Transforming growth factor-b1, -b2, and -b3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes. J Biol Chem 267:19482–19488, 1992 14. Franzen P, ten Dijke P, Ichijo H, Yamashita H, Schulz P, Heldin CH, Miyazono K: Cloning of a TGF b type I receptor that forms a heteromeric complex with the TGF b type II receptor. Cell 75:681–692, 1993 15. Taketazu F, Kato M, Gobl A, Ichijo H, ten Dijke P, Itoh J, Kyogoku M, Ronnelid J, Miyazono K, Heldin CH, Funa K: Enhanced expression of transforming growth factor-bs and transforming growth factor-b type II receptor in the synovial tissues of patients with rheumatoid arthritis. Lab Invest 70:620–630, 1994 16. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV: Human transforming growth factor-b complementary DNA sequence and expression in normal and transformed cells. Nature 316:701–705, 1985 17. Lin HY, Wang XF, Ng-Eaton E, Weinberg RA, Lodish HF: Expression cloning of the TGF-b type II receptor, a functional transmembrane serine/threonine kinase. Cell 68:775–785, 1992 18. Brinker JM, Gudas LJ, Loidl HR, Wang SY, Rosenbloom J, Kefalides NA, Myers JC: Restricted homology between human alpha 1 type IV and other procollagen chains. Proc Natl Acad Sci USA 82:3649–3653, 1985 19. Kornblihtt AR, Vibe-Pedersen K, Baralle FE: Isolation and characterization of cDNA clones for human and bovine fibronectins. Proc Natl Acad Sci USA 80:3218–3222, 1983 20. Ercolani L, Florence B, Denaro M, Alexander M: Isolation and complete sequence of a functional human glyceraldehyde-3phosphate dehydrogenase gene. J Biol Chem 263:15335–15341, 1988 21. Norton PA, Hynes RO: Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol Cell Biol 7:4297–4307, 1987 22. Blaza E, Borsi L, Allemanni G, Zardi L: Transforming growth factor b regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS Lett 228:42–44, 1988 23. Borsi L, Castellani P, Risso AM, Leprini A, Zardi L: Transforming growth factor-b regulates the splicing pattern of fibronectin messenger RNA precursor. FEBS Lett 261:175–178, 1990 24. Viedt C, Burger A, Hansch GM: Fibronectin synthesis in tubular

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25.

26. 27.

28.

Kaneto et al: TGF-b1 in human obstructive nephropathy

epithelial cells: Up-regulation of the EDA splice variant by transforming growth factor b. Kidney Int 48:1810–1817, 1995 Burger A, Wagner C, Viedt C, Reis B, Hug F, Hansch GM: Fibronectin synthesis by human tubular epithelial cells in culture: Effects of PDGF and TGF-b on synthesis and splicing. Kidney Int 54:407–415, 1998 Kuncio GS, Neilson EG, Haverty T: Mechanisms of tubulointerstitial fibrosis. Kidney Int 39:550–556, 1991 Niemir ZI, Stein H, Noronha IL, Kruger C, Andrassy K, Ritz E, Waldherr R: PDGF and TGF-b contribute to the natural course of human IgA glomerulonephritis. Kidney Int 48:1530–1541, 1995 Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H, Maki S: Transforming growth factor-b protein and mRNA in glomeruli in normal and diseased human kidneys. Lab Invest 68:154–163, 1993

29. Schreiner GF, Harris KPG, Purkerson ML, Klahr S: Immunological aspects of acute ureteral obstruction: Immune cell infiltrate in the kidney. Kidney Int 34:487–493, 1988 30. Yamamoto T, Noble NA, Miller DE, Boder WA: Sustained expression of TGF-b1 underlies development of progressive kidney fibrosis. Kidney Int 45:916–927, 1994 31. Munger JS, Harpel JG, Gleizes PE, Mazzieri R, Nunes I, Rifkin DB: Latent transforming growth factor-b: Structural features and mechanisms of activation. Kidney Int 51:1376–1382, 1997 32. Wright EJ, McCaffrey TA, Robertson AP, Vaughan ED Jr, Felsen D: Chronic unilateral ureteral obstruction is associated with interstitial fibrosis and tubular expression of transforming growth factor-b. Lab Invest 74:528–537, 1996 33. Horvath LZ, Friess H, Schilling M, Borisch B, Deflorin J, Gold LI, Korc M, Buchler MW: Altered expression of transforming growth factor-bs in chronic renal rejection. Kidney Int 50:489–498, 1996