Quantification of TGF-beta 1 mRNA along rat nephron in obstructive nephropathy

Kyoichi Fukuda, Koji Yoshitomi, Taihei Yanagida, Masanori Tokumoto, and Hideki Hirakata

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and transforming growth factor-beta 1 (TGF-beta 1) is thought to play an important role in this process. Although increased TGF-beta 1 mRNA expression in the obstructed kidney has been demonstrated, the source of the increased TGF-beta 1 remains to be elucidated. To determine the precise localization of TGF-beta 1 in the obstructed kidney, we examined TGF-beta 1 mRNA expression using in situ hybridization and competitive RT-PCR in rats with UUO. In situ hybridization demonstrated that TGF-beta 1 mRNA expression was preferentially increased in tubular epithelial cells and to a lesser degree in infiltrating macrophages in obstructed kidneys. Quantitative analysis using competitive RT-PCR in microdissected nephron segments revealed that levels of TGF-beta 1 mRNA in obstructed kidneys relative to control kidneys increased significantly in proximal tubules, thick ascending limbs of Henle, and distal convoluted tubules, whereas those in glomeruli and collecting ducts did not change significantly. Of the tubular segments, the proximal tubules appeared to predominantly contribute to increased TGF-beta 1 mRNA. Our findings suggest that renal tubules, particularly proximal tubules, are the main contributors to increased TGF-beta 1 mRNA expression in obstructed kidneys and to the subsequent interstitial fibrosis.

unilateral ureteral obstruction; interstitial fibrosis; microdissection; competitive polymerase chain reaction; transforming growth factor-beta 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTERSTITIAL FIBROSIS IS A common consequence of progressive renal disease. Because previous studies have revealed that tubulointerstitial changes correlate with decrements in glomerular filtration rate in a variety of renal diseases (20), interstitial fibrosis is considered to play a major role in progression of chronic renal disease. Unilateral ureteral obstruction (UUO) is a well-characterized model of experimental hydronephrosis, which results in tubulointerstitial fibrosis of the obstructed kidney (14, 19). In general, fibrosis develops as a result of an imbalance between extracellular matrix (ECM) synthesis and matrix degradation. Several investigators have examined the mechanisms underlying fibrosis in the UUO model, and numerous cytokines have been implicated as mediators of interstitial fibrosis in this model (15). Among these cytokines, transforming growth factor-beta 1 (TGF-beta 1) is recognized as a major regulator of ECM deposition and fibrogenesis (16). TGF-beta 1 stimulates ECM protein synthesis and inhibits matrix degradation by increasing the activity of protease inhibitors and decreasing the amount of proteases (25). In addition, TGF-beta 1 stimulates synthesis of matrix protein receptors such as integrins and osteopontin (17).

In the UUO model, increased TGF-beta 1 expression in the obstructed kidney has been reported by a number of investigators (7, 13, 32). However, the intrarenal localization of increased TGF-beta 1 expression remains to be clearly identified. Therefore, we designed this study to determine which sites mainly express TGF-beta 1 in the obstructed kidneys of rats with UUO. For this purpose, we examined the expression of TGF-beta 1 mRNA using in situ hybridization in kidney tissue sections and competitive RT-PCR in microdissected nephron segments. In preliminary experiments, we noted a progressive development of interstitial fibrosis from the onset of ureteral ligation, which often made microdissection of the nephron segments from the obstructed kidneys difficult. Previous studies have reported that the TGF-beta 1 mRNA level in obstructed kidneys increased significantly compared with that in control kidneys as early as 3 days post-ureteral ligation, and, in addition, the level was as high as the maximal level during UUO (13). Thus we performed the main experiments at 3 days after the initiation of UUO and assessed TGF-beta 1 expression.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Experimental protocol. Male Sprague-Dawley (SD) rats, weighing 200-250 g, were used in the present study. After induction of general anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), rats were subjected to either UUO or sham operation. Ureteral obstruction was performed by ligating the left ureter with 4-0 silk through a small suprapubic incision (19). Sham operation consisted of a similar suprapubic incision and visualization of the left ureter without further manipulation. Sham-operated animals were used to obtain control kidneys. Rats were killed at 3, 7, and 14 days after operation for histological examination of kidney tissue. A group of rats was killed 3 days after an operation for microdissection of nephron segments. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kyushu University.

Routine histological examination. Kidney tissues for light microscopy were fixed in neutral formalin and embedded in paraffin, and 2-µm-thick sections were stained with periodic acid-Schiff reagent.

Serial section analysis. To confirm TGF-beta 1 protein expression, we carried out a serial section analysis of TGF-beta 1 mRNA and TGF-beta 1 protein. In addition, to determine whether the TGF-beta 1-expressing cells were infiltrating macrophages, we carried out another serial section analysis of TGF-beta 1 mRNA and macrophage cell marker antigen. Kidney tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer overnight, dehydrated with a graded ethanol series and chloroform, and embedded in paraffin. Two serial 3-µm-thick sections were prepared and mounted on 2% 3-amino-propyl-triethoxysilane-coated slides. All treatments were performed under ribonuclease-free conditions.

Each section was then individually reacted with either a TGF-beta 1 cRNA probe by in situ hybridization or anti-TGF-beta 1 antibody, and ED-1 antibody by the immunoperoxidase technique as described below.

Immunohistochemical staining. Kidney sections were deparaffinized, immersed in methanol containing 0.3% H2O2 for 30 min, and digested with proteinase K (5 µg/ml) (Boehringer Mannheim Biochemica, Mannheim, Germany) at 37°C for 5 min (TGF-beta 1) or 15 min (ED-1). Sections were then immunostained for TGF-beta 1 with TGF-beta 1(V), a rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA), using biotinylated goat anti-rabbit IgG, or immunostained for macrophages with ED-1, a mouse monoclonal IgG antibody (Serotec, Oxford, UK), using biotinylated rabbit anti-mouse IgG. Sections were developed using 3,3'-diaminobenzidine (Nichirei, Tokyo, Japan) as the chromogen and were counterstained with 1% methyl green solution. To quantify interstitial macrophage infiltration, numbers of ED-1-positive cells in the cortex were counted in 20 consecutive fields and averaged (cells/high-power field).

In situ hybridization. Digoxigenin-labeled single-strand RNA probes were prepared using the DIG RNA labeling kit (Boehringer Mannheim) according to the instructions provided by the manufacturer. A 404-bp fragment was excised from a 1.6-kb TGF-beta 1 cDNA (provided by Dr. T. Nakamura, Osaka University) with SmaI and PstI and subcloned into the SmaI-PstI site of pBluescript II SK(-) transcription vector that contains promoters for T7 and T3 RNA polymerase (Stratagene, La Jolla, CA). Antisense and sense probes were obtained by in vitro transcription of this plasmid linearized with BamHI or EcoRI with the use of T7 or T3 RNA polymerase, respectively. The specificity of the 404-bp cDNA was verified by DNA sequencing.

In situ hybridization was performed as previously described (9). In brief, kidney sections were deparaffinized, rehydrated, and then digested with proteinase K (5 µg/ml) at 37°C for 15 min. The sections were refixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 15 min and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. They were dehydrated with a graded ethanol solution and air-dried. The hybridization solution contained 50% deionized formamide, 10 mM Tris · HCl (pH 7.6), 1× Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA (pH 8.0), 200 µg/ml of yeast tRNA, and 2.0 µg/ml RNA probe. Hybridization solution (50 µl) was applied to each section, and each was coverslipped. Hybridization was performed in a moist chamber humidified by 50% formamide at 50°C for 16 h. Sections were then washed briefly in 5× SSC and then in 2× SSC containing 50% formamide for 30 min at 50°C. In some of the sections, RNase A treatment (20 µg/ml RNase A in 10 mM Tris · HCl, pH 8.0, 1 mM EDTA) was performed at 37°C for 30 min to exclude any false-positive readings due to mishybridization. Sections were washed in 2× and 0.2× SSC twice for 20 min at 50°C. Blocking was accomplished by 2% skim milk for 1 h. Sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:1,000 dilution; Boehringer Mannheim) for 30 min at room temperature. After being washed in buffer 1 (100 mM Tris · HCl, pH 7.5, 150 mM NaCl) twice for 15 min, and in buffer 2 (100 mM Tris · HCl pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 5 min, visualization was carried out with nitroblue tetrazolium salt, 5-bromo-4-chloro-3-indolyl phosphate, and 1 mM levamisole (Sigma, St. Louis, MO) in buffer 2. Controls were obtained by replacing the antisense probe with a sense probe under the same conditions.

Microdissection of nephron segments. Rats were anesthetized intraperitoneally with pentobarbital sodium 50 mg/kg body wt. The kidneys were perfused in situ via the aorta with ice-cold microdissection solution (Hanks' balanced-salt solution, pH 7.4; GIBCO-BRL, Grand Island, NY), containing 1 mg/ml collagenase type I (Sigma) and 1 mg/ml BSA. The left kidney was removed and cut into 1-2-mm-thick coronal slices. The kidney slices were incubated at 37°C for 30 min in the dissection solution containing collagenase and BSA. The incubated tissues were washed with cold dissection solution containing 10 mM vanadyl ribonucleoside complex (GIBCO-BRL) to inhibit RNA degradation (27).

Tubular segments were microdissected under a stereomicroscope using fine forceps. We obtained the following structures: glomerulus (Glm), proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb of Henle (mTAL), cortical thick ascending limb of Henle (cTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). In general, 30 glomeruli or 5-10 mm of tubule segments were pooled to constitute one sample. The time period for dissection was limited to within 1 h after digestion with collagenase. The microdissected samples were rinsed, snap-frozen, and stored at -80°C until RNA extraction.

RNA extraction and RT. Total RNA was extracted from the microdissected tissues or whole kidney tissues using the acid guanidinium thiocyanate-phenol-chloroform extraction method (6) with Isogen (Nippon Gene, Tokyo, Japan). The final RNA pellets from the tissues were resuspended in diethylpyrocarbonate-treated water and quantified spectrophotometrically with the absorbance at 260 nm (A260). First-strand cDNA was synthesized from 100 ng of total RNA using 200 U of Moloney murine leukemia virus RT (Superscript II; GIBCO-BRL) in a total volume of 20 µl. The reaction mixture consisted of (in mM) 50 Tris · HCl (pH 8.3), 75 KCl, 3 MgCl2, 2.5 dithiothreitol, and 1 each of dNTPs as well as 1 U/µl RNase inhibitor and 2.5 µM oligo(dT)12-18 primer. The mixture was incubated at room temperature for 10 min and at 42°C for 45 min, and the reaction was terminated by heating to 70°C for 15 min. After being chilled on ice, 2 U RNaseH (GIBCO-BRL) were added and incubated at 37°C for 20 min to remove the RNA template. The synthesized cDNA was then used in a PCR amplification procedure.

Oligonucleotide primers. For rat TGF-beta 1, the specific primers used were sense 5'-GGACTACTACGCCAAAGAAG-3' (bp 715-734) and antisense 5'-TCAAAAGACAGCCACTCAGG-3' (bp 989-1,008). The anticipated PCR product was 294 bp in length. For rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the specific primers used were sense 5'-TGAGA- ATGGGAAGCTG-3' (bp 251-266) and antisense 5'-TTGGG- GGTAGGAACAC-3' (bp 766-781). The anticipated PCR product was 531 bp in length. To verify the authenticity of the PCR products, the amplified products were gel purified, subcloned into pBluescript II SK(-), and validated by DNA sequencing using the dideoxyribonucleotide sequencing method.

Competitive mutant template preparation. Competitive mutant DNA templates were prepared as described previously (5). The 254-bp template was generated from rat TGF-beta 1 cDNA (24) by deleting a 40-bp segment using the following primers: sense 5'-GGACTACTACGCCAAAGAAG-3' and antisense 5'-TCAAAAGACAGCCACTCAGGCAGGAATTGTTGCTATATTT-3'. The 450-bp template was generated from rat GAPDH cDNA (31) by deleting an 81-bp segment by using the following primers: sense 5'-TGAGAATGGGAAGCTG-3' and antisense 5'-TTGGGGGTAGGAACACTGATGTTCTGG- GCTGC-3'. Mutant templates shared the same primer template with the target cDNA sequence but contained a smaller intervening sequence. Mutant fragments were gel purified and quantified spectrophotometrically. The authenticity of the PCR amplified products was verified by DNA sequencing.

Competitive PCR. To quantify TGF-beta 1 mRNA, competitive PCR was performed as described previously (10). Six equal aliquots (1 µl) of sample cDNA were co-amplified with the dilution series of mutant template prepared for TGF-beta 1 in a total volume of 50 µl. The reaction mixture comprised 20 pmol of each primer, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 µM each of dNTPs, and 1.25 U of Taq DNA polymerase. After the mixture was heated at 95°C for 5 min, the PCR reaction was run for 35 cycles with a denaturing phase at 95°C for 1 min, an annealing phase at 54°C for 1 min, and an extension phase at 72°C for 1 min. The reaction was followed by a final elongation step at 72°C for 7 min. Controls, with no added RT, were run in parallel to rule out genomic contamination. No negative controls generated PCR products (data not shown). The amplified products were size fractionated by electrophoresis on 3% NuSieve-agarose gels (FMC Bioproducts, Rockland, ME), and product bands were visualized by ultraviolet fluorescence after ethidium bromide staining. The intensity of the luminescence was measured by a charge-coupled device image sensor and quantified (29).

To normalize variations in RNA extraction and efficiency of reverse transcription, competitive PCR for GAPDH was also performed. Six equal aliquots (1 µl) of sample cDNA, derived from the same cDNA solution as used in TGF-beta 1 quantification, were coamplified with the dilution series of mutant template prepared for GAPDH using PCR conditions as described for TGF-beta 1, except that the annealing temperature was 55°C and 30 cycles were performed. The amplified products were size fractionated by electrophoresis and quantified in the same way.

Statistical analysis. Data are expressed as means ± SE. Differences between groups were assessed by the unpaired t-test. Statistical significance was defined as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Morphological findings after initiation of UUO. Light microscopy of periodic acid-Schiff-stained renal sections from the obstructed kidneys of rats with UUO showed that the cortical and medullary interstitial spaces progressively increased due to edema with infiltration of mononuclear cells after the initiation of UUO, as reported previously (19, 26). A large number of renal tubules were dilated, and in some the epithelium was flattened. In contrast, sections from the control kidneys of sham-operated rats appeared normal. Immunohistochemical labeling revealed that the number of ED-1-positive macrophages increased 3 days after UUO in kidneys with ureteral obstruction, suggesting that the infiltrating mononuclear cells were, at least in part, ED-1-positive macrophages. Quantitatively, the number of macrophages in the obstructed kidneys was 8.4-fold greater than that in control kidneys (Table 1).

                              
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Table 1.   Number of cortical interstitial macrophages in obstructed kidneys of rats with UUO and control kidneys of sham-operated rats at 3 days after operation

Localization of TGF-beta 1 mRNA by in situ hybridization. In control kidneys of sham-operated rats, TGF-beta 1 mRNA expression was detected in glomeruli and tubules, predominantly in distal tubules (Fig. 1A). Glomerular TGF-beta 1 mRNA expression was localized mainly in mesangial cells and glomerular epithelial cells (Fig. 1B). In obstructed kidneys of rats at 3 and 7 days after UUO, increased TGF-beta 1 mRNA expression was observed. TGF-beta 1 mRNA was predominantly expressed by renal tubular cells in the cortex (Fig. 1, C and E) and outer medulla (Fig. 1D) but was also present in the peritubular interstitial cells. After 2 wk, although many collapsed tubules were observed, the predominance of tubular expression of TGF-beta 1 mRNA was still evident (Fig. 1F). A control experiment with a sense probe did not show any tissue staining (Fig. 1G).


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Fig. 1.   In situ hybridization of transforming growth factor-beta 1 (TGF-beta 1) mRNA in control kidneys of sham-operated rats (A, B, G) and obstructed kidneys of rats with unilateral ureteral obstruction (UUO; C-F). In the control kidneys, TGF-beta 1 mRNA is expressed mainly in glomeruli and distal tubules (A), and glomerular expression is localized to epithelial cells and mesangial cells (B). Increased expression of TGF-beta 1 mRNA is noted in renal tubular cells in the cortex (C) and outer medulla (D) of the obstructed kidneys at 3 days after operation. Tubular expression of TGF-beta 1 mRNA is pronounced at 7 days as well (E), and the feature is still present at 14 days (F). Hybridization with a sense probe shows no staining (G). Magnification: A, C-G, ×200; B, ×400.

Localization of TGF-beta 1 protein by immunohistochemistry. To confirm TGF-beta 1 protein expression, we performed immunolabeling with anti-TGF-beta 1 antibody in the consecutive section of in situ hybridization. Immunohistochemical staining showed that the nephron segments expressing TGF-beta 1 mRNA were also positive for TGF-beta 1 protein in both control kidneys of sham-operated rats (Fig. 2, A and B) and obstructed kidneys of rats at 3 days after UUO (Fig. 2, C and D).


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Fig. 2.   In situ hybridization of TGF-beta 1 mRNA (A, C, E) and immunohistochemistry of TGF-beta 1 protein (B, D) or ED-1 (a marker of macrophages; F) in serial sections. Tubules positive for TGF-beta 1 mRNA are also positive for TGF-beta 1 protein both in control kidneys (A, B) and in obstructed kidneys at 3 days after UUO (C, D). Although TGF-beta 1 mRNA is predominantly expressed in tubular cells (E), immunostaining shows that clusters of ED-1-positive interstitial cells are also positive for TGF-beta 1 mRNA (F, arrows). Magnification: A-D, ×200; E, F, ×400.

Identification of interstitial cells expressing TGF-beta 1 mRNA. To characterize the fewer cells expressing TGF-beta 1 in the interstitium, we performed immunolabeling for macrophages with ED-1 in the consecutive section of in situ hybridization. Immunohistochemical staining showed that, although infiltrating macrophages also expressed TGF-beta 1 mRNA (Fig. 2, E and F), the intensity of the signal in macrophages was not as strong as that in renal tubular cells. Therefore, the majority of TGF-beta 1 mRNA-expressing cells in obstructed kidneys were renal tubular cells.

Appearance of microdissected renal tubules. It was considerably more difficult to dissect tubules in obstructed kidneys compared with control kidneys, suggesting that UUO induced interstitial fibrosis in obstructed kidneys as early as 3 days after ureteral ligation as reported previously (28). The proximal tubules (PCT and PST) and DCT from obstructed kidneys were slightly dilated in diameter compared with those from control kidneys, and the epithelia of these tubules were slightly flattened. The collecting tubules (CCD, OMCD, and IMCD) from obstructed kidneys were markedly dilated in diameter, and the epithelia of these tubules were also flattened compared with those from control kidneys (Fig. 3). In contrast, there was no evidence of dilatation in the thick ascending loops of Henle from obstructed kidneys. Glomeruli appeared to be unchanged after UUO.


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Fig. 3.   Profiles of microdissected proximal straight tubule (PST) and cortical collecting duct (CCD) from the control kidneys of sham-operated rats and those from the obstructed kidneys of rats at 3 days after UUO. The lumens are dilated and epithelial cells are flattened in tubules from the obstructed kidneys compared with those in tubules from the control kidneys. All tubules are shown at the same magnification. Note that these post-UUO changes are mild in PST and marked in CCD.

Quantification of TGF-beta 1 mRNA along nephron segments. For quantification of TGF-beta 1 mRNA expression in microdissected nephron segments and whole kidney, quantitative competitive RT-PCR was performed. Two- or threefold dilutions of DNA templates (termed "competitor") were used to compete for PCR amplification of reverse-transcribed sample cDNAs (termed "target"). A plot of the ratio of competitor to target product vs. the known concentration of input competitor was linear when plotted on a log-log scale (Fig. 4). The r-value for all competitive PCR linear regression curves ranged from 0.92 to 0.99. At the point where competitor and target product are in equivalence (i.e., competitor-to-target ratio = 1.0), the starting amount of TGF-beta 1 cDNA is equal to the known starting amount of the competitor. Final quantitative data were arbitrarily expressed as the ratio of TGF-beta 1 to GAPDH derived from the same cDNA samples (Fig. 5).


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Fig. 4.   Quantitative competitive RT-PCR analysis of TGF-beta 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from representative experiments in PST at 3 days after UUO. Aliquots of cDNA prepared from microdissected PST were each coamplified with a dilution series of mutant template either for 35 cycles with TGF-beta 1-specific primers (A) or for 30 cycles with GAPDH-specific primers (B). The products were separated on 3% NuSieve-agarose gels and visualized by ethidium bromide staining. Corresponding amounts of initial mutant templates (×10-3 attomol) are indicated above each lane. Molecular weight (MW) standards are the 100-bp ladder from GIBCO-BRL. The log of the ratio of band intensity of PCR product generated from competitor to that from target was plotted as a function of the log of the initial amount of competitor. TGF-beta 1 target, 294 bp; competitor, 254 bp; GAPDH target, 531 bp; competitor, 450 bp.



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Fig. 5.   Relative quantification of TGF-beta 1 cDNA along the nephron segments from the control kidneys of sham-operated rats and those from the obstructed kidneys of rats at 3 days after UUO. Competitive RT-PCR was used to quantify the amounts of TGF-beta 1 mRNA in the microdissected structures as described in MATERIALS AND METHODS and Fig. 4. Quantitative results were arbitrarily expressed as the ratio of TGF-beta 1 to GAPDH. See text for abbreviations of different nephron segments. Values are means ± SE of 4 separate experiments. *P < 0.05 vs. sham-operated rats.

On a whole-kidney basis, the level of TGF-beta 1 mRNA in obstructed kidneys of rats with UUO increased significantly compared with that in control kidneys of sham-operated rats 3 days after the operation (UUO: 44.2 ± 2.4 × 10-3, n = 4; sham: 21.4 ± 1.9 × 10-3, n = 4). On a nephron-segment basis, TGF-beta 1 mRNA expression was constitutively expressed all along the nephron segments in control kidneys of sham-operated rats. The level of TGF-beta 1 mRNA in the glomeruli was at least one order of magnitude higher than that in the tubules. Of the tubular segments, the level of TGF-beta 1 mRNA in the proximal tubules was relatively lower than those in the distal tubules. In obstructed kidneys of rats with UUO, the levels of TGF-beta 1 mRNA expression increased significantly in PCT, PST, mTAL, cTAL, and DCT but not in glomeruli, CCD, OMCD, or IMCD. The largest increase in TGF-beta 1 mRNA expression was observed in the proximal tubules (PCT and PST) from obstructed kidneys, reaching 7.0- and 6.9-fold of that of control kidneys, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main finding of the present study is that the increased TGF-beta 1 mRNA expression after UUO is predominantly localized to renal tubular cells in the obstructed kidney. The in situ hybridization study clearly demonstrated that UUO increased TGF-beta 1 mRNA expression in the obstructed kidney, which was associated with TGF-beta 1 protein expression, and that the main sites of increased TGF-beta 1 expression in obstructed kidneys were renal tubules in the cortex and outer medulla. Although increased TGF-beta 1 expression in obstructed kidneys has been reported by many investigators (13, 32), the intrarenal localization of TGF-beta 1 in obstructed kidneys is still controversial. In previous reports, Diamond et al. (7) noted highly significant increments in renal cortical TGF-beta 1 mRNA levels in obstructed kidneys and localized TGF-beta 1 protein to peritubular cells of the renal interstitium by immunolabeling, suggesting the infiltrating renal interstitial macrophage as a cellular source for increased TGF-beta 1 expression. In contrast, other investigators reported that the increased TGF-beta 1 in obstructed kidneys was confined to renal tubular cells, using immunohistochemistry (35) or RT-PCR (13). With the use of in situ hybridization, we detected TGF-beta 1 mRNA expression in the peritubular interstitial cells in addition to renal tubular cells. Thus, to confirm TGF-beta 1 mRNA expression in infiltrating macrophages as reported in the former study, we performed immunolabeling for macrophages with ED-1 and in situ hybridization of TGF-beta 1 in the serial section. Our results showed that TGF-beta 1 mRNA expression was also localized to a lesser degree to ED-1-positive infiltrating macrophages but that the predominant localization was in renal tubular cells. This finding confirms that the main cells expressing TGF-beta 1 mRNA in the obstructed kidney are not the interstitial infiltrating macrophages but renal tubular cells.

To determine the main segment contributing to increased TGF-beta 1 mRNA expression, we applied a competitive RT-PCR method to microdissected nephron segments. In control kidneys of sham-operated rats, TGF-beta 1 mRNA was found to be constitutively expressed all along the nephron segments, consistent with our previous report (1). Quantitative data showed that the abundance of TGF-beta 1 mRNA in proximal tubules was lower than that in the other nephron segments, which was consistent with our results using in situ hybridization. In rats with UUO, our results showed that TGF-beta 1 mRNA levels in PCT and PST from obstructed kidneys were markedly increased and that those in mTAL, cTAL, and DCT from obstructed kidneys were moderately increased compared with those in the corresponding segments from control kidneys. In contrast, TGF-beta 1 mRNA levels in glomeruli, CCD, OMCD, and IMCD from obstructed kidneys did not change significantly. Although the degree of TGF-beta 1 mRNA increase in proximal tubules was most striking, the expression level in this segment was not higher than in any other tubular segments, as the baseline level of TGF-beta 1 mRNA in proximal tubules was relatively low. Regardless of this result, proximal tubular cells may contribute predominantly to increased TGF-beta 1 mRNA expression in obstructed kidneys. The relative kidney tissue volume occupied by proximal tubules (PCT and PST) has been reported as ~66% of whole kidney tissue in normal rats (21). If the relative volume for the proximal tubules does not change during UUO, the increase in TGF-beta 1 (normalized by GAPDH) mRNA after UUO in the proximal tubules amounted to ~11.8 × 10-3, which can be estimated to occupy over half that in the whole kidney (~22.8 × 10-3) (Fig. 6). This indicates, therefore, that the proximal tubular cells predominantly contribute to the increase in TGF-beta 1 mRNA expression in obstructed kidneys of rats with UUO. This finding also implies that the major tubules highly expressing TGF-beta 1 in the cortex and outer medulla could be PCT and PST, respectively.


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Fig. 6.   Comparison of the relative amount of TGF-beta 1 mRNA derived from proximal tubules per unit tissue mass of whole kidney in the obstructed kidneys of rats with UUO and control kidneys of sham-operated rats. The relative amounts of TGF-beta 1 mRNA derived from proximal tubules per unit mass of whole kidney were estimated using the mean values shown in Fig. 5, assuming that the relative tissue mass occupied by PCT and PST is 47% and 19% of whole kidney tissue, respectively. Data are expressed as the ratio of TGF-beta 1 to GAPDH mRNA (×10-3). It is assumed that the ratio of TGF-beta 1 to GAPDH mRNA indicates the amount of TGF-beta 1 mRNA transcripts per unit tissue mass, considering the GAPDH gene as a "housekeeping" gene, which is expressed in any tissue at a constant level. Thus these data represent only a crude approximation of their relative amount. Note that proximal tubules account for over half of the increase in TGF-beta 1 mRNA content of whole kidney after UUO.

In this study, we used a competitive RT-PCR method to quantify TGF-beta 1 mRNA. Although RT-PCR is a powerful tool for detecting small amounts of RNA, it provides only qualitative results due to the exponential nature of the amplification process. To generate quantitative data, the concept of competitive RT-PCR, which is not cycle dependent, has been developed (2, 10, 33). The principle of this method is based on the coamplification of the sequence to be quantified (the target) with a known amount of another sequence (the competitor), which is flanked by the same primers as the target. The competitor is added to the sample as cRNA before the RT step or as cDNA after the RT step. The RNA competitor approach potentially makes it possible to determine the absolute amounts of target RNA. However, this method cannot check any variations in RNA extraction efficiency and RNA quantification. Indeed, the initial amplifiable RNA amounts determined by spectrophotometric analysis were rather variable among samples in our experience, because this analysis cannot distinguish between undegraded and degraded RNA or between genomic DNA and RNA. To circumvent these problems, we chose a DNA competitor and sequentially performed two competitive PCRs using each DNA competitor as follows. We first quantified the amounts of TGF-beta 1 mRNA, and, second, quantified the amounts of mRNA of the "housekeeping gene" GAPDH as an internal standard; then we normalized the results of TGF-beta 1 mRNA quantification to correct variations in RNA quantification and reverse transcription. Thus our data provide only an estimate of the relative number of copies of TGF-beta 1 mRNA normalized by GAPDH gene transcripts. Nevertheless, the results fulfill our purpose of determining differences between samples from control and obstructed kidneys.

Although the present data detected the cellular source and abundance of TGF-beta 1 mRNA in the obstructed kidney, the mechanism of TGF-beta 1 mRNA upregulation remains unclear. Recent studies in the UUO model have revealed that the renin-angiotensin system (RAS) could play a key role in the pathogenesis of tubulointerstitial fibrosis (8, 22, 23). It has been suggested that the intrarenal RAS is upregulated in the obstructed kidney and that increased angiotensin II stimulates TGF-beta 1 synthesis, thereby causing interstitial fibrosis. The effect of angiotensin II on upregulation of TGF-beta 1 appears to be mediated through an AT1 receptor, as administration of an AT1-receptor antagonist blunted the increase in TGF-beta 1 mRNA and ameliorated the degree of interstitial fibrosis (12) as did admininstration of the angiotensin-converting enzyme (ACE) inhibitor (13). Indeed, AT1 receptors are expressed all along the tubular segments in the rat kidney, with the highest abundance in proximal tubules (3, 18, 30). In addition, our data indicate that the tubular segment expressing AT1 receptors most abundantly coincides with the segment in which TGF-beta 1 mRNA increased most markedly after UUO, namely, the proximal tubule. This finding suggests, therefore, that the increase in tubular expression of TGF-beta 1 mRNA could be stimulated by angiotensin II through AT1 receptors in the obstructed kidney in the UUO model. This is supported by an in vitro study in which angiotensin II induced TGF-beta 1 expression in proximal tubular cells (34). In addition, recent evidence has identified proximal tubules as a de novo source for all components of the intrarenal RAS, i.e., angiotensinogen, renin, and ACE (4, 11). Thus angiotensin II may act in an autocrine or paracrine manner in the proximal tubules to upregulate TGF-beta 1. Taken together, prior reports and our new data indicate that the proximal tubules are the main source of both intrarenal RAS and TGF-beta 1 in obstructed kidneys, such that they may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in the setting of UUO.

In summary, we have clearly determined in the present study the sites and relative abundance of TGF-beta 1 mRNA expression along the nephron segments in obstructed kidneys of rats with UUO. Our results suggest that the main source for increased expression of TGF-beta 1 mRNA in the obstructed kidney is the renal tubular cells, particularly proximal tubular cells, and that these cells could thereby contribute to subsequent interstitial fibrosis.


    ACKNOWLEDGEMENTS

We thank Dr. T. Nakamura, Osaka University, for providing TGF-beta 1 cDNA and H. Noguchi for helpful technical assistance.


    FOOTNOTES

This study was supported by a research grant from the Glomerular Injury Research Committee of the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan.

Address for reprint requests and other correspondence: K. Fukuda, Dept. of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan (E-mail: kfukuda{at}intmed2.med.kyushu-u.ac.jp).

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 14 August 2000; accepted in final form 3 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 281(3):F513-F521
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