Background

Severe proteinuria not only indicates the presence of progressive glomerular disease, but also causes tubular epithelial cells to produce inflammatory mediators leading to tubulointerstitial (TI) injury. We investigated the role of nuclear factor-B (NF-B) in tubular epithelial cells in the development of proteinuria-induced TI injury.

Methods

To specifically inhibit NF-B activation, a recombinant adenovirus vector expressing a truncated form of IB (AdexIBN) was injected into renal arteries of protein-overloaded rats, a model of TI injury characterized by infiltration of mononuclear cells and fibrosis.

Results

Activation of NF-B in the renal cortex, observed in protein-overloaded rats treated with a control vector, recombinant lacZ adenovirus, was prevented in AdexIBN-injected rats. Microscopic examination revealed AdexIBN treatment to markedly attenuate proteinuria-induced TI injury. Increased immunostaining of vascular cell adhesion molecule-1, transforming growth factor-, and fibronectin in TI lesions also was suppressed by AdexIBN injection.

Conclusions

These findings provide evidence of the critical role of NF-B activation in TI injury and suggest the therapeutic potential of adenovirus-mediated IBN gene transfer into the kidney as a means of interrupting the process of TI damage.

METHODS

Recombinant adenovirus vector

A recombinant adenovirus vector was constructed that expressed the non-degraded form of the NF-B inhibitor IB (Adex1CAKT IBN; abbreviated AdexIBN) as previously described²¹. This non-degraded IB (IBN) lacked the 54 amino acids of the NH₂-terminus of wild-type human IB (MAD3). A modification of the cosmid-terminal protein complex method established by Dr. I. Saito (Tokyo University, Tokyo, Japan)²² was used to construct this adenovirus vector. Purified virus stocks were prepared by cesium chloride (CsCl) step gradient centrifugation, as previously described²³. A control vector, recombinant lacZ adenovirus (AdexlacZ) containing the CAG promotor, lacZ gene, which encodes Escherichia coli-galactosidase, and polyA signal sequences, was kindly supplied by Dr. I. Saito²⁴.

Animals and experimental design

Female Wistar rats (six weeks old) were purchased from Charles River Japan (Tokyo, Japan) and housed individually in cages. They were fed CA-1 rat chow (27% protein; Clea Japan, Inc., Tokyo, Japan) and given free access to water. One week after a right uninephrectomy, the rats were laparotomized under general anesthesia, achieved with intraperitoneal injections of pentobarbital (10 mg/kg). After clipping the aorta both proximally and distally to the renal artery, AdexlacZ (1.0 10⁸ PFU/mL) or AdexIB (5.0 10⁷ PFU/mL) dissolved in 1 mL saline was injected into the renal artery and the clip was released three minutes after the injection. Control rats received injection of 1 mL saline without the virus.

In experiments determining the duration and localization of -galactosidase expression, animals were killed by complete exsanguination under general anesthesia at day 7, 14, 21, or 28 after the injection of AdexlacZ. At the time of sacrifice, the remaining kidney was perfused with saline, decapsulated, and processed for X-gal staining.

To detect gene transfer of AdexIBN into the renal cortex, rats were sacrificed at day 1, 4, or 7 after AdexIBN infection. The renal cortex was excised from the perfused kidney, homogenized, quickly frozen in liquid nitrogen, and stored at -80°C for reverse transcription-polymerase chain reaction (RT-PCR) analysis. Rats injected with saline instead of virus were used as the control in this set of experiments.

In experiments investigating the effects of AdexIBN administration on tubulointerstitial injury, uninephrectomized rats received daily intraperitoneal 2 g injections of bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO, USA) starting one week after adenoviral infection. Control rats not injected with adenovirus were divided into two groups, a group injected with 2 g BSA and a group injected with an equivalent volume of saline. Rats were sacrificed at one, two, or three weeks after starting the BSA injections. After perfusion and decapsulation, the remaining kidney was bivalved. One section was cut into small pieces of cortex, snap frozen in liquid nitrogen, and stored at -80°C for determination of NF-B activity. The other section was processed for histological and immunohistochemical analyses.

For determination of 24-hour urinary protein excretion and 24-hour creatinine clearance, fasting animals were placed in individual metabolic cages to collect urine. Blood was collected at the time of sacrifice for determinations of serum albumin, creatinine, and total cholesterol. Blood pressure was measured by tail-cuff plethysmography.

All procedures used in the animal experiments complied with the standards described in the Guidelines for the Care and Use of Laboratory Animals in Keio University School of Medicine.

X-gal staining

Coronal slices of each kidney were placed in O.C.T. embedding compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), snap-frozen in liquid nitrogen, and stored at -80°C. Frozen sections, 8 m in thickness, were cut with a cryostat and placed on poly-L-lysine-coated slides. The slices were fixed in Buffer A (0.2% glutaraldehyde, 0.1 mol/L potassium phosphate buffer, pH 7.4, 5 mmol/L EGTA, 2 mmol/L MgCl₂) at room temperature for five minutes. After three rinses with Buffer B (0.1 mol/L potassium phosphate buffer, pH 7.4, 0.02% Nonidet P40, 0.01% sodium-deoxycholate, 5 mmol/L EGTA, 2 mmol/L MgCl₂), the tissues were stained with X-gal solution [10 mmol/L K₃Fe(CN)_6, 10 mmol/L K₄Fe(CN)₆, 0.5 mg/mL 5-bromo-4-chloro-3-indolyl -D-galactopyranoside; Sigma Chemical Company] in Buffer B (pH 7.4) at 37°C for 10 hours.

RT-PCR analysis

Total RNA was extracted from renal cortices with an RNA extraction kit, ISOGEN (Nippon Gene Co., Tokyo, Japan), according to the manufacturer's instructions. RNA isolated from each group was treated with RNase-free DNase, and cDNA was synthesized with a commercial kit (Ready To Go™ T-Primed First-Standard Kit; Amersham Pharmacia Biotech Inc., Tokyo, Japan). The cDNA product was amplified by PCR with the use of primers for AdexIBN (sense primer, 5'-CTCCAGCAGACTCCACTCCACT-3', and antisense primer, 5'-ACACCAGCCACCACCTTCTGAT-3'), yielding a 712 bp fragment. The PCR was initiated by a five minute incubation at 95°C, followed by 35 cycles of one minute at 95°C, one minute 30 seconds at 60°C, and one minute 30 seconds at 72°C. The resulting reaction products were analyzed by gel electrophoresis (1% agarose) using a 100 bp ladder as a size marker.

Histological analysis

Coronal sections of renal tissue were immersion-fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with periodic acid Schiff (PAS) to determine cellular infiltration and Masson's trichrome for fibrotic changes, and then viewed with a microscope. The severity of tubulointerstitial scarring and glomerulosclerosis was graded semiquantitatively (1 through 4) in a blinded manner, and the mean score was calculated according to a previously described scoring method^25,26. Tubulointerstitial scarring was scored as follows: 1 = normal tubules and interstitium; 2 = mild tubular atrophy and interstitial fibrosis; 3 = moderate tubular atrophy and dilation with marked interstitial fibrosis; 4 = end-stage kidney with extensive interstitial fibrosis and few remaining atrophic tubules. A score was given to each microscopic field viewed at a magnification of 200. The glomerulosclerosis scoring system was as follows: 1 = normal glomeruli; 2 = presence of mild segmental glomerulosclerosis affecting <25% of the glomerular tuft; 3 = moderate segmental sclerosis affecting 25 to 50% of the glomerular tuft; 4 = diffuse severe glomerulosclerosis affecting>50% of the tuft, including glomeruli with total tuft obliteration, fibrosis, and obsolescence. The scores for tubulointerstitial scarring and glomerulosclerosis in each rat were obtained by the examination of 25 to 50 cortical fields and glomeruli, respectively, per kidney. The mean number of interstitial mononuclear cells was calculated in a blinded manner by averaging the total number of mononuclear cells in the interstitium in 30 randomly selected high-power (200) cortical fields as previously described²⁷.

Immunohistochemistry of VCAM-1, transforming growth factor-, and fibronectin

Expression of VCAM-1, transforming growth factor- (TGF-), and fibronectin was detected by immunostaining according to a previously described method²⁸, with minor modifications. Coronal sections of renal tissue were immersion-fixed in 4% buffered-paraformaldehyde for 12 hours and washed with 10%, 15%, and 20% sucrose in phosphate-buffered saline (PBS) for four hours each time and then embedded in O.C.T. These sections were snap-frozen in liquid nitrogen, and stored at -80°C. Frozen sections, 8 m in thickness, were cut with a cryostat and air dried. After being washed in PBS (pH 7.4), the sections were blocked sequentially with 0.3% H₂O₂ in methanol and 2% normal goat serum. The sections were incubated at room temperature for two hours with rabbit anti-VCAM-1 (dilution 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-rat TGF- (dilution 1:500; R&D Systems, Minneapolis, MN, USA), or rabbit anti-rat fibronectin (dilution 1:500; Chemicon International, Temecula, CA, USA) polyclonal antibodies as the primary antibodies. After three rinses with PBS, goat anti-rabbit biotinylated secondary antibody at 1:1000 dilutions was applied to the sections for 60 minutes. The sections were then reacted with streptavidin-biotinylated peroxidase complex for 30 minutes, and stained with tetramethylbenzidine for horseradish peroxidase histochemistry. After washing and cover slipping on glycerol, the sections were examined under a microscope within 24 hours. For evaluation of the immunostaining of TGF- and fibronectin, each tubulointerstitial grid field was graded semiquantitatively (0 through 4) in a blinded manner and the mean score was calculated according to a previously described scoring method^29,30,31.

Extraction of nuclear proteins from renal cortex homogenates

Nuclear protein extracts from cortical tissue were prepared according to a previously described method³², with minor modifications⁸. Two hundred mg of cortical tissue were homogenized with a glass Teflon homogenizer in 400 L of ice-cold buffer A [10 mmol/L HEPES, pH 7.9, 10 mmol/L mol/L KCl, 2 mmol/L MgCl_2, and 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA)], a protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim, Germany)] followed by addition of 65 L of 2% Nonidet-P40. The mixture was vortexed, and then centrifuged at 13,000 g for five minutes. The supernatant was removed, and the pellet was resuspended in 60 L of Buffer B [50 mmol/L HEPES, 10% (vol/vol) glycerol, 300 mmol/L NaCl, 50 mmol/L KCl, a protease inhibitor cocktail tablet]. The mixture was centrifuged at 13,000 g for 10 minutes. The suspernatant included nuclear protein, and was diluted to a standard concentration of 3 g/L. The protein concentrations were determined by the Bradford method (Bio-Rad Laboratories. Hercules, CA, USA)³³.

Electrophoretic mobility shift assay and densitometry

Double stranded NF-B consensus oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI, USA) were end labeled with [³²P]--ATP (Amersham Life Science Inc. Sydney, Australia). Unincorporated label was removed with a QIAquick spin column (Qiagen K.K., Valencia, CA, USA). The binding reaction was performed for 30 minutes at room temperature and the binding mixture contained 5 g of nuclear protein extract, 2 L of gel shift binding 5 buffer [20% glycerol, 5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 2.5 mmol/L dithiothreitol (DTT), 250 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 0.25 mg/mL poly[dl-dC]-poly[dl-dC] and 1 L of ³²P-labeled (50,000 cpm counting) oligonucleotides in a total volume of 10 L. In the competition assays, a 100-fold excess of unlabeled NF-B consensus or mutant NF-B oligonucleotides (5'-AGTTGAGGCAACGGTCCCAGGC-3') was added to labeled NF-B consensus oligonucleotides. After the addition of 1 L of gel-loading buffer (250 mmol/L Tris-HCl, pH 7.5, 0.2% bromophenol blue, 40% glycerol), the DNA-protein complexes were resolved by electrophoresis on a 7% polyacrylamide gel in TBE buffer as previously described³⁴. The gel was run at 150 V for 90 minutes and then dried at 80°C with a gel drier. Autoradiographs were prepared by exposing the dried gel to X-ray film with intensifying screens for three to six hours at room temperature. The density of specific NF-B complex was determined with a laser scanning densitometer and image analysis software (BAStation; Fuji Photo Film Co., Ltd., Tokyo, Japan) as previously described³⁵.

Western blot analysis of VCAM-1

Vascular cell adhesion molecule-1 protein levels in cortical tissue were determined according to a previously described method¹⁶, with minor modifications. In brief, cortical tissue was homogenized in Tris buffer with proteinase inhibitors (Roche Molecular Biochemicals). After determination of the protein concentration with the Bio-Rad protein assay kit, protein samples (40 g) were mixed with reducing buffer, heated at 100°C for five minutes and then subjected to 7.5% SDS-PAGE. The separated proteins were electrophoretically transferred to nitrocellulose membranes. The blots were blocked in 5% nonfat milk and incubated for four hours at 22°C with the primary polyclonal antibody against VCAM-1 (dilution 1:100; Santa Cruz Biotechnology), and then for one hour with a secondary antibody conjugated to horseradish peroxidase (dilution 1:500; Amersham Life Science). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Life Science). Densitometric analysis was performed with the NIH image program (Bethesda, MD, USA).

Statistics

All data are expressed as means SEM. Multiple parametric comparisons were evaluated by analysis of variance (ANOVA), followed by Fisher's protected least significant difference test. The scores for tubulointerstitial scarring, glomerulosclerosis, and immunostaining were compared by Kruskal-Wallis test, followed by the Mann-Whitney U test. Values of P < 0.05 were considered statistically significant.

RESULTS

In vivo gene transfer of -galactosidase into tubular cells

As previously described²⁰, injection of AdexlacZ into renal artery resulted in the expression of -galactosidase in tubular epithelial cells at day 7, as shown in Figure 1a. The expression of -galactosidase gradually decreased at days 14 and 21 Figure 1b, c and only few -galactosidase-positive cells were found at day 28 (data not shown). The expression of -galactosidase was not observed in either glomerular or interstitial areas at any time. No -galactosidase-transduced cells could be detected in other organs including liver Figure 1d and heart (data not shown). There was no X-gal staining in the cortex of rats injected with saline instead of AdexlacZ Figure 1e.

Figure 1.

In vivo gene transfer of -galactosidase into proximal tubular cells. AdexlacZ was injected into the renal artery and the expression of -galactosidase was detected as a blue area in tubular epithelial cells 7 (A), 14 (B), and 21 (C) days after adenoviral injection (original magnification 100). There was no X-gal staining in the liver of a rat 7 days after injection of AdexlacZ into the renal artery (D, original magnification 100) or in the renal cortex of a rat 7 days after injection of saline into the renal artery (E, original magnification 100).

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Expression of IBN in the renal cortex

Transcription of AdexIBN was detected by RT-PCR using specific primers. RNA extracted from the cortices at one, four, and seven days after AdexIBN injection was reverse transcribed and amplified using specific primers. As shown in Figure 2, the 712 bp fragment was amplified from AdexIBN-injected group samples, but not from control samples. Although the intensity of the band tended to be decreased on day 7, a faint band of AdexIBN still could be detected. No bands of this size were obtained from the AdexIBN-injected group samples without reverse transcriptase.

Figure 2.

Reverse transcription-polymerase chain reaction (RT-PCR) of renal cortical mRNA for IBN transcripts. RT-PCR was performed with total RNA extracted from the renal cortices of rats 1, 4, and 7 days after injection of AdexIBN or saline (control) in the presence and absence of reverse transcriptase (RT). As a positive control, the product from AdexIBN itself was included (P).

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Time course of cortical NF-B activation in proteinuric rats

Nuclear factor-B DNA-binding activities were assessed in whole cortical nuclear extracts from rats with and without protein overload by EMSA Figure 3. In rats without a protein overload, the incubation of cortical nuclear extracts with labeled consensus NF-B oligonucleotides produced weak bands (Figure 3a, lanes 1 and 2). The activation of NF-B was induced at one week, peaked at two weeks, and persisted for at least three weeks after initiating protein overload in AdexlacZ-treated rats (Figure 3a, lanes 3 to 6). By contrast, in AdexIBN-injected rats, NF-B activation was markedly reduced (Figure 3a, lanes 7 to 10). As shown in Figure 3b, the NF-B/DNA bands were abolished by the unlabeled consensus oligonucleotides, but not by the mutant oligonucleotides. Treatment of nuclear extracts with a specific anti-p65 antibody resulted in a supershift of the NF-B/DNA bands, demonstrating the presence of p65 in the bands Figure 3c. By contrast, antibody to p50 did not produce any change in the NF-B/DNA bands. Densitometric analysis revealed the treatment of proteinuric rats with AdexIBN to prevent the cortical activation of NF-B at one and three weeks Figure 4. The cortical activation of NF-B in the proteinuric rats that had not been injected with adenovirus was similar in extent to the activation in the AdexlacZ group at one week.

Figure 3.

Time course of nuclear factor-B (NF-B) activity in protein-overloaded rats. A representative autoradiogram of an electrophoretic mobility shift assay for NF-B in nuclear extracts from the renal cortex at 0 to 3 weeks after the start of daily bovine serum albumin (BSA) injections is shown (A). Lanes 1 and 2, rats at 0 and 3 weeks of daily intraperitoneal saline injection (control); lanes 3 to 6, AdexlacZ-treated rats at 0 to 3 weeks of protein overload; lanes 7 to 10, AdexIBN-treated rats at 0 to 3 weeks of protein overload. Competition assay was performed to determine the binding specificity of the NF-B oligonucleotides (B). The binding reactions were performed with nuclear proteins from AdexlacZ-treated rats at 3 weeks of protein overload (lane 1), in the presence of a 100-fold excess of unlabeled consensus (lane 2) or mutant (lane 3) oligonucleotide competitors. Nuclear extracts obtained from AdexlacZ-treated rats at 3 weeks of protein overload were incubated with or without (lane 1) anti-p50 (lane 2) or anti-p65 (lane 3) antibody and analyzed for NF-B binding activity. Brackets indicate the positions of specific NF-B complex. An arrowhead indicates the position of supershifted complex.

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Figure 4.

Densitometric analysis of the autoradiographic results at one (A) and three (B) weeks of protein overload. Values obtained from electrophoretic mobility shift assays for NF-B in nuclear extracts from the renal cortex at one and three weeks after the start of daily bovine serum albumin (BSA) injections were normalized and expressed as percentages of the control. Control rats were injected with saline daily. Rats loaded with BSA had been injected with saline, AdexlacZ or AdexIBN one week before the start of protein overload. Data are means SEM from 4 rats. *P < 0.05 vs. control values. #P < 0.05 vs. values of saline-treated rats with BSA loading. **P < 0.05 vs. values of AdexlacZ-treated rats.

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Expression of VCAM-1

To investigate whether AdexIBN attenuated induction of an NF-B dependent molecule, we examined the cortical expression of VCAM-1 in the early phase of the renal injury. Western blot analysis revealed that protein overload induced marked increases in VCAM-1 protein in the renal cortical tissue of saline- and AdexlacZ-injected rats Figure 5. As shown in Figure 6, the up-regulation of VCAM-1 was mainly observed in proximal tubular cells, with marked induction in the basolateral portion of the epithelium. Occasional expression of VCAM-1 also was observed in glomerular and interstitial areas. The levels of VCAM-1 protein were significantly lower in rats injected with AdexIBN Figure 5 and Figure 6.

Figure 5.

Cortical expression of VCAM-1 after one week of protein overload. Protein levels of VCAM-1 in the cortical tissue were determined by Western blotting and are shown in the upper panel. Lanes 1 and 2, rats after one week of daily intraperitoneal saline injections; lanes 3 and 4, saline-injected rats after one week of protein overload; lanes 5 and 6, AdexlacZ-injected rats after one week of protein overload; lanes 7 and 8, AdexIBN-injected rats after one week of protein overload. As shown in the lower panel, values obtained by densitometric analysis of Western blots for VCAM-1 were normalized and expressed as percentages of the control. Data are means SEM from 3 rats. *P < 0.05 vs. control values; #P < 0.05 vs. values of saline-injected rats with protein overload; **P < 0.05 vs. values of AdexlacZ-injected rats.

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Figure 6.

Representative photomicrographs of VCAM-1 immunostaining in kidney sections after one week of protein overload. (A) Rats without protein overload. (B) Saline-injected rats with protein overload. (C) AdexlacZ-injected rats with protein overload. (D) AdexIBN-injected rats with protein overload (original magnification 200).

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Biochemical profile

As shown in Figure 7, protein-overloaded rats developed significant proteinuria. The levels of urinary protein excretion did not differ significantly among groups injected with saline, AdexlacZ, and AdexIBN during the course of BSA overload. Kidney weight and the ratio of kidney/body weight were significantly greater in protein-overloaded rats that had been injected with saline or AdexlacZ than in the control group Table 1. The increase in the ratio of kidney/body weight was significantly attenuated in the AdexIBN-treated group compared to the saline- and AdexlacZ-treated groups. Serum albumin levels of rats with protein overload were significantly higher than those of controls. Blood pressure, total cholesterol, serum creatinine, and 24-hour creatinine clearance did not differ significantly among the groups at sacrifice.

Figure 7.

Time course of urinary protein excretion of rats with (+) or without (-; ) bovine serum albumin (BSA) loading. Rats loaded with BSA had been injected with saline (), AdexlacZ (), or AdexIBN() one week before the start of protein overload. *P < 0.05, vs. control values.

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Table 1 - Biochemical profile of rats with or without (control) protein overload for 3 weeks.

Full table

Light microscopy studies

In accordance with previous reports^5,18, light microscopic analysis revealed protein overload to induce marked tubulointerstitial injury in the renal cortices of saline- and AdexlacZ-injected rats at three weeks Figure 8. The tubular changes consisted of tubular cell brush border loss, cellular atrophy, and basement membrane thickening. The interstitial space was expanded due to an increase in mononuclear inflammatory cell infiltration, interstitial edema, and fibrosis. In contrast, kidneys injected with AdexIBN showed minor tubulointerstitial injury as compared to the saline- and AdexlacZ-injected groups. Sections were scored according to the severity of tubulointerstitial scarring and glomerulosclerosis at three weeks after protein overload Figure 9. While protein overload induced a marked increase in the tubulointerstitial scarring score in the saline- and AdexlacZ-treated groups, the increase in the score was significantly attenuated in rats infected with AdexIBN. There was a slight increase in the glomerulosclerosis score in proteinuric rats, but no significant differences were observed among the groups. The number of interstitial mononuclear cells was increased in the saline- and AdexlacZ-treated groups, while the increase was markedly attenuated in rats injected with AdexIBN Figure 9.

Figure 8.

Representative photomicrographs of periodic acid-Schiff (A, C, E, and G) and Masson's trichrome (B, D, F, and H) staining of kidney sections at 3 weeks after the start of protein overload. (A and B) Rats without protein overload. (C and D) Saline-injected rats with protein overload. (E and F) AdexlacZ-injected rats with protein overload. (G and H) AdexIBN-injected rats with protein overload (original magnification 200).

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Figure 9.

Tubulointerstitial scarring and glomerulosclerosis scores and number of interstitial mononuclear cells in rats with or without protein overload for 3 weeks. Rats with protein overload had been injected with saline, AdexlacZ, or AdexIBN one week before the start of protein overload. Data are means SEM from 5 rats. *P < 0.05 vs. control values; #P <0.05 vs. values of saline-injected rats with protein overload; **P < 0.05 vs. values of AdexlacZ-injected rats.

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Expression of TGF- and fibronectin

Since increased levels of interstitial TGF- and fibronectin were reportedly associated with interstitial fibrosis in uninephrectomized rats with protein overload⁵, we next investigated the expressions of these proteins. Strong focal and segmental staining for TGF- and fibronectin was observed in the interstitial space in AdexlacZ-infected rats, whereas staining was much less intense in AdexIBN-infected rats Figure 10. No significant staining was observed without primary antibodies (data not shown). Semiquantitative scoring revealed that tubulointerstitial immunostaining scores for TGF- and fibronectin were significantly lower in rats treated with AdexIBN than in AdexlacZ-treated rats Figure 11. Weak staining for TGF- and fibronectin in the glomeruli of proteinuric rats also was identified, but there were no differences between the AdexlacZ and AdexIBN-infected groups.

Figure 10.

Representative photomicrographs of immunostaining for TGF- (A, B, and C) and fibronectin (D, E, and F) in kidney sections at three weeks after protein overload. (A and D) Rats without protein overload. (B and E) AdexlacZ-treated rats with protein overload. (C and F) AdexIBN-treated rats with protein overload (original magnification 100).

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Figure 11.

Immunostaining scores for TGF- and fibronectin in the tubulointerstitial space of rats with or without protein overload. Rats with protein overload had been injected with AdexlacZ or AdexIBN one week before the start of protein overload. Data represent mean values SEM from 4 rats. *P < 0.05 vs. control values; **P < 0.05 vs. values of AdexlacZ-treated rats.

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DISCUSSION

This study demonstrates that NF-B activation in the renal cortex plays a critical role in tubulointerstitial injury induced by proteinuria. The present results also suggest the possibility of AdexIBN being utilized as a therapeutic tool.

To investigate the process of tubulointerstitial injury associated with proteinuria, we selected a non-immunogenic rat model of tubulointerstitial injury induced by protein overload. Since the levels of protein excretion did not differ between the AdexIBN and AdexlacZ groups, tubular epithelial cells were considered to have been subjected to the same protein load in both groups. This finding indicates that our experimental conditions were appropriate for investigating the role of NF-B activation in the formation of tubulointerstitial injury induced by protein overload.

Previous reports have demonstrated that the distribution of gene products transduced by adenovirus in the kidney depends on the conditions of viral administration and on the transfected species. When the adenoviral vector is infused via the rat ureter, tubular cells in the papilla and medulla are the predominant sites of transduction²⁰. While Sukhatme's group successfully transduced a reporter gene in rat renal vasculature by infusing the adenovirus via the renal artery with venous clamping and cold incubation³⁶, interstitial cells in the cortex are mainly transfected when a similar method is applied to dogs³⁷. Glomerular cells have been reported to be transfected also when porcine kidney is continuously perfused for two hours³⁸. In agreement with the finding by Moullier et al²⁰, we observed that the injection of adenovirus into the rat renal artery with arterial clamping resulted in a selective gene transfer into proximal tubular cells. Although Moullier et al reported a rather heterogeneous transduction of -galactosidase in tubular cells, we observed a homogeneous distribution of -galactosidase-positive cells in renal cortex. The reason for the discrepancy between these results is unclear. However, the status of the intrarenal hemodynamics seems to be particularly important in determining the distribution of adenoviral infection, since co-administration of vasodilators with adenovirus has been reported to induce a significant change in the distribution of transferred gene products in the kidney³⁶. Thus, the altered renal circulation caused by the heminephrectomy that was performed prior to transfection in our study may have contributed to the homogeneous distribution of adenovirus in the renal cortex.

Treatment with AdexIBN prevented the NF-B activation observed in AdexlacZ-injected rats throughout the course of protein overload. Expression of IBN transcripts in the renal cortices of the AdexIBN-injected group was confirmed by RT-PCR analysis up until 7 days after the administration of the adenovirus. However, the IBN mRNA levels seemed to be decreased on day 7. These results suggest that IBN protein levels sufficient to prevent NF-B activation may be maintained during the course of the renal injury despite the decrease in the mRNA levels in the early stage. In rats with protein overload, tubular cells have been shown to be the major source of NF-B during the early stage of interstitial injury, beginning as early as 24 hours after the start of protein overload¹⁰. This report, together with the present results concerning the distribution of -galactosidase in tubular cells, supports the idea that AdexIBN treatment prevents NF-B activation in tubular cells during the early stage of interstitial injury induced by protein overload. This notion is supported further by the present finding that the increase in tubular levels of VCAM-1, the expression of which is controlled by NF-B in renal epithelial cells³⁹, was attenuated by AdexIBN after one week of protein overload.

The present study demonstrates that the inhibition of the NF-B pathway in the renal cortex by AdexIBN attenuates tubulointerstitial injury, including interstitial infiltration of mononuclear cells, in proteinuric rats. Since VCAM-1 mediates localization and stimulation of inflammatory cells^40,41 and its expression is related to the degree of tubulointerstitial injury in human glomerulonephritis⁴², inhibition of VCAM-1 expression by AdexIBN probably contributes to the attenuation of tubulointerstitial injury to some extent. In addition to VCAM-1, other NF-B dependent molecules, such as MCP-1 and RANTES^6,7, may play some role in the development of NF-B dependent tubulointerstitial injury induced by protein overload, although the precise contributions of the various proinflammatory molecules remain to be elucidated.

The reduced ratio of kidney weight/body weight in rats treated with AdexIBN, as compared to the saline and AdexlacZ groups, indicates that the inhibition of NF-B activation attenuates renal hypertrophy in proteinuric rats. In line with this observation, the histological analysis revealed that interstitial fibrosis in proteinuric rats was reduced in rats treated with AdexIBN. These effects of AdexIBN in part may be due to a reduction in the interstitial expression of TGF-, a profibrogenic cytokine, and fibronectin, an interstitial matrix protein, as demonstrated by immunostaining. Since interstitial inflammatory cells have been shown to produce TGF- in rats with protein overload⁵, the attenuated infiltration of mononuclear cells is likely to contribute to the reduction in TGF- staining observed in the AdexIBN group.

Largo et al reported the up-regulation of angiotensin-converting enzyme and angiotensinogen in proximal tubules of rats made proteinuric with protein overload, and suggested local production of angiotensin II to play a role in the tubulointerstitial injury in this model²⁸. Angiotensin II appears to participate in renal interstitial fibrosis by stimulating production of TGF- and fibronectin in renal interstitial cells⁴³. Since transcription of angiotensinogen gene is controlled by NF-B⁴⁴, decreased local generation of angiotensin II also may contribute to the attenuated expression of TGF- and fibrosis in the AdexIBN group.

Possible involvement of NF-B activation in tubulointerstitial injury in proteinuric rats was suggested in a recent report demonstrating that the administration of pyrrolidine dithiocarbamate (PDTC), an antioxidant, inhibits renal NF-B activation and tubulointerstitial injury induced by adriamycin⁸, an oxidant known to deplete cellular glutathione⁴⁵. The specificity of PDTC as an inhibitor of NF-B is questionable, however, since PDTC increases intracellular glutathione levels⁴⁶ and also acts as a metal chelator⁴⁷. In this regard, the present study provides direct evidence that NF-B activation is involved in tubulointerstitial injury associated with proteinuria.

In conclusion, our present study shows that the adenovirus-mediated gene transfer of mutant IB prevented tubulointerstitial injury induced by protein overload. This result demonstrates the important role of NF-B activation in tubulointerstitial injury and also suggests the possibility of using gene therapy targeting NF-B for the treatment of tubulointerstitial injury associated with glomerulonephritis.

References

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Acknowledgments

This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Health Science Research Grants from the Ministry of Health, Labor and Welfare, and a Grant for Fundamental Research Program for Advanced Medical Apparatus Undertaken in Cooperation with Medical and Engineering Researchers from the New Energy and Industrial Technology Development Organization (NEDO). Portions of this work were presented at the 33rd Annual Scientific Meeting of the American Society of Nephrology, Toronto, Canada.