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Am J Physiol Renal Physiol 294: F508-F517, 2008. First published December 19, 2007; doi:10.1152/ajprenal.00306.2007
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Protective effects of low-dose carbon monoxide against renal fibrosis induced by unilateral ureteral obstruction

Lin Wang,1 Ji-Yang Sophie Lee,1 Joon Hyeok Kwak,1 Yanjuan He,1 Sung Il Kim,1 and Mary E. Choi1,2

1Department of Medicine, Renal Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 2Renal Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts

Submitted 5 July 2007 ; accepted in final form 19 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tubulointerstitial fibrosis is a hallmark of chronic progressive kidney disease leading to end-stage renal failure. An endogenous product of heme oxygenase activity, carbon monoxide (CO), has been shown to exert cytoprotection against tissue injury. Here, we explored the effects of exogenous administration of low-dose CO in an in vivo model of renal fibrosis induced by unilateral ureteral obstruction (UUO) and examined whether CO can protect against kidney injury. UUO in mice leads to increased extracellular matrix (ECM) deposition and tubulointerstitial fibrosis within 4 to 7 days. Kidneys of mice exposed to low-dose CO, however, had markedly reduced ECM deposition after UUO. Moreover, low-dose CO treatment inhibited the induction of {alpha}-smooth muscle actin ({alpha}-SMA) and major ECM proteins, type 1 collagen and fibronectin, in kidneys after UUO. In contrast, these anti-fibrotic effects of CO treatment were abrogated in mice carrying null mutation of Mkk3, suggesting involvement of the MKK3 signaling pathway in mediating the CO effects. Additionally, in vitro CO exposure markedly inhibited TGF-β1-induced expression of {alpha}-SMA, collagen, and fibronectin in renal proximal tubular epithelial cells. Our findings suggest that low-dose CO exerts protective effects, via the MKK3 pathway, to inhibit development of renal fibrosis in obstructive nephropathy.

transforming growth factor-β1; obstructive nephropathy; MAPK; MKK3; HO-1

MANY CHRONIC KIDNEY diseases, regardless of the nature of the initial injury, progress to end-stage renal disease (ESRD) with irreversible loss of tissue and function. Tubulointerstitial fibrosis is a hallmark of chronic progressive kidney disease and is thought to be the final common mechanism that leads to ESRD (14). The pathogenesis of tubulointerstitial fibrosis is characterized by relentless production and deposition of extracellular matrix (ECM) proteins, such as fibronectin and collagens within the interstitium of the kidney, and strongly correlates with deterioration of renal function (19). A better understanding of the nature of chronic kidney disease leading to prevention and effective therapy is critical, such as strategies that suppress elaboration of ECM and thereby inhibiting the pathogenesis of tubulointerstitial fibrosis and prevent development of ESRD.

Recent investigations suggest that carbon monoxide (CO) has cytoprotective effects in tissue injury. CO is a gaseous molecule with well-known toxicity and lethality to living organisms when exposed to very high concentrations. However, CO is also generated endogenously in mammalian cells primarily through the catalysis of heme by heme oxygenase (HO) and has been implicated to play key physiological roles, including functioning as a chemical messenger in neuronal transmission and modulating vascular contractility (26, 38, 45, 51–53). HO-1, the stress-inducible form of heme oxygenase, has been shown to play a vital function in providing cytoprotection against oxidative stress and maintaining cellular homeostasis (33). Studies suggest that CO, a major catalytic byproduct of HO activity, mediates the protective functions of HO-1.

CO, when administered at low concentrations, has been shown to provide potent cytoprotective effects in several models of tissue injury such as ischemia-perfusion lung injury, endotoxic shock, hyperoxic lung injury, aeroallergen-induced airway inflammation, and suppress rejection in mouse-to-rat cardiac xenotransplantation, and can fully substitute for the cytoprotective effects otherwise observed with HO-1 (2, 5, 32, 38–40). However, few studies have examined the role of CO in kidney injury, and the functional role of CO in the context of renal fibrosis has not been previously examined. In the present study, we sought to determine whether inhalation of low-dose CO can provide protection against kidney injury by inhibiting ECM production and the development of renal tubulointerstitial fibrosis utilizing an in vivo model of renal fibrosis induced by unilateral ureteral obstruction (UUO). The mitogen-activated protein kinase (MAPK) is a major intracellular signal transduction pathway regulating stress responses, and here we investigated whether the MAPK is involved in mediating the CO effects and cellular response to kidney injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Recombinant human transforming growth factor-β1 (TGF-β1; rhTGF-β1) was purchased from R & D Systems (Minneapolis, MN). Anti-type 1 collagen antibodies were obtained from Calbiochem (San Diego, CA) and anti-fibronectin antibodies from BD Transduction Laboratories (San Jose, CA). Anti-TGF-β types I and II receptor antibodies and anti-{alpha}-tubulin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies directed against {alpha}-smooth muscle actin ({alpha}-SMA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from American Research Products (Belmont, MA) and Biodesign International (Saco, ME), respectively.

Animals. Male C57BL6 mice (8–12 wk of age) were purchased from Jackson Laboratory (Bar Harbor, ME), and MKK3 null (Mkk3–/–) mice and control wild-type (Mkk3+/+) mice were previously described (24, 50). The animals were maintained in laminar flow cages in a specific pathogen-free animal facility at the University of Pittsburgh and fed a standard diet and water ad libitum. Experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

UUO and in vivo CO exposures. UUO was performed by completely ligating the left ureter in mice. Sham-operated mice had the same surgical procedure except for the ureter ligation. For in vivo CO exposures, 1% CO mixed with room air (RA; 21% oxygen) was directed into a 3.7-ft3 glass exposure chamber at a flow rate of 12 l/min (56). A CO analyzer (Interscan, Chatsworth, CA) was used to continuously maintain the CO level at 250 ppm in the chamber. The mice were preexposed to 250 ppm concentration of CO for 2 h before undergoing UUO or sham operation, and then continuously exposed to 250 ppm CO for the indicated time periods after the surgical procedures. The groups of mice not undergoing CO exposures were maintained in normal RA. The animals (n = 6 for each group of animals) were killed at 4 and 7 days after UUO or sham operation, and the kidneys were harvested for histology and Western blot analyses. Kidney tissues were immediately fixed in 10% neutral buffered formalin, embedded in paraffin, and 4-µm sections were stained with hematoxilin and eosin (H&E), periodic acid-Schiff (PAS), or Masson's trichrome (Research Histology Services, University of Pittsburgh Department of Pathology). Portions of the kidney tissues were also snap-frozen in liquid nitrogen for protein extraction.

In vitro CO exposures. Cell cultures were exposed to low-dose exogenous CO at concentrations of 250 ppm supplied in balanced air, containing 5% CO2 for buffering requirements, and delivered into humidified modular chambers maintained at 37°C as previously described (56). CO levels in the chambers were monitored using a CO analyzer (Interscan). Gas samples were introduced to the analyzer through a port in the top of the chamber at a rate of 1 l/min and were analyzed by electrochemical detection, with a sensitivity of 10–600 ppm. Concentration levels were measured hourly and there were no fluctuations in the CO concentrations after the chamber had equilibrated (~5 min).

Cell culture. Human proximal tubular cells (HK-2) were obtained from ATCC (Manassas, VA) and cultured in DMEM/F-12 medium containing insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and 10% FBS. Mouse proximal tubular epithelial cells from MKK3 null (Mkk3–/–) mice and control wild-type (Mkk3+/+) mice were prepared from kidney cortical sections subjected to differential sieving technique and collagenase digestion, as previously described (50). Tubular fragments were suspended in gradient buffer (42% Percoll in Krebs-Henseleit buffer) and subjected to centrifugation, and layer containing proximal tubules was collected and checked for purity by microscopy. The cells were cultured in DMEM/F-12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, 25% HEPES, 1 ng/ml prostaglandin, 5 x 10–11 M triiodothyronine, 5 x 10–8 M hydrocortisone, and 25 ng/ml mouse epidermal growth factor and incubated in a humidified atmosphere of 5% CO2-95% air at 37°C. In experiments involving treatment with exogenous TGF-β1, cells grown to subconfluence were rendered quiescent in serum-free medium for 24 h, followed by treatment with human TGF-β1 (2 ng/ml). In experiments involving exogenous CO treatment, cells were preexposed to 250 ppm CO for 2 h before treatment with or without exogenous TGF-β1 and then continuously exposed to 250 ppm CO for the indicated time periods.

Western blot analysis. For protein extraction, kidney tissues were cut into small pieces and placed in radioimmune precipitation assay buffer (RPA; 1% phosphate-buffered saline, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, 1 mM NaF) and homogenized on ice. The tissue lysates were incubated on ice for 30 min and then centrifuged at 14,000 g for 15 min at 4°C. The protein concentrations were determined by BCA protein assay reagent kit (Pierce, Rockford, IL). Eighty-microgram protein samples were loaded onto 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies (anti-fibronectin, anti-type 1 collagen, anti-{alpha}-SMA, and anti-TGF-β type I or type II receptor; 1:1,000 dilution) overnight on a rocker at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Signal development was carried out using LumiGLO (New England Biolabs) and exposure to X-ray film. The same membranes were reblotted with anti-GAPDH antibodies to control for relative equivalence of protein loading. Quantitative densitometry analysis was performed using Image J software (Research Services Branch, National Institutes of Health) and data are presented as means ± SE (groups of 6 animals) of the ratio of target protein to GAPDH normalized to the sham-operated control mice. Statistical significance of the experimental data (n = 6) was determined by the Student's t-test for paired data. P values <0.05 were considered significant.

For protein extraction from the in vitro experiments, cells were washed with ice-cold phosphate-buffered saline, followed by lysis in RPA buffer. The cell lysates were passed through 21-gauge needles several times and then centrifuged for 15 min at 13,000 g at 4°C. The protein concentrations were determined by BCA protein assay reagent kit (Pierce). Eighty-microgram protein samples were analyzed by 10% SDS-PAGE and immunoblotting was carried out as described above. Signal development was carried out using LumiGLO (New England Biolabs, Ipswich, MA) and exposure to X-ray film. The same membranes were reblotted with anti-GAPDH or anti-{alpha}-tubulin antibodies to control for protein loading. All of the experiments were repeated at least three times with essentially the same results, and representative blots are shown.

Northern blot analysis. Total RNA was isolated by cell lysis with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and size-fractionated (15 µg/lane) on a 1% agarose, 2% formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA (pH 7.2), followed by transfer and UV cross-linking to nylon membranes (Gene Screen Plus; Dupont). The blots were prehybridized for 2 h in Church Gilbert's hybridization buffer (Quality Biological) and hybridized overnight in the same solution containing the appropriate 32P-labeled cDNA probes at 65°C. The blots were then washed two times in solution containing 0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) for 30 min each at 65°C, followed by 15-min washes with solution containing 1% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) at 65°C. The blots were exposed to Kodak X-AR film. The human pro-{alpha}1(I) collagen cDNA and fibronectin cDNA were obtained from ATCC and previously described (49). To control for relative equivalence of RNA loading, the same blots were hybridized with 32P-labeled oligonucleotide probes corresponding to the 18S rRNA as previously described (49). The blots shown are representative of three separate sets of experiments.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Inhaled low-dose CO protects against renal fibrosis in a murine UUO model. To assess the functional effects of inhaled low-dose CO on renal fibrosis, the kidneys of male C57BL6 mice subjected to UUO or sham operation and exposed to either normal RA or continuous inhaled CO (250 ppm) were examined for histopathology (Fig. 1). Masson's trichrome stain of representative kidney sections demonstrates increased ECM deposition within the tubulointerstitium 4 and 7 days after undergoing UUO in mice maintained in normal RA (Fig. 1, C and E, respectively). However, exposure to low-dose CO (250 ppm) suppressed the tubulointerstitial ECM deposition at both 4 and 7 days after UUO (Fig. 1, D and F, respectively). No gross alterations were observed in CO-treated control sham-operated mice (Fig. 1B) compared with those sham-operated mice maintained in normal RA (Fig. 1A).


Figure 1
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  		Fig. 1. Inhaled low-dose carbon monoxide (CO) suppresses extracellular matrix (ECM) accumulation and tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO). Masson's trichrome stain of kidney sections from C57BL6 mice 4 days (UUO 4d: C, D) and 7 days (UUO 7d: E, F) after undergoing UUO, and sham-operated mice (Sham: A, B), exposed to inhaled CO treatment (CO; B, D, F) or control room air (RA; A, C, E). Blue staining demonstrates ECM deposition in the cortical sections of the kidney. Original magnification x200.

 
CO inhibits expression of major ECM proteins, fibronectin, type 1 collagen, and {alpha}-SMA induced by UUO. We examined kidney tissue lysates obtained from sham-operated control mice and from mice 4 and 7 days after undergoing UUO and maintained in normal RA or exposed to low-dose CO (250 ppm). Levels of fibronectin and type 1 collagen were assessed by Western blot analysis and representative blots are shown in Fig. 2, A and C. Quantitative analysis by densitometry shows significant increases in the expression of fibronectin and type 1 collagen in the kidneys of mice at day 4 following UUO (Fig. 2B), and further increased at day 7 after undergoing UUO (Fig. 2D) compared with control sham-operated mice. In contrast, in mice exposed to low-dose CO (250 ppm) significant reductions in the fibronectin and type 1 collagen expression were observed at both 4 and 7 days after UUO compared with the counterpart mice in RA.


Figure 2
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  		Fig. 2. Inhaled low-dose CO inhibits the expression of fibronectin (FN) and type 1 collagen (Col-1) in the kidney induced by UUO. Eighty micrograms of kidney tissue lysates from C57BL6 mice 4 (A) and 7 days (C) after undergoing UUO and exposed to inhaled CO or RA, and sham-operated control mice, were assayed for FN and Col-1 content by Western blot analysis with respective antibodies. Immunoblotting with anti-GAPDH antibodies was used as sample loading controls. Each lane represents protein from the kidney of a single animal. Densitometry (B, D) for quantitative analysis of expression levels of FN and Col-1 protein represents means ± SE (n = 6) for each group of animals; band density was quantified as the ratio to GAPDH and normalized to the sham-operated control mice (*P < 0.05 vs. sham-operated mice; **P < 0.05 vs. UUO mice in RA).

 
We also assessed expression levels of {alpha}-SMA protein, a marker of tubular epithelial-myofibroblast transdifferentiation, and a representative Western blot is shown in Fig. 3A. Quantitative densitometry analysis shows significantly increased expression of {alpha}-SMA in the kidneys of mice at day 4 following UUO compared with control sham-operated mice, and these effects were inhibited by inhaled low-dose CO (250 ppm) treatment (Fig. 3B). As with the histological findings (Fig. 1, A and B), no differences in the expression levels of fibronectin, collagen, or {alpha}-SMA were observed in the kidneys of control sham-operated mice exposed to low-dose CO (250 ppm) compared with sham-operated mice maintained in normal RA (data not shown).


Figure 3
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  		Fig. 3. Inhaled low-dose CO inhibits {alpha}-smooth muscle actin ({alpha}-SMA) expression in the kidney induced by UUO. A: 80 µg of kidney tissue lysates from C57BL6 mice 4 days after undergoing UUO alone and maintained in RA or UUO with inhaled CO, and sham-operated control mice, were subjected to Western blot analysis using anti-{alpha}-SMA antibodies. Immunoblotting with anti-GAPDH antibodies was used as sample loading controls. Each lane represents protein from the kidney of a single animal. B: densitometry for quantitative analysis of expression levels of {alpha}-SMA protein represents means ± SE (n = 6) for each group of animals; band density was quantified as the ratio to GAPDH and normalized to the sham-operated control mice (*P < 0.05 vs. sham-operated mice; **P < 0.05 vs. UUO mice in RA).

 
CO suppresses UUO-induced TGF-β receptor expression in the kidney. We determined the effects of low-dose CO on the expression of TGF-β signaling receptors type I and type II (8). Kidney tissue lysates obtained from sham-operated control mice and from mice 4 days after undergoing UUO and maintained in normal RA or exposed to low-dose CO (250 ppm) were analyzed by Western blot analysis. A representative blot shown in Fig. 4A and quantitative densitometry analysis in Fig. 4B demonstrate that 4-day UUO resulted in marked increases in the expression of TGF-β type I and type II receptors in the kidney and CO treatment significantly suppressed this induction.


Figure 4
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  		Fig. 4. TGF-β type I receptor (TβR-I) and type II receptor (TβR-II) expression in the kidney induced by UUO is suppressed by inhaled low-dose CO treatment. A: 80 µg of kidney tissue lysates from C57BL6 mice 4 days after undergoing UUO alone and maintained in RA or UUO with inhaled CO, and sham-operated control mice, were subjected to Western blot analysis using anti-TβR-I or anti-TβR-II antibodies. Immunoblotting with anti-GAPDH antibodies was used as sample loading controls. Each lane represents protein from the kidney of a single animal. B: densitometry for quantitative analysis of expression levels of TβR-I and TβR-II protein represents means ± SE (n = 6) for each group of animals; band density was quantified as the ratio to GAPDH and normalized to the sham-operated control mice (*P < 0.05 vs. sham-operated mice; **P < 0.05 vs. UUO mice in RA).

 
MKK3 signaling pathway is required for the anti-fibrotic effects of CO. We next examined the role of the MAPK kinase 3 (MKK3) in mediating the CO effects in UUO using mice carrying a null mutation of the Mkk3 gene. Mkk3 null mice (Mkk3–/–) and wild-type Mkk3+/+ mice were subjected to UUO or sham operation and exposed to either normal RA or continuous inhaled CO as previously described above. Representative kidney tissue sections, shown in Fig. 5, illustrate increased ECM deposition within the tubulointerstitium 4 days after undergoing UUO in both the wild-type mice and Mkk3–/– null mice maintained in normal RA (Fig. 5, C and D) compared with sham-operated wild-type (Fig. 5A) and Mkk3–/– null mice (Fig. 5B). However, in the wild-type mice exposed to low-dose CO (250 ppm), marked reduction in the ECM deposition was observed 4 days after UUO, while CO treatment failed to inhibit ECM deposition after UUO in the Mkk3–/– null mice (Fig. 5, E and F, respectively).


Figure 5
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  		Fig. 5. Inhaled low-dose CO fails to inhibit the ECM accumulation induced by UUO in Mkk3–/– null mice. Masson's trichrome stain of kidney sections from Mkk3–/– null mice (Mkk3–/–: B, D, F) and wild-type Mkk3+/+ mice (WT: A, C, E) 4 days after undergoing UUO with (UUO+CO: E, F) or without (UUO: C, D) inhaled CO treatment, and sham-operated mice (Sham: A, B). Blue staining demonstrates ECM deposition. Original magnification x200.

 
Accordingly, we also assessed levels of fibronectin, type 1 collagen, and {alpha}-SMA protein in the kidney tissues lysates by Western blot analysis and representative blots are shown in Fig. 6A and quantitative densitometry analysis in Fig. 6B. UUO in the Mkk3–/– mice resulted in increased expression of fibronectin, type 1 collagen, and {alpha}-SMA, similar to UUO in the wild-type mice compared with sham-operated control mice. As with the histology data, while CO treatment significantly inhibited the induction of fibronectin, type 1 collagen, and {alpha}-SMA after UUO in the wild-type mice, similar inhibition was not observed in the Mkk3–/– mice. No differences were seen in kidney tissue lysates obtained from Mkk3–/– mice 4 days after undergoing UUO and maintained in normal RA or exposed to low-dose CO (250 ppm). Thus, our findings indicating that the anti-fibrotic effects of CO treatment were abrogated in mice carrying null mutation of Mkk3 suggest an involvement of the MKK3 signaling pathway in mediating the CO effects.


Figure 6
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  		Fig. 6. Inhaled low-dose CO fails to inhibit the expression of FN, Col-1, and {alpha}-SMA induced by UUO in Mkk3–/– mice. A: 80 µg of kidney tissue lysates from wild-type Mkk3+/+ mice and Mkk3–/– null mice 4 days after undergoing UUO alone and maintained in RA or UUO with inhaled CO, and sham-operated control mice (Ctl), were subjected to Western blot analysis using respective antibodies against FN, Col-1, and {alpha}-SMA. As loading controls, the same cell lysates were subjected to immunoblotting with anti-GAPDH antibodies. B: densitometry for quantitative analysis of expression levels of FN, Col-1, and {alpha}-SMA protein represents means ± SE (n = 6) for each group of animals; band density was quantified as the ratio to GAPDH and normalized to the sham-operated control mice (*P < 0.05 vs. sham-operated mice; **P < 0.05 vs. UUO mice in RA).

 
CO inhibits TGF-β1-induced fibronectin, type 1 collagen, and {alpha}-SMA expression in renal proximal tubular epithelial cells. To further examine the mechanisms of CO effects, we examined in vitro effects of low-dose CO (250 ppm) exposure on human kidney proximal tubular epithelial cells that are a prototypic target cell type in renal tubulointerstitial injury in UUO (19). Western blot analysis demonstrates that stimulation of HK-2 cells with TGF-β1 (2 ng/ml) resulted in the induction of {alpha}-SMA expression up to 24 h after stimulation with TGF-β1 before declining (Fig. 7A) and that low-dose CO (250 ppm) treatment (representative blot for 24-h time point shown) suppressed TGF-β1-induced {alpha}-SMA protein expression in HK-2 cells (Fig. 7B). We next determined whether CO exerted similar inhibitory effects on TGF-β1-induced fibronectin and type 1 collagen expression in HK-2 cells. As shown in Fig. 8A, Northern blot analysis showed that stimulation with TGF-β1 (2 ng/ml) for 12–72 h increased both fibronectin and type 1 collagen mRNA expression in HK-2 cells in a time-dependent fashion. Low-dose CO (250 ppm) exposure for 12–72 h (representative blots for 72-h time point shown) resulted in significant inhibition of TGF-β1-induced fibronectin and type 1 collagen mRNA and protein expression in HK-2 cells (Fig. 8, B and C). To determine whether inhibiting the MKK3 pathway will abrogate the anti-fibrotic actions of CO, we utilized primary cultures of proximal tubular epithelial cells from MKK3 null (Mkk3–/–) mice and control wild-type (Mkk3+/+) mice. As shown in Fig. 8D, low-dose CO (250 ppm) treatment failed to inhibit TGF-β1-induced fibronectin and type 1 collagen protein expression in Mkk3–/– tubular cells, suggesting that the anti-fibrotic effects of CO are mediated via the MKK3 signaling pathway.


Figure 7
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  		Fig. 7. CO inhibits TGF-β1-induced {alpha}-SMA expression in renal proximal tubular epithelial cells. A: time course of induction of {alpha}-SMA by TGF-β1 in human proximal tubular cells (HK-2) cells. Total cell lysates isolated from HK-2 cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) for the indicated time periods were subjected to Western blot analysis using anti-{alpha}-SMA antibodies. Immunoblotting with anti-{alpha}-tubulin antibodies was used as protein loading controls. B: low-dose CO treatment suppresses TGF-β1-induced {alpha}-SMA expression in HK-2 cells. Cell lysates isolated from HK-2 cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) with (+) or without (–) 250 ppm CO for 24 h were subjected to Western blot analysis with anti-{alpha}-SMA antibodies. Immunoblotting with anti-{alpha}-tubulin antibodies was used as protein loading controls. Expression levels of {alpha}-SMA protein were quantitated by densitometry and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments (*P < 0.05 vs. untreated cells; **P < 0.05 vs. TGF-β1-treated cells under RA).

 

Figure 8
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  		Fig. 8. CO inhibits TGF-β1-induced FN and Col-1 expression in renal proximal tubular epithelial cells. A: time course of induction of FN and pro-{alpha}1(I) collagen mRNA by TGF-β1 in HK-2 cells. Total RNA isolated from HK-2 cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) for the indicated time periods was subjected to Northern blot analysis with 32P-labeled cDNA probes corresponding to FN and pro-{alpha}1(I) collagen. 18S rRNA hybridization signals served as normalization for RNA loading. B: low-dose CO treatment suppresses TGF-β1-induced FN and Col-1 expression in HK-2 cells. Left: total RNA isolated from HK-2 cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) with (+) or without (–) 250 ppm CO for 72 h was subjected to Northern blot analysis with 32P-labeled cDNA probes corresponding to FN or pro-{alpha}1(I) collagen [Pro-{alpha}1(I) Col]. 18S rRNA hybridization signals served as normalization for RNA loading. C: Western blot analysis. Total cell lysates obtained from HK-2 cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) with (+) or without (–) 250 ppm CO for 72 h were subjected to Western blot analysis with anti-FN and anti-Col-1 antibodies. Immunoblotting with anti-{alpha}-tubulin antibodies was used as protein loading controls. Expression levels of FN or Col-1 protein were quantitated by densitometry and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments (*P < 0.05 vs. untreated cells; **P < 0.05 vs. TGF-β1-treated cells under RA). D: CO fails to inhibit TGF-β1-induced FN and Col-1 expression in Mkk3–/– renal proximal tubular epithelial cells. Total cell lysates obtained from MKK3 null (Mkk3–/–) mouse or wild-type (WT) mouse proximal tubular epithelial cells incubated in the absence (–) or presence (+) of exogenous TGF-β1 (2 ng/ml) with (+) or without (–) 250 ppm CO for 72 h were subjected to Western blot analysis with anti-FN and anti-Col-1 antibodies. Immunoblotting with anti-{alpha}-tubulin antibodies was used as protein loading controls. Expression levels of FN or Col-1 protein were quantitated by densitometry and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments (*P < 0.05 vs. untreated cells; **P < 0.05 vs. TGF-β1-treated cells without CO).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study investigated the functional effects of CO on renal injury leading to tubulointerstitial fibrosis. Kidneys with ureteral obstruction develop progressive tubulointersitial damage. UUO is a well-established in vivo model of renal fibrosis (19). We show that UUO induced through complete ligation of the left ureter in mice led to increased ECM deposition within the tubulointerstitium 4 and 7 days after undergoing UUO, and correlated with strongly induced expression of major ECM proteins, fibronectin, and type 1 collagen, as well as {alpha}-SMA, a marker of activated myofibroblasts, in the kidney. Administration of inhaled low-dose CO (250 ppm) markedly reduced the deposition of ECM after UUO in the obstructed kidney and inhibited UUO-induced fibronectin, type 1 collagen, and {alpha}-SMA expression, suggesting that inhaled CO can inhibit development of renal fibrosis in UUO.

CO is a low-molecular weight gas, well-known for its toxicity at high concentrations, which can lead to hemoglobin saturation, tissue hypoxia, and injury. Deleterious toxicity of CO leading to death occurs in the range of 50–80% carboxyhemoglobin (COHb) saturation (48). However, CO at 250 ppm is well below the concentration of 3,000 ppm used in humans during measurement of diffusing capacity of the lung for CO (DLCO) in pulmonary function testing. Furthermore, human and animal studies used CO concentration in the range of 250 ppm up to 1,500 ppm without adverse effects (27, 42, 55). CO is also generated endogenously in mammalian cells primarily as a major byproduct of heme degradation catalyzed by HO (33, 45). Of the isoforms of HO identified, HO-1 is highly inducible, whereas HO-2 is constitutively expressed. HO-3 is the least understood and may be a pseudogene, and at least in the rat, it has been suggested that there is no functional HO-3 gene (12). HO-1 can be induced by a variety of agents causing oxidative stress including heavy metals, cytokines, hormones, endotoxin, and heat shock (1, 3, 6, 17, 25). Emerging body of evidence indicates that HO-1, besides its role in heme degradation, serves as a key biological molecule in the adaptation response and defense against oxidative stresses, and it provides cytoprotection in a variety of in vivo and in vitro models of cellular and tissue inflammation and injury (33). Although the precise molecular mechanism by which HO-1 confers protection against cellular stress is still incompletely understood, there is evidence in the literature to support the role of the catalytic byproducts of HO reaction, in particular CO, in mediating the protective function of HO-1.

A number of recent studies demonstrating that exogenous low-dose CO at similar concentrations to those used here can provide potent cytoprotective effects have been reported in models of lung and hepatic injury, airway inflammation, and cardiac transplantation (2, 32, 39, 57). In the bleomycin-induced lung injury model, overexpression of HO-1 gene or administration of exogenous CO inhibits development of pulmonary fibrosis (46, 56). However, few studies examined the role of HO-1 and CO in kidney injury, and the functional role of CO in the context of renal fibrosis has not been previously examined. HO-1 overexpression or CO administration has been shown to protect experimental kidney transplants from ischemia-reperfusion injury (4, 30). Moreover, the administration of CO donor compounds, namely carbon monoxide-releasing molecules (CORM-3), protected against cisplatin nephrotoxicity and ischemia-reperfusion-induced renal injury (44, 47). A recent report also demonstrated that hemin, a chemical inducer of HO-1, decreased renal injury and interstitial fibrosis in UUO (18). Our report is the first to demonstrate that inhaled CO can inhibit development of renal fibrosis in UUO.

Oxidative stress has been proposed as one major cause responsible for the initiation and progression of chronic kidney diseases. In response to oxidative stress, induction of anti-oxidant enzymes is thought to occur as a mechanism to protect cellular functions and maintain in vivo homeostasis. HO-1 is one of the most potent anti-oxidant enzymes known. A number of laboratories have now demonstrated that induction of endogenous HO-1 or exogenous administration of HO-1 via gene transfer provided protection against oxidative stresses in various in vivo and in vitro models (7, 21, 34). The importance of HO-1 in the host's defense against oxidant stress is underscored by studies in HO-1 null (HO-1–/–) mice, which exhibit increased susceptibility to oxidative stress such as LPS (36, 37). Furthermore, the only documented human case of HO-1 deficiency exhibited significant phenotypic changes reflective of homeostasis imbalance, with extensive endothelial cell damage, anemia, iron deposition, hyperbilirubinemia, and HO-1-deficient cells derived from the patient demonstrated increased susceptibility to oxidative stress (54). Recent studies showed that oxidative stress plays an important role in a variety of pathological processes including pulmonary and hepatic fibrosis (10, 31, 41). The role of oxidative stress has also been implicated in renal injury in the UUO model (16). Deficiency of an anti-oxidant enzyme catalase enhanced tubulointerstitial injury and fibrosis in UUO in mice (43). On the other hand, anti-oxidant agents, such fluvastatin and bioflavonoids, inhibited renal fibrosis in UUO (15, 28). Therefore, anti-oxidant actions may represent one plausible mechanism by which CO inhibits renal tubulointerstitial fibrosis in UUO.

CO has previously been shown to exert some of its actions via the MAPK pathways. For example, CO acts via the activation of p38 MAPK pathway to protect against TNF-{alpha}-induced apoptosis in endothelial cells (5), and the MKK3 is required for the anti-inflammatory effects of CO and protection against oxidant-induced lung injury (35). However, in rat aortic smooth muscle cells, the anti-apoptotic actions of CO involve the activation of soluble guanylate cyclase, but not p38 MAPK (23). CO protection of the lung against ischemia-reperfusion injury involves guanylate cyclase and cGMP (11), whereas CO provides protection in a murine model of sepsis through modulation of inflammatory cytokine production via the JNK signaling pathway (29). These findings suggest that the intracellular signaling pathways mediating CO effects are cell type and context specific. Here, we examined the role of the MKK3 signaling pathway in mediating the CO effects in UUO. MKK3 is one of the immediate upstream MAPK kinase required for activation of p38 MAPK (49, 50). In our present study, the anti-fibrotic effects of CO treatment were abrogated in mice carrying a null mutation of MAPK kinase 3 (Mkk3–/–). CO failed to inhibit the expression of {alpha}-SMA and production of collagen and fibronectin in UUO in Mkk3–/– mice compared with wild-type mice. Moreover, CO failed to inhibit TGF-β1-induced fibronectin and type 1 collagen expression in cultured Mkk3–/– tubular cells. Our findings indicate that the anti-fibrotic effects of CO are mediated via the MKK3 signaling pathway.

The potent profibrogenic actions of TGF-β1 are well-established. Induction of TGF-β1 expression has been previously demonstrated in the UUO model and this is thought to be the key mediator of renal tubulointerstitial fibrosis (9, 13, 20, 22). Our studies show that the expression of TGF-β type I and type II receptors is markedly induced in the kidney after UUO, and CO treatment significantly suppresses this induction, indicating a potential contribution to enhance profibrotic actions of TGF-β1 through upregulation of its signaling receptors. Additionally, we examined CO effects in vitro in human kidney proximal tubular epithelial cells to determine whether low-dose CO (250 ppm) exerted an inhibitory effect on the induction of ECM proteins in HK-2 cells stimulated with TGF-β1. Our data demonstrate that in vitro CO exposure markedly suppressed TGF-β1-induced expression of {alpha}-SMA, type 1 collagen, and fibronectin in HK-2 cells, which may represent a plausible mechanism for the anti-fibrotic effects of CO. In summary, we show that administration low-dose CO (250 ppm) inhibits UUO-induced ECM elaboration in mice in vivo and inhibits TGF-β1-stimulated ECM expression in renal proximal tubular epithelial cells in vitro. Taken together, our findings suggest that low-dose CO can provide protection against renal injury by exerting anti-fibrotic effects, via the MKK3 signaling pathway, to inhibit development of renal fibrosis in obstructive nephropathy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Institutes of Health Grant R01-DK-57661 from National Institute of Diabetes and Digestive and Kidney Diseases and the M. James Scherbenske Grant from the American Society of Nephrology to M. E. Choi.


   ACKNOWLEDGMENTS
 
We thank E. Ifedigbo for assistance with animal breeding and the CO exposure experiments; and C. Dai for helpful guidance with the UUO technique.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Choi, Brigham and Women's Hospital, Renal Division, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115 (e-mail: mchoi{at}rics.bwh.harvard.edu)

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.


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 RESULTS
 DISCUSSION
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 REFERENCES
 

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