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Department of Nephrology (T.D.H., R.M., G.J.B.), Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; and Departments of Medicine (T.D.H., R.M., G.J.B.) and Biochemistry and Molecular Biology (I.M., G.W.T., C.S.S.), Howard Florey Institute of Experimental Physiology and Medicine (I.M., C.Z., G.W.T., C.S.S.), University of Melbourne, Victoria 3010, Australia
Address all correspondence and requests for reprints to: Chrishan S. Samuel, Ph.D., Howard Florey Institute, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: c.samuel{at}hfi.unimelb.edu.au.
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Abstract |
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-smooth muscle actin (-SMA) staining (myofibroblasts) and interstitial collagen sub-types (SDS-PAGE; types I, III, and V), whereas gelatin zymography demonstrated increased expression of matrix metalloproteinase-2 after surgery. By d 10 after UUO, there was a 5-fold decrease in RLX mRNA expression (quantitative RT-PCR) in RLX+/+ animals. Total collagen and -SMA expression were significantly greater in the obstructed kidneys of RLX–/– mice 3 d after UUO (both P < 0.05 vs. RLX+/+ D3 after UUO), but comparable to that in RLX+/+ animals 10 d after UUO. Administration of recombinant H2 relaxin to RLX–/– mice 4 d before UUO ameliorated the increase in collagen and -SMA expression (both P < 0.05 vs. untreated RLX–/– mice) by d 3 after UUO. Expression of monocyte chemoattractant protein-1 and macrophage infiltration (inflammation) in addition to that of matrix metalloproteinases was unaffected by genotype after UUO. These combined data demonstrate that endogenous RLX acts as a modulating factor in tubulointerstitial fibrosis, a hallmark of progressive renal disease. This is likely to be via direct effects on renal myofibroblast function. |
Introduction |
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-smooth muscle actin (-SMA) (3). Their accumulation is a key event, as they have consistently been shown to be the most prodigious matrix producers. Finally, newly synthesized matrix is remodeled by various proteases, the net accumulation being the result of less degradation than synthesis (1). However, it is increasingly recognized that a number of naturally occurring renoprotective factors limit fibrogenesis, with tissue homeostasis being a balance between profibrotic signals and naturally occurring inhibitors (4). The most studied of these, hepatocyte growth factor (HGF) (5) and bone morphogenic protein 7 (BMP-7) (6) down-regulate profibrotic TGF-ß1 signaling by interfering with Smad signal transduction. It is our contention that the polypeptide hormone relaxin is likewise an important modulator of fibrosis in the kidney at several levels (7).
Relaxin is a small peptide hormone structurally related to the insulin family of peptides and is primarily produced by the ovary and/or placenta in pregnancy and the prostate of mammals (8). Humans and higher primates have three relaxin genes, designated H1, H2, and H3 relaxin, whereas rodents contain two relaxin genes, relaxin (equivalent to H2 relaxin) and relaxin-3 (equivalent to H3 relaxin). H2 relaxin or relaxin (in rodents) are the major sources of circulating relaxin. Normally associated with reproduction, relaxin has been implicated in a number of pregnancy-related functions involving extracellular matrix (ECM) turnover and collagen degradation (8). It is now becoming evident that the ability of relaxin to reduce matrix synthesis and increase ECM degradation has important implications elsewhere (9). Recombinant H2 relaxin has been shown to reduce fibrosis in a number of organs (8, 10), specifically ameliorating progression of interstitial and glomerular fibrosis in several experimental models of renal disease (11, 12, 13) and down-regulating the activation of renal myofibroblasts in vitro (14).
Furthermore, recent studies of the natural history of relaxin-deficient mice (RLX–/–) provide valuable insights into the importance of endogenous relaxin (15). Male RLX–/– mice developed an age-related progressive fibrosis in several organs including the kidney (15), lung (16), heart (17), skin (18), prostate, and testis (19). The increased renal fibrosis resulted in a 25–30% rise in renal collagen concentration and increased renal dysfunction by 12-months of age, although correcting relaxin deficiency with exogenous recombinant H2 relaxin was able to reverse this effect within 14 d (15).
Therefore, a growing body of evidence suggests that relaxin is potentially a counterbalance to the profibrotic factors released after injury (7). Reduced levels of endogenous anti-fibrotic factors may thus accelerate fibrosis and its destructive consequences. To investigate this, the present study examined the significance of endogenous relaxin in the pathogenesis of an experimental model of renal tubulointerstitial fibrosis (20), the final common pathway to all progressive kidney disease. Therefore, unlike previous work, this study focused on the role of endogenous relaxin in regulating the renal response to injury.
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Materials and Methods |
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Animals and experimental protocol
The time course of progressive fibrosis postunilateral ureteric obstruction (UUO) was examined and compared in relaxin-deficient and replete mice. The experimental model was chosen on the basis that UUO is a widely used model of primary tubulointerstitial disease, occurs independently of species and strain, and demonstrates changes that mimic the pathology of human progressive renal disease (20, 22).
All 4- to 6-wk-old male relaxin wild-type (RLX+/+) and RLX–/– littermate mice used in this study were generated from relaxin heterozygous (RLX+/–) parents, and were housed and fed in a controlled environment as detailed before (15). UUO surgery was performed under general anesthesia. The left ureter was ligated with 5.0 surgical silk while the contralateral ureter remained intact. The incision was sutured and mice were allowed to recover after temgesic administration (Buprenorphine; Reckitt Benckiser, West Ryde, New South Wales, Australia). These experiments were approved by the Howard Florey Institute’s Animal Ethics Committee, which adheres to the Australian Code of Practice for the Care and Use of Laboratory Animals for Scientific Purposes.
Tissue collection
Mice (n = 8–12 per genotype and time point) were killed by an overdose of anesthetic before the obstructed and contralateral kidneys were removed at d 0 (control), 3, or 10 after surgery. These time points were based upon pilot studies, which showed early and chronic tubulointerstitial fibrosis at d 3 and 10, respectively. Kidney tissues were weighed and used for various analyses including real-time PCR (relaxin and relaxin-3 mRNA), RT-PCR [monocyte chemoattractant protein-1 (MCP-1) mRNA], hydroxyproline analysis (total collagen content), SDS-PAGE (interstitial collagen types), and histological analyses (myofibroblast accumulation, collagen types, and macrophage infiltration). In each case, the tissue was divided into equal transverse sections, with equivalent pieces being allocated to individual assays.
Human recombinant relaxin treatment of relaxin-deficient mice
In separate experiments, 4- to 6-wk-old male RLX–/– mice (n = 10) were anesthetized and implanted with sc osmotic mini-pumps (model 1007D; Alzet, Cupertino, CA) as described before (15, 16). The osmotic mini-pumps were administered with either 20 mM sodium acetate buffer, pH 5.0 (vehicle; n = 5), or 0.5 mg/kg·d H2 relaxin in vehicle (n = 5) over 7 d, 4 d before UUO surgery. The dose of H2 relaxin used was previously shown to produce circulating relaxin levels of approximately 20 ng/ml (16), which is within the levels found in pregnant rodents (8, 23, 24). At the completion of the 7-d H2 relaxin treatment period (3 d after UUO surgery) animals were killed before kidney tissue was collected as above.
Tissue RNA extraction, real-time PCR analysis of relaxin and relaxin-3 mRNA expression, and RT-PCR analysis of MCP-1 mRNA in the kidney
To determine whether endogenous relaxin mRNA expression was altered during chronic renal disease, obstructed and contralateral kidney tissues from control (d 0), 3-, and 10-d post-UUO RLX+/+ mice (n = 4–6 per group) were collected and stored at –80 C. To determine whether endogenous relaxin-3 mRNA expression was altered during chronic renal disease, obstructed and contralteral kidneys from control (d 0), 3-, and 10-d post-UUO RLX+/+ and RLX–/– mice (n = 4–6 per group) were collected and stored at –80 C. To examine the effects of endogenous relaxin deficiency on acute inflammation, kidney tissues from mice at 0-, 3-, and 10-d post-UUO RLX+/+ and RLX–/– mice (n = 4–5 samples per genotype and time point) were collected and stored at –80 C. All tissues were then homogenized with TRIzol reagent (Invitrogen, Carlsbad, CA) before the RNA was extracted from all samples according to the manufacturer’s instructions.
For real-time PCR analysis of relaxin and relaxin-3 mRNA expression, total RNA (1 µg) from each sample was used for the RT reaction, as described previously (16, 25). The following mouse relaxin primers and probes were designed to span the intron of the respective genes: relaxin forward primer 5'-AGGCAAGCCACTGAAGTTGTG-3'; relaxin reverse primer 5'-GCCTTGAGCTCTTCAGACAAATTT-3'; relaxin probe (6-carboxyfluorescein) 5'-CAAAGATGCAGAGCCTTTCGATACGACG-3'; relaxin-3 forward primer 5'-CAATACAGACCACCTGGCCA-3'; relaxin-3 reverse primer 5'-TTTGGTTAGGGCCAGCCAC-3'; relaxin-3 probe (6-carboxyfluorescein) 5'-CTGGATGAAGCGGTGGGCTCCA-3'.
A second transcription reaction used 30 ng RNA and a series of kidney RNA dilutions (100–0.001 ng) for the endogenous reference (18s ribosomal RNA) PCR, as described before (25). The standard curve method (as outlined in the ABI User Bulletin no. 2) was used in this study as previously described (25).
RT-PCR was also used to determine the effects of relaxin deficiency on MCP-1 mRNA in the kidney, using previously detailed primer sequences and PCR conditions (26). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in separate PCR to control for quality and equivalent loading of the cDNA. The MCP-1 to GAPDH ratio was obtained from densitometry of the PCR products and is expressed as arbitrary units.
Hydroxyproline analysis of kidney collagen
A similar portion of the obstructed and contralateral kidneys of RLX+/+ and RLX–/– mice, at d 0, 3, and 10 after surgery (n = 4–7 per genotype and time point) were weighed (wet weight) and lyophilized to dry weight, to determine their hydroxyproline content, as described previously (27). Hydroxyproline values were then converted to collagen content by multiplying by a factor of 6.94 (28) and further expressed as a percentage of the tissue dry weight (collagen concentration).
SDS-PAGE analysis of kidney collagen types
The interstitial collagen types present in the post-UUO kidney were determined by limited pepsin digestion (enzyme to substrate ratio, 1:10) as described before (15). The pepsin-digested collagen chains were analyzed by interrupted electrophoresis with delayed reduction of the type III collagen disulfide bonds to separate the 1(III) collagen chains from 1(I) collagen chains (29). The gels were stained overnight at 4 C with 0.1% (wt/vol) Coomassie brilliant blue R-250 and destained as described previously (15).
Immunohistochemical staining for myofibroblasts, collagen III, and macrophages in the kidney
Histochemical staining with antisera against -SMA (EPOS conjugated; Dako Cytomation, Glostrup, Denmark), collagen III (Southern Biotechnology, Birmingham, AL), and f4/80 (Serotec, Kidlington, Oxford, UK) was used to identify myofibroblasts (30), collagen III deposition (30), and monocyte/macrophage infiltration (26), respectively. In each case, 4% paraformaldehyde (-SMA, collagen III)- or methyl carnoys (f4/80)-fixed tissue and paraffin-embedded sections were dewaxed, rehydrated, and incubated with appropriate antiserum. Unconjugated primary antisera were detected using an appropriate biotinylated secondary antibody and avidin DH-biotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA), as described previously (30). Peroxidase activity was demonstrated using a freshly prepared substrate solution of 3.3'-diaminobenzidine tetrahydrochloride (Dako Cytomation). Cortical interstitial immunoperoxidase staining for -SMA, collagen III, and f4/80 was quantified by point counting (30) using an eye piece graticule with equidistant intersecting lines; results were expressed as the percentage of intersections with positive staining divided by the total number of grid intersections. Results are expressed as the percentage of total area stained.
Gelatin zymography of kidney matrix metalloproteinase (MMP)-2 and MMP-9
The MMPs from the obstructed and contralateral kidneys of RLX+/+ and RLX–/– mice 0, 3, and 10 d after UUO were extracted as described before and analyzed by zymography for gelatinase expression and activity (31). The glycosylated, latent, and active forms of MMP-2 and MMP-9 were determined, while densitometry of the combined latent and active forms of MMP-2 and MMP-9 was performed using a Bio-Rad GS 710 Densitometer (Richmond, CA) and Quantity-One software (Bio-Rad). The mean ± SE density of MMP-2 and MMP-9 were graphed and expressed as the relative ratio of the values in the d-0 RLX+/+ group, which was expressed as 1.
Statistical analysis
The results were analyzed by one-way ANOVA using the Newman-Keuls test for multiple comparisons between groups. All data in this paper are presented as the mean ± SEM, with P < 0.05 described as statistically significant.
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), which were modestly decreased by 36% (P = 0.14) and by 5- to 6-fold (P < 0.02) in the obstructed, but not unobstructed (contralateral) kidneys of wild-type mice 3 and 10 d after UUO, respectively (Fig. 1A).
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) to that of renal relaxin mRNA, whereas expression was 36% less (P < 0.05) in control RLX–/– mice. In both genotypes, a biphasic response was observed after UUO; relaxin-3 expression decreased at d 3 (P < 0.05 vs. control) before increasing above basal levels by d 10 (P < 0.05 vs. control). The gene expression of relaxin-3 in contralateral kidneys was qualitatively similar, although mRNA levels only returned to basal levels at d 10.
The effects of relaxin deficiency on inflammation after UUO
Low mRNA levels of the chemoattractant MCP-1 (corrected by GAPDH loading) were measured in the kidneys of control RLX+/+ and RLX–/– mice and in the unobstructed kidneys of mice, 3 and 10 d after UUO (Fig. 2A). In the obstructed kidneys of RLX+/+ and RLX–/– mice, there was a transient but equivalent 6- to 7-fold increase in the levels of MCP-1 mRNA at 3 d after UUO. By d 10 after UUO, MCP-1 mRNA levels were similar to that measured in the kidneys of control animals (Fig. 2A). Likewise, control RLX+/+ and RLX–/– mice demonstrated low and equivalent numbers of interstitial macrophages (Fig. 2B), which were markedly increased 6-fold in the obstructed kidneys of both groups of animals by 3 d after UUO (P < 0.01 vs. d 0 values), and more than 20-fold by d 10 (P < 0.001 vs. d 0 values). Thus, the absence of relaxin did not significantly alter inflammation in the obstructed kidney of mice after UUO.
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-SMA (Fig. 3A), and hence myofibroblasts, were measured in control and unobstructed kidneys of both RLX+/+ and RLX–/– mice. UUO in RLX+/+ mice resulted in a 3-fold increase in myofibroblast accumulation by 3 d after UUO (P < 0.05 vs. d 0), significantly less than the 4.5-fold increase seen in RLX–/– mice (P < 0.01 vs. d 0; P < 0.05 vs. d-3 obstructed RLX+/+; Fig. 3A). By 10 d after UUO, there was a 6.5-fold increase in kidney -SMA staining in both RLX+/+ and RLX–/– mice (Fig. 3A), which was not statistically different between the two groups. These findings demonstrated that the absence of relaxin led to the acceleration of myofibroblast accumulation in the early phases of tubulointerstitial fibrosis.
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-SMA was significantly less in RLX–/– mice treated with H2 relaxin compared with untreated RLX–/– mice (Fig. 3B). Indeed, H2 relaxin treatment of animals was able to decrease -SMA expression to levels below that found in the obstructed kidneys of RLX+/+ animals, albeit not down to levels detected in the corresponding contralateral kidneys of these mice (Fig. 3B). As expected, there was no significant difference in myofibroblast accumulation in the kidneys of untreated and vehicle-treated RLX–/– mice, 0 and 3 d after UUO (data not shown). Furthermore, no significant differences in -SMA staining (Fig. 3B) were detected in the contralateral kidneys of RLX+/+, untreated/vehicle-treated RLX–/–, and H2 relaxin-treated mice, 3 d after surgery.
The effects of relaxin deficiency on total renal collagen concentration after UUO
The extent to which interstitial renal fibrosis had developed after UUO in RLX+/+ and RLX–/– mice, was determined by total collagen concentration (Fig. 4A). Equivalent (baseline) levels of collagen concentration (Fig. 4A) were detected in the kidneys of control RLX+/+ and RLX–/– mice (at d 0) and in the unobstructed kidneys of both groups of mice, 3 and 10 d after UUO. In 3-d obstructed kidneys of RLX+/+ mice, a 45% increase in collagen concentration (P < 0.05 vs. d 0 values) was measured, significantly less than the 2-fold increase in RLX–/– mice at this time point (P < 0.01 vs. d 0; P < 0.05 vs. d-3 obstructed RLX+/+; Fig. 4A). By d 10 after UUO, a 3.5-fold increase in collagen concentration in both RLX+/+ and RLX–/– mice was measured (P < 0.001 vs. d-0 values; P < 0.05 vs. d-3 obstructed kidney values), which was not statistically different between the two groups. These findings indicated that the absence of relaxin led to the acceleration of collagen accumulation in early phases of tubulointerstitial fibrosis in this model.
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) in the obstructed kidneys of these animals compared with respective levels measured in untreated/vehicle-treated RLX–/– mice. However, although collagen levels were equivalent to RLX+/+ mice, they did not reach levels seen in the contralateral control kidneys. No significant differences in collagen accumulation were detected in the kidneys of untreated and vehicle-treated RLX–/– mice, 0 and 3 d after UUO (data not shown). Furthermore, no significant differences in collagen concentration (Fig. 4B) were detected in the contralateral kidneys of RLX+/+, untreated/vehicle-treated RLX–/–, and H2 relaxin-treated mice, 3 d after surgery.
The effects of relaxin deficiency on accumulation of interstitial collagen types after UUO
SDS-PAGE analysis was used to qualitatively examine the accumulation of collagen isotypes (Fig. 5A). Of the isotypes examined, collagen I was the most abundant, with a detectable increase at d 3 after UUO. However, the increase was greater (P < 0.05) in RLX–/– animals, compared with that measured in RLX+/+ mice at this time point. Conversely, collagen III and V chains were barely detectable at d 0 and 3, suggesting temporal differences in collagen regulation. By d 10, all collagen isotypes had increased markedly, although collagen I remained the most abundant. However, there was no difference in collagen I, III, or V expression between RLX–/– and RLX+/+ animals. The composition of ECM in contralateral unobstructed kidneys was similar to control d-0 tissue throughout.
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). By 10 d after surgery, collagen III staining in obstructed kidneys was 20- to 25-fold greater than that seen in control and d-3 animals (Fig. 5B), with no difference between genotypes. Little collagen III staining was seen in the contralateral kidneys at both time points.
The effects of relaxin deficiency on renal MMP-2 and MMP-9 expression and activation after UUO
Gelatin zymography of MMP extracts was used to analyze relaxin-induced changes in MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) expression and activity after UUO (Fig. 6A). Shown is a representative zymograph of duplicate samples per group and further experiments (of n = 4–6 samples per group) gave similar results. These MMPs were predominantly expressed in their latent forms in control and 3-d obstructed/contralateral kidneys (Fig. 6A). By 10 d after UUO, MMP-9 was still primarily expressed in its latent form, whereas most of the MMP-2 detected was present in its activated form (Fig. 6A), which correlated to the severity of disease at this time point.
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) demonstrated equivalent levels of MMP-9 expression in control and 3-d obstructed and unobstructed kidneys. MMP-9 expression was significantly elevated (P < 0.05) in the obstructed kidneys of both RLX+/+ and RLX–/– mice, and to a further extent in the unobstructed kidneys of both groups of mice by 10 d after UUO, compared with that measured at d 0 (Fig. 6B). Basal MMP-2 levels were significantly increased (P < 0.05) in control RLX–/– mice, compared with that detected in control RLX+/+ mice (Fig. 6C). MMP-2 expression was further elevated in the obstructed, but not contralateral kidneys of 3-d after UUO RLX+/+ and RLX–/– mice (P < 0.01 vs. d 0 RLX+/+ levels). However, MMP-2 expression was significantly decreased (P < 0.05) in the unobstructed kidneys of RLX–/– mice, compared with values from unobstructed kidneys of 3-d after UUO RLX+/+ mice (Fig. 6C). By 10 d after UUO, there was a 5- to 10-fold increase in MMP-2 expression in both RLX+/+ and RLX–/– mice, which was predominantly detected in its active form (Fig. 6, A and C), compared with levels detected at d 0 and 3 after UUO (all P < 0.01). Again, this increase in MMP-2 expression was not observed in the contralateral kidneys of both groups of mice (Fig. 6C). These results demonstrate that MMP-2 is progressively up-regulated and activated in interstitial renal disease. |
Discussion |
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UUO is a widely used experimental model of primary tubulointerstitial disease. Permanent obstruction leads to an inevitable accumulation of ECM in the interstitium. In wild-type animals, up-regulation of the proinflammatory cytokine MCP-1 and accumulation of interstitial macrophages and myofibroblasts was seen within 3 d of obstruction. Consistent with this, our biochemical studies indicated that UUO was paralleled by a progressive increase in both total collagen and individual collagen types (I, III, V) commonly found in interstitial fibrosis. A serendipitous finding was that the accumulation of the various collagen types studies was temporally distinct, with an increase in collagen I at d 3 occurring before an increase in collagen III and V. Although the significance of this is unclear, it does imply that collagen isotypes are differentially regulated.
Injury to the kidney can be initiated via diverse mechanisms, such as ischemia, genetic and metabolic defects, immune mechanisms, and mechanical injury. Based on the disease kinetics, renal injury is classified as being either acute or chronic. Although there can be some level of endogenous tissue repair after acute injury (22), chronic renal disease is generally considered irreversible; the switch from repair to scarring is poorly understood.
However, it does seem increasingly likely that an imbalance of profibrotic and antifibrotic signals may have an important role in determining the pathological fate of the kidney (5, 6). For instance, BMP-7 is a member of the TGFß superfamily that antagonizes TGFß1 signaling by activating Smad6. Reduced expression accompanies several models of chronic kidney disease, whereas recombinant BMP-7 is renoprotective (6). Like BMP-7, HGF abrogates the cellular effects of TGFß1 (5): suppressing recruitment and activation of fibroblasts, reducing matrix production, and increasing its degradation (5). Recombinant HGF limits progression, whereas inhibition of endogenous HGF enhances renal fibrosis (1).
In the present study, we have focused on the role of endogenous relaxin in this process. Therefore, studies in wild-type mice were undertaken to examine the regulation of relaxin-1 before and during fibrosis. Although the renal copy number was low, our findings suggest that the renal concentration of relaxin peptide is the product of both circulating peptide and local transcription. Furthermore, quantitative-PCR studies indicated that progression of disease was accompanied by a modest reduction in renal relaxin-1 mRNA expression, similar to that seen with HGF (5). Controversy surrounds the existence and significance of local relaxin synthesis. Previous work suggests that local organ synthesis may supplement the low circulating levels of relaxin found in male rodents (23, 24).
Obstructed kidneys from RLX–/– mice had more myofibroblasts at 3 but not 10 d postobstruction, although sc administration of exogenous relaxin was sufficient to abrogate changes in myofibroblast burden. Conversely, deficiency of relaxin had no effect on expression of MCP-1 or macrophage recruitment. Therefore, these findings suggest that relaxin directly inhibits myofibroblast-based fibrogenesis, and that this effect is not an indirect consequence of less inflammation. This is consistent with our in vitro work (14) where renal myofibroblasts were found to express the relaxin receptor LGR7 (9), whereas recombinant H2 relaxin decreased collagen synthesis and inhibited -SMA expression in myofibroblasts.
The effect of relaxin on myofibroblast recruitment was paralleled by similar changes in collagen accumulation. Notably, the early increase in both total collagen and collagen isotypes was more marked in the absence of relaxin; the magnitude of the increase in collagen was greater in RLX–/– animals at 3 d after UUO, but similar to RLX+/+ animals at d 10. Furthermore, the reintroduction of H2 relaxin into RLX–/– mice via osmotic mini-pumps ameliorated the increase in collagen. These findings substantiate the renoprotective importance of relaxin, even at the low levels found endogenously.
Control (d 0) relaxin-deficient animals had more MMP-2 expression, but equivalent MMP-2 activity to that measured in their wild-type counterparts, a previously unrecognized part of their renal phenotype. Despite MMP-2 activity increasing 10-fold by d 10 after UUO, there was no difference between RLX+/+ and RLX–/– animals, consistent with the equivalent increase in collagen concentration at this time point. Therefore, the pathological significance of the dramatic increase in MMP-2 is not immediately apparent, but is consistent with the multifactorial role of MMP-2 in the progression of disease (32). There were also some modest changes in MMP-2 activity in the unobstructed kidneys. Activity of the metalloproteinase MMP-9 was unchanged in obstructed kidneys, although there was a significant and unexpected increase in MMP-9 activity in the contralateral unobstructed kidney of both genotypes. The reason and significance of this is unclear, but presumably relates to the compensatory mechanisms after unilateral obstruction. Several studies have demonstrated that exogenous relaxin treatment promotes MMP expression to breakdown collagen associated with fibrosis (14, 33) and to facilitate tumor cell attachment, migration, and invasion (34, 35). However, our findings and that of others (11) suggest that the ability of relaxin to inhibit the progression of fibrosis in the kidney is not primarily mediated via a gelatinase-induced breakdown of ECM/collagen, but rather through inhibition of myofibroblast-induced collagen synthesis and secretion.
In conclusion, relaxin is emerging as an important endogenous renoprotective factor, with its most prominent effects seen during early phases of tubulointerstitial fibrosis. However, low endogenous levels of relaxin were ultimately not able to prevent fibrosis in a model as rapidly progressive as this. Our study highlights the importance of the anti-fibrotic autocoids in homeostasis and the regulation of fibrogenesis. A better understanding of these factors is an important part of understanding the pathogenesis of chronic kidney disease.
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Acknowledgments |
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Footnotes |
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A portion of this work was presented in the conference proceedings of the 4th International Conference on Relaxin and Related Peptides (September 2004), Jackson Hole, WY; and in abstract form at the Annual Meeting of the American Society of Nephrology (November 2005), Philadelphia, PA, and the Annual Meeting of The Endocrine Society (June 2006), Boston, MA.
All authors have nothing to declare.
First Published Online November 9, 2006
1 T.D.H. and I.M. contributed equally to this work. 
Abbreviations: BMP-7, Bone morphogenic protein 7; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HGF, hepatocyte growth factor; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; -SMA, -smooth muscle actin; UUO, unilateral ureteric obstruction.
Received June 16, 2006.
Accepted for publication November 2, 2006.
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B pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol 15:1514–1529This article has been cited by other articles:
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  Y. Moodley, D. Atienza, U. Manuelpillai, C. S. Samuel, J. Tchongue, S. Ilancheran, R. Boyd, and A. Trounson Human Umbilical Cord Mesenchymal Stem Cells Reduce Fibrosis of Bleomycin-Induced Lung Injury Am. J. Pathol., July 1, 2009; 175(1): 303 - 313. [Abstract] [Full Text] [PDF]
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 T. D. Hewitson Renal tubulointerstitial fibrosis: common but never simple Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1239 - F1244. [Abstract] [Full Text] [PDF]
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 Q. Xu, E. D. Lekgabe, X.-M. Gao, Z. Ming, G. W. Tregear, A. M. Dart, R. A. D. Bathgate, C. S. Samuel, and X.-J. Du Endogenous Relaxin Does Not Affect Chronic Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis Endocrinology, February 1, 2008; 149(2): 476 - 482. [Abstract] [Full Text] [PDF]
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 P. Boor, K. Sebekova, T. Ostendorf, and J. Floege Treatment targets in renal fibrosis Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3391 - 3407. [Full Text] [PDF]
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