Endogenous Relaxin Is a Naturally Occurring Modulator of ...

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Endocrinology 148(2):660 – 669 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0814

Endogenous Relaxin Is a Naturally Occurring Modulator of Experimental Renal Tubulointerstitial Fibrosis Tim D. Hewitson,* Ishanee Mookerjee,* Rosemary Masterson, Chongxin Zhao, Geoffrey W. Tregear, Gavin J. Becker, and Chrishan S. Samuel 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 Relaxin is a naturally occurring regulator of collagen turnover. In this study, we determined the role of endogenous relaxin in the pathogenesis of primary tubulointerstitial fibrosis after unilateral ureteric obstruction (UUO). Four- to 6-wk-old relaxin (RLX) gene-knockout (RLXⴚ/ⴚ) and agematched wild-type (RLXⴙ/ⴙ) mice, with equivalent baseline collagen levels, were subjected to UUO. Obstructed and contralateral kidneys were collected at d 0, 3, and 10 after surgery and analyzed for changes in inflammatory and fibrosis-related markers. UUO was associated with a progressive increase in fibrosis in all obstructed, but not contralateral kidneys. The increase in total collagen (hydroxyproline analysis) was associated with more ␣-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 RLXⴙ/ⴙ animals. Total collagen and ␣-SMA expression were significantly greater in the obstructed kidneys of RLXⴚ/ⴚ mice 3 d after UUO (both P < 0.05 vs. RLXⴙ/ⴙ D3 after UUO), but comparable to that in RLXⴙ/ⴙ animals 10 d after UUO. Administration of recombinant H2 relaxin to RLXⴚ/ⴚ mice 4 d before UUO ameliorated the increase in collagen and ␣-SMA expression (both P < 0.05 vs. untreated RLXⴚ/ⴚ 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. (Endocrinology 148: 660 – 669, 2007)

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brotic TGF-␤1 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 (11–13) 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

LL PROGRESSIVE KIDNEY diseases share the process of tubulointerstitial fibrosis as a component of their pathology (1, 2). Injury to the tubules and interstitium may be either a primary insult or secondary to glomerular injury and results in the sequential but overlapping events of infiltration of inflammatory cells (neutrophils, macrophages, T cells) and generation of profibrotic cytokines. This inevitably leads to recruitment of renal myofibroblasts, activated fibroblastic cells characterized by their expression of the protein ␣-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 profiFirst Published Online November 9, 2006 * 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.

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Hewitson et al. • Relaxin Inhibits Tubulointerstitial Fibrosis

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 antifibrotic 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. Materials and Methods Relaxin Recombinant human gene-2 (H2) relaxin was generously provided by BAS Medical Inc. (San Mateo, CA) and is bioactive in mice (15, 16, 18, 21).

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), SDSPAGE (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䡠d H2 relaxin in vehicle (n ⫽

Endocrinology, February 2007, 148(2):660 – 669

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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 ␮g) 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 (6carboxyfluorescein) 5⬘-CAAAGATGCAGAGCCTTTCGATACGACG3⬘; 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

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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 (BioRad). 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 NewmanKeuls 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.

Results Endogenous relaxin-1 mRNA expression in progressive tubulointerstitial disease

In the absence of a suitable antibody to mouse relaxin, we used quantitative RT-PCR to identify any temporal changes in local relaxin expression during progressive fibrosis. Control RLX⫹/⫹ mice had low levels of renal relaxin mRNA (relaxin to 18s ratio: 3.8 ⫻ 10⫺4; Fig. 1A), 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). Quantitative PCR was also used to determine whether the local expression of renal relaxin-3 was able to compensate for the loss of relaxin. Control RLX⫹/⫹ mouse kidneys had similar levels of renal relaxin-3 mRNA (relaxin-3 to 18s ratio: 5.9 ⫻ 10⫺4; Fig. 1B) 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

FIG. 1. Real-time PCR analysis of relaxin (A) and relaxin-3 (B) mRNA expression in the obstructed (OB) or unobstructed (UO) kidneys of RLX⫹/⫹ and RLX⫺/⫺ mice at d 0 (control), 3, and 10 after UUO. The mRNA levels of relaxin and relaxin-3 are expressed as a ratio of gene to 18s values from each sample, respectively. Results are expressed as the mean ⫾ SEM; numbers in parentheses represent number of animals used per group. a, P ⬍ 0.05 vs. d 0 RLX⫹/⫹ values; b, P ⬍ 0.05 vs. values from the UO kidneys of RLX⫹/⫹ mice 10 d after UUO; c , P ⬍ 0.05 vs. d 0 RLX⫺/⫺ values; d, P ⬍ 0.05 vs. values from the UO kidneys of RLX⫺/⫺ mice 10 d after UUO.

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. The effects of relaxin deficiency on renal myofibroblast accumulation after UUO

Equivalent levels of interstitial ␣-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



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FIG. 2. Effects of relaxin deficiency on inflammation. A, RT-PCR of MCP-1 mRNA (corrected for GAPDH) was performed on cDNA samples from RLX⫹/⫹ and RLX⫺/⫺ mouse kidney tissues, obtained at d 0 (control), 3, and 10 after UUO. The mean ⫾ SE ratio of MCP-1/GAPDH was then graphed based on densitometry readings of the corresponding PCR products; numbers in parentheses represent number of animals used per group. B, Immunostaining of macrophage infiltration in the kidneys of RLX⫹/⫹ and RLX⫺/⫺ mice. Shown is a representative figure of macrophage infiltration (brown staining) within the cortical interstitium of RLX⫹/⫹ and RLX⫺/⫺ mice at d 0 and in the obstructed (OB) kidneys of these mice at d 3 and 10 after UUO. Quantitative analysis of macrophage infiltration staining was expressed as the mean ⫾ SEM f4/80 staining expressed as percentage total area (at each time point); numbers in parentheses represent number of animals used per group. a, P ⬍ 0.01 and b , P ⬍ 0.001 vs. values from control kidneys. UO, Unobstructed.

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. As an additional control, we reconstituted relaxin-deficient mice by administering recombinant H2 relaxin by osmotic mini-pump. Again we examined the recruitment of

interstitial myofibroblasts 3 d after UUO, at a time when the absence of endogenous relaxin led to a greater burden of myofibroblasts. Immunohistochemical staining for ␣-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 myo-

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FIG. 3. Effects of relaxin deficiency on myofibroblast accumulation. Shown is a representative figure of ␣-SMA expression (brown staining) within the cortical interstitium of RLX⫹/⫹ and RLX⫺/⫺ mice at d 0 and in the obstructed (OB) kidneys of these mice at d 3 and 10 after UUO. A, Quantitative analysis of ␣-SMA staining in the OB and unobstructed (UO) kidneys of d 0 (control), 3, and 10 after UUO RLX⫹/⫹ and RLX⫺/⫺ mice was expressed as a mean ⫾ SE percentage of the total area (n ⫽ 4 – 6 animals per group and time point). a , P ⬍ 0.05; b, P ⬍ 0.01 vs. d 0 values; c, P ⬍ 0.01 vs. respective UO kidney values; d, P ⬍ 0.05 vs. OB kidney values of 3-d after UUO RLX⫹/⫹ mice; e, P ⬍ 0.05 vs. OB kidney values of 3-d after UUO RLX⫺/⫺ mice. B, Quantitative immunostaining of ␣-SMA was determined in the unobstructed (UO) and obstructed (OB) kidneys of RLX⫹/⫹, untreated/vehicle-treated RLX⫺/⫺, and H2 relaxintreated RLX⫺/⫺ mice (n ⫽ 5–7 per group). a, P ⬍ 0.05 vs. UO kidney values of RLX⫹/⫹ mice 3 d after UUO; b, P ⬍ 0.05 vs. UO kidney values of RLX⫺/⫺ mice 3 d after UUO; c, P ⬍ 0.05 vs. OB kidney values of RLX⫹/⫹ mice 3 d after UUO; d, P ⬍ 0.05 vs. OB kidney values of untreated/vehicle-treated RLX⫺/⫺ mice 3 d after UUO.

fibroblast 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. Once again, as an additional control, we examined the net accumulation of collagen in relaxin-deficient mice reconstituted with



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FIG. 4. Effects of relaxin deficiency on collagen concentration. A, Collagen concentration (collagen content expressed as percentage of the dry weight tissue) in the obstructed (OB) and unobstructed (UO) kidney of RLX⫹/⫹ and RLX⫺/⫺ mice, after UUO was derived from the hydroxyproline data (n ⫽ 5–7 animals per group). a, P ⬍ 0.05; b, P ⬍ 0.01 vs. d 0 values; c, P ⬍ 0.01 vs. respective UO kidney values; d, P ⬍ 0.05; e, P ⬍ 0.01 vs. OB kidney values of 3-d after UUO RLX⫹/⫹ mice; f , P ⬍ 0.01 vs. OB kidney values of 3-d after UUO RLX⫺/⫺ mice. B, Collagen concentration was also determined in the unobstructed (UO) and obstructed (OB) kidneys of RLX⫹/⫹, untreated/vehicle-treated RLX⫺/⫺, and H2 relaxin-treated RLX⫺/⫺ mice (n ⫽ 5–11 per group). a, P ⬍ 0.05 vs. UO kidney values of RLX⫹/⫹ mice 3 d after UUO; b , P ⬍ 0.05 vs. UO kidney values of RLX⫺/⫺ mice 3 d after UUO; c, P ⬍ 0.05 vs. OB kidney values of RLX⫹/⫹ mice 3 d after UUO; d, P ⬍ 0.05 vs. OB kidney values of untreated/vehicle-treated RLX⫺/⫺ mice 3 d after UUO.

H2 relaxin. H2 relaxin treatment of RLX⫺/⫺ mice caused a significant decrease in collagen concentration (P ⬍ 0.05; Fig. 4B) 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.

Consistent with this, little collagen III immunostaining was seen in obstructed kidneys from RLX⫺/⫺ and RLX⫹/⫹ mice at d 0 and 3 d after UUO (Fig. 5B). 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 MMP9 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. Densitometric analysis of the combined latent and active forms of MMP-9 (Fig. 6B) 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

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FIG. 5. Effects of relaxin deficiency on interstitial collagen types. A, SDSPAGE analysis of pepsin-digested interstitial collagen shows the maturely cross-linked insoluble interstitial collagen types. Type I collagen monomers are represented by the ␣1(I) and ␣2(I) chains; type III collagen monomers are represented by the ␣1(III) chains, and type V collagen monomers are represented by the ␣1(V) and ␣2(V) chains. Type I collagen dimers are represented by ␤11 [dimers of two ␣1(I) subunits] and ␤12 [dimers of ␣1(I) and ␣2(I) monomers]. Densitometric analysis of type I collagen was also performed and expressed as the mean ⫾ SE. OD collagen I (per genotype and time point). a , P ⬍ 0.05 vs. d 0 values; b, P ⬍ 0.05 vs. d 3 OB kidney values of RLX⫹/⫹ mice; c , P ⬍ 0.001 vs. d 0 and 3 values. B, Immunostaining of collagen III (brown staining) is also shown in the obstructed kidneys of RLX⫹/⫹ and RLX⫺/⫺ mice at d 0, 3, and 10 after UUO. Point counting techniques were used to determine the mean ⫾ SEM type III collagen in each group, which was expressed as a percentage of the fractional area of renal cortex; numbers in parentheses represent number of animals used per group. c , P ⬍ 0.001 vs. d 0 and 3 values.

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



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FIG. 6. Expression and activity of MMP-2 and MMP-9. A, A representative gelatin zymography of MMP-2 and MMP-9 shows the glycosylated, latent, and active forms of each enzyme in the obstructed and unobstructed kidneys of RLX⫹/⫹ and RLX⫺/⫺ mice, at d 0, 3, and 10 after UUO. Additional zymographs (of n ⫽ 4 – 6 samples per group in total) gave similar results. The combined latent and active forms of MMP-9 (B) and MMP-2 (C) were quantitated by densitometry scanning (in arbitrary units) and presented as the relative mean ⫾ ⫹/⫹ SEM of values in the d-0 RLX (control) mouse group, which was expressed as 1.0. To correct for interassay variation (in density measurements), the d-0 (control) samples from RLX⫹/⫹ and RLX⫺/⫺ mice were loaded on each gel analyzed. Numbers in parentheses represent number of animals used per group. a, P ⬍ 0.05 and b, P ⬍ 0.01 vs. d 0 RLX⫹/⫹ values; c, P ⬍ 0.05 vs. obstructed (OB) kidney values of 3-d postUUO RLX⫹/⫹ mice; d, P ⬍ 0.05 vs. d-0 values; e, P ⬍ 0.05 vs. unobstructed (UO) kidney values of 3-d post-UUO RLX⫹/⫹ values; f, P ⬍ 0.01 vs. OB kidney values of 3-d post-UUO mice.

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

This study has provided novel insights into the role of endogenous relaxin in the pathogenesis of early and chronic tubulointerstitial fibrosis after injury. Accumulation of both interstitial myofibroblasts and collagen was greater in relaxin-deficient animals at 3 d after UUO compared with that detected in wild-type animals, with no difference between groups at 10 d after UUO. This was paralleled by a significant decrease in relaxin mRNA expression in normal mice 10 d after UUO. Because no changes in inflammation were detected between relaxin-null and wild-type mice 3 d after UUO, our findings are consistent with relaxin directly inhibiting myofibroblast-induced collagen production, rather than indirectly via effects on inflammation. Although this study was not able to differentiate the contributions of locally produced vs. circulating relaxin to chronic renal disease in wild-type mice (due to the lack of a suitable antibody to

mouse relaxin), our combined data suggests that endogenous relaxin plays an important protective role in the kidney, particularly in early phases of tubulointerstitial fibrosis. Furthermore, even though a modest increase in relaxin-3 mRNA was detected in the 10-d obstructed kidneys of mice, this was not sufficient to inhibit the progression of tubulointerstitial fibrosis, consistent with our previous findings in other organs associated with fibrosis (16). UUO is a widely used experimental model of primary tubulointerstitial disease. Permanent obstruction leads to an inevitable accumulation of ECM in the interstitium. In wildtype 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 sig-

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nificance 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 TGF␤ superfamily that antagonizes TGF␤1 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 TGF␤1 (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 wildtype 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, quantitativePCR 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. Acknowledgments We sincerely thank Mr. Daniel Scott (Howard Florey Institute) and Dr. Laura Parry (Department of Zoology, University of Melbourne) for relaxin real-time PCR primer and probe design. Received June 16, 2006. Accepted November 2, 2006. 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: [email protected]. This study was supported by funding from BAS Medical Inc. I.M. is the recipient of a Howard Florey Institute Postgraduate Scholarship, R.M. is the recipient of a National Health and Medical Research Council Postgraduate Biomedical Scholarship, while G.W.T. is a National Health and Medical Research Council Senior Principle Research Fellow. 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.

References 1. Eddy AA 2005 Progression in chronic kidney disease Adv Chronic Kidney Dis 12:353–365


2. Becker GJ, Hewitson TD 2000 The role of tubulointerstitial injury in chronic renal failure. Curr Opin Nephrol Hypertens 9:133–138 3. Hewitson TD, Becker GJ 1995 Interstitial myofibroblasts in IgA glomerulonephritis. Am J Nephrol 15:111–117 4. Liu Y 2006 Renal fibrosis: New insights into the pathogenesis and therapeutics. Kidney Int 69:213–217 5. Liu Y 2004 Hepatocyte growth factor in kidney fibrosis: therapeutic potential and mechanisms of action. Am J Physiol Renal Physiol 287:F7–F16 6. Zeisberg M, Muller GA, Kalluri R 2004 Are there endogenous molecules that protect kidneys from injury? The case for bone morphogenic protein-7 (BMP7). Nephrol Dial Transplant 19:759 –761 7. Samuel CS, Hewitson TD 2006 Minireview: relaxin in cardiovascular and renal disease. Kidney Int 69:1498 –1502 8. Sherwood OD 2004 Relaxin’s physiological roles and other diverse actions. Endocr Rev 25:205–234 9. Ivell R, Einspanier A 2002 Relaxin peptides are new global players. Trends Endocrinol Metab 31:343–348 10. Samuel CS 2005 Relaxin: antifibrotic properties and effects in models of disease. Clin Med Res 3:241–249 11. Garber SL, Mirochnik Y, Brecklin CS, Unemori EN, Singh AK, Slobodskoy L, Grove BH, Arruda JA, Dunea G 2001 Relaxin decreases renal interstitial fibrosis and slows progression of renal disease. Kidney Int 59:876 – 882 12. Garber SL, Mirochnik Y, Brecklin C, Slobodskoy L, Arruda JA, Dunea G 2003 Effect of relaxin in two models of renal mass reduction. Am J Nephrol 23:8 –12 13. McDonald GA, Sarkar P, Rennke H, Unemori E, Kalluri R, Sukhatme VP 2003 Relaxin increases ubiquitin-dependent degradation of fibronectin in vitro and ameliorates renal fibrosis in vivo. Am J Physiol Renal Physiol 285:F59 –F67 14. Masterson R, Hewitson TD, Kelynack KJ, Martic M, Parry L, Bathgate R, Darby I, Becker G 2004 Relaxin downregulates renal fibroblast function and stimulates matrix remodelling in vitro. Nephrol Dial Transplant 19:544 –552 15. Samuel CS, Zhao C, Bond CP, Hewitson TD, Amento EP, Summers RJ 2004 Relaxin-1-deficient mice develop an age-related progression of renal fibrosis. Kidney Int 65:2054 –2064 16. Samuel CS, Zhao C, Bathgate RAD, Bond CP, Burton MD, Parry LJ, Summers RJ, Tang MLK, Amento EP, Tregear GW 2003 Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis. FASEB J 17:121–123 17. Du XJ, Samuel CS, Gao XM, Zhao L, Parry LJ, Tregear GW 2003 Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype. Cardiovasc Res 57:395– 404 18. Samuel CS, Zhao C, Yang Q, Wang H, Tian H, Tregear GW, Amento EP 2005 The relaxin gene knockout mouse: a model of progressive scleroderma. J Invest Dermatol 125:692– 699 19. Samuel CS, Tian H, Zhao L, Amento EP 2003 Relaxin is a key mediator of prostate growth and male reproductive tract development. Lab Investigation 83:1055–1067


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20. Diamond JR, Ricardo SD, Klahr S 1998 Mechanisms of interstitial fibrosis in obstructive nephropathy. Semin Nephrol 8:594 – 602 21. Ferraiolo BL, Cronin M, Bakhit C, Roth M, Chestnut M, Lyon R 1989 The pharmacokinetics and pharmacodynamics of a human relaxin in the mouse pubic symphysis bioassay. Endocrinology 125:2922–2926 22. Cochrane AL, Kett MM, Samuel CS, Campanale NV, Anderson WP, Hume DA, Little MH, Bertram JF, Ricardo SD 2005 Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 16:3623–3630 23. O’Byrne EM, Steinetz BG 1976 Radioimmunoassay (RIA) of relaxin in sera of various species using an antiserum to porcine relaxin. Proc Soc Exp Biol Med 152:272–276 24. Sherwood OD, Crnekovic VE 1979 Development of a homologous radioimmunoassay for rat relaxin. Endocrinology 104:893– 897 25. Siebel AL, Gehring HM, Nave CD, Bathgate RA, Borchers CE, Parry LJ 2002 The upregulation of mesotocin receptors in the tammar wallaby myometrium is pregnancy-specific and independent of estrogen. Biol Reprod 66:1237–1243 26. Esteban V, Lorenza O, Ruperez M, Suzuki Y, Mezzano S, Blanco J, Kretzler M, Sugaya T, Edigo J, Ruiz-Ortega M 2004 Angiotensin II, via AT1 and AT2 receptors and NK-␬B pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol 15:1514 –1529 27. Bergman I, Loxley R 1963 Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Anal Chem 35:1961– 1965 28. Gallop PM, Paz MA 1975 Posttranslational protein modifications, with special attention to collagen and elastin. Physiol Rev 55:418 – 487 29. Sykes B, Puddle B, Francis M, Smith R 1976 The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 72:1472–1480 30. Hewitson TD, Wu H, Becker GJ 1995 Interstitial myofibroblasts in experimental renal infection and scarring. Am J Nephrol 15:411– 417 31. Woessner JF Jr 1995 Quantification of matrix metalloproteinases in tissue samples. Methods Enzymol 248:510 –528 32. Cheng S, Lovett DH 2003 Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation. Am J Pathol 162:1937–1949 33. Lekgabe ED, Kiriazis H, Zhao C, Xu Q, Moore XL, Su Y, Bathgate RAD, Du XJ, Samuel CS 2005 Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats. Hypertension 46:412– 418 34. Silvertown JD, Summerlee AJ, Klonisch T 2003 Relaxin-like peptides in cancer. Int J Cancer 107:513–519 35. Kamat AA, Feng S, Agoulnik IU, Kheradmand F, Bogatcheva NV, Coffey D, Sood AK, Agoulnik AI 2006 The role of relaxin in endometrial cancer. Cancer Biol Ther 5:71–77

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