Relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to up-regulate matrix metalloproteinases: the additional involvement of iNOS.

The hormone, relaxin, inhibits aberrant myofibroblast differentiation and collagen deposition by disrupting the TGF-ß1/Smad2 axis, via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), extracellular signal-regulated kinase (ERK)1/2 phosphorylation (pERK) and a neuronal nitric oxide (NO) synthase (nNOS)-NO-cyclic guanosine monophosphate (cGMP)-dependent pathway. However, the signalling pathways involved in its additional ability to increase matrix metalloproteinase (MMP) expression and activity remain unknown. This study investigated the extent to which the NO pathway was involved in human gene-2 (H2) relaxin's ability to positively regulate MMP-1 and its rodent orthologue, MMP-13, MMP-2 and MMP-9 (the main collagen-degrading MMPs) in TGF-ß1-stimulated human dermal fibroblasts and primary renal myofibroblasts isolated from injured rats; by gelatin zymography (media) and Western blotting (cell layer). H2 relaxin (10-100 ng/ml) significantly increased MMP-1 (by ~50%), MMP-2 (by ~80%) and MMP-9 (by ~80%) in TGF-ß1-stimulated human dermal fibroblasts; and MMP-13 (by ~90%), MMP-2 (by ~130%) and MMP-9 (by ~115%) in rat renal myofibroblasts (all p<0.01 vs untreated cells) over 72 hours. The relaxin-induced up-regulation of these MMPs, however, was significantly blocked by a non-selective NOS inhibitor (L-nitroarginine methyl ester (hydrochloride); L-NAME; 75-100 µM), and specific inhibitors to nNOS (N-propyl-L-arginine; NPLA; 0.2-2 µM), iNOS (1400W; 0.5-1 µM) and guanylyl cyclase (ODQ; 5 µM) (all p<0.05 vs H2 relaxin alone), but not eNOS (L-N-(1-iminoethyl)ornithine dihydrochloride; L-NIO; 0.5-5 µM). However, neither of these inhibitors affected basal MMP expression at the concentrations used. Furthermore, of the NOS isoforms expressed in renal myofibroblasts (nNOS and iNOS), H2 relaxin only stimulated nNOS expression, which in turn, was blocked by the ERK1/2 inhibitor (PD98059; 1 µM). These findings demonstrated that H2 relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to mediate its anti-fibrotic actions, and additionally signals through iNOS to up-regulate MMPs; the latter being suppressed by TGF-ß1 in myofibroblasts, but released upon H2 relaxin-induced inhibition of the TGF-ß1/Smad2 axis.

Relaxin and castration in male mice protect from, but testosterone exacerbates, age-related cardiac and renal fibrosis, whereas estrogens are an independent determinant of organ size.

This study determined the effects of castration and hormone replacement therapy on the age-related cardiac and renal pathology of male relaxin gene-knockout (RlnKO) and age-matched wild-type (RlnWT) mice and that of aged male aromatase knockout (ArKO) mice, which lack estrogens and have 5-10 times the androgen levels of male wild-type mice. One-month-old RlnWT and RlnKO mice were bilaterally gonadectomized or sham operated and maintained until 12 months. Subgroups of castrated animals received testosterone or 17ß-estradiol treatment from 9 to 12 months. Male ArKO mice and aromatase wild-type mice were aged to 12 months. Collected heart and kidney tissues were assessed for changes in organ size and fibrosis. Castration reduced body, heart, left ventricle, and kidney weights in both RlnKO and RlnWT mice, and the cardiac/renal fibrosis that was seen in sham RlnKO animals (all P < 0.05 vs. respective sham). Testosterone normalized organ weights and organ weight to body weight ratio of castrated animals and increased cardiac/renal collagen concentration to levels measured in or beyond that of sham RlnKO mice (all P < 0.05 vs. respective castrated mice). Furthermore, expression of TGF-ß1, mothers against decapentaplegic homolog 2 (Smad2), and myofibroblast differentiation paralleled the above changes (all P < 0.05 vs. respective castrated mice), whereas matrix metalloproteinase-13 was decreased in testosterone-treated RlnKO mice. Conversely, 17ß-estradiol only restored changes in organ size. Consistent with these findings, intact ArKO mice demonstrated increased cardiac/renal fibrosis in the absence of changes in organ size. These findings suggest that relaxin and castration protect, whereas androgens exacerbate, cardiac and renal fibrosis during ageing, whereas estrogens, in synergy with relaxin, regulates age-related changes in organ size.

Relaxin remodels fibrotic healing following myocardial infarction.

In the setting of myocardial infarction (MI), implanted stem cell viability is low and scar formation limits stem cell homing, viability, and integration. Thus, interventions that favorably remodel fibrotic healing may benefit stem cell therapies. However, it remains unclear whether it is feasible and safe to remodel fibrotic healing post-MI without compromising ventricular remodeling and dysfunction. This study, therefore, determined the anti-fibrotic and other effects of the hormone, relaxin in a mouse model of MI. Adult male mice underwent left coronary artery ligation-induced MI and were immediately treated with recombinant human relaxin (MI+RLX) or vehicle (MI+VEH) over 7 or 30 days, representing time points of early and mature fibrotic healing. Cardiac function was assessed by echocardiography and catheterization, while comprehensive immunohistochemistry, morphometry, and western blotting were performed to explore the relaxin-induced mechanisms of action post-MI. RLX significantly inhibited the MI-induced progression of cardiac fibrosis over 7 and 30 days, which was associated with a reduction in TGF-ß1 expression, myofibroblast differentiation, and cardiomyocyte apoptosis in addition to a promotion of matrix metalloproteinase-13 levels and de novo blood vessel growth (all P<0.05 vs respective measurements from MI+VEH mice). Despite the evident fibrotic healing post-MI, relaxin did not adversely affect the incidence of ventricular free-wall rupture or the extent of LV remodeling and dysfunction. These combined findings demonstrate that RLX favorably remodels the process of fibrotic healing post-infarction by lowering the density of mature scar tissue in the infarcted myocardium, border zone, and non-infarcted myocardium, and may, therefore, facilitate cell-based therapies in the setting of ischemic heart disease.

H3 relaxin demonstrates antifibrotic properties via the RXFP1 receptor.

Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor ß1 (TGF-ß1) administration to ventricular fibroblasts significantly increased Smad2 phosphorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metalloproteinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-ß1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-ß1-stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-ß1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-ß1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin.

Relaxin plays an important role in the regulation of airway structure and function.

Relaxin is a reproductive hormone with pleiotropic actions. In addition to airway fibrosis, relaxin deficiency results in airway structural changes (epithelial thickening) and increased lung recoil, suggesting that relaxin may impact other aspects of airway/lung structure and function beyond its ability to regulate collagen turnover. Furthermore, these structural changes associated with relaxin deficiency show marked similarity to the structural changes seen in asthma. The current study investigated the broader role of relaxin in regulating airway structure and function and examined the relationship between airway inflammation, structural changes, and airway hyperresponsiveness (AHR) using an ovalbumin (OVA)-induced model of allergic airways disease (AAD). The model of AAD was applied to 12-month-old relaxin-deficient (Rln(-/-)) mice with established airway fibrosis and age-matched wild-type (Rln(+/+)) controls. OVA-treated Rln(+/+) mice (induced inflammation) developed increased epithelial thickening (P < 0.05) and AHR (P < 0.05) but not airway fibrosis, compared with saline-treated Rln(+/+) controls. Saline-treated Rln(-/-) mice had significantly increased lung collagen deposition (existing fibrosis) and epithelial thickening and remarkably were found to have increased AHR that was equivalent to that in OVA-treated Rln(+/+) mice (all P < 0.05 vs. saline-treated Rln(+/+) controls). OVA-treated Rln(-/-) mice (existing fibrosis and induced inflammation) had increased airway/lung fibrosis (P < 0.05) but equivalent airway inflammation and AHR compared with OVA-treated Rln(+/+) animals. These findings demonstrate for the first time a role for relaxin in the regulation of airway responses using Rln(-/-) mice and suggest that airway fibrosis and/or epithelial thickening can result in increased AHR equivalent to that induced by airway inflammation in AAD.

Improved chemical synthesis and demonstration of the relaxin receptor binding affinity and biological activity of mouse relaxin.

The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.

Endogenous relaxin is a naturally occurring modulator of experimental renal tubulointerstitial fibrosis.

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 (RLX(-/-)) and age-matched wild-type (RLX(+/+)) 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 alpha-smooth muscle actin (alpha-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 alpha-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 alpha-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.

Atrial electrical and structural abnormalities in an ovine model of chronic blood pressure elevation after prenatal corticosteroid exposure: implications for development of atrial fibrillation.

AIMS: Elevated blood pressure (EBP) is the most prevalent and potentially modifiable risk factor for AF, yet little is known of its atrial effects. We aimed to characterize the atrial electrical and structural changes in a chronic ovine model of EBP after prenatal corticosteroid exposure. METHODS AND RESULTS: Twelve sheep with chronically EBP (mean arterial pressure 94+/-3 mmHg) and six controls (71+/-4 mmHg, P<0.01) underwent acute open chest electrophysiologic and pathologic studies. We measured refractoriness at the atrial appendages at 3 cycle lengths (CL); conduction velocities at Bachmann's bundle, both atrial appendages and free walls at 4 CLs; conduction heterogeneity; atrial wavelength and AF duration. We performed light microscopy (LM) and electron microscopy (EM) and collagen and apoptosis studies. EBP was associated with widespread conduction abnormalities, shortening of atrial wavelength, and increased AF. There was no significant change in refractoriness. LM demonstrated atrial myocyte hypertrophy and myolysis in all EBP sheep and focal scarring in six. EM demonstrated mitochondrial and nuclear enlargement and increased collagen fibrils in EBP sheep, findings not present in any controls. Atrial collagen and apoptosis were increased in EBP animals. CONCLUSION: This study demonstrates that chronically, EBP is associated with significant atrial electrical and structural remodelling. These changes may explain the increased propensity to atrial arrhythmias observed with long-standing EBP.

The effects of relaxin and estrogen deficiency on collagen deposition and hypertrophy of nonreproductive organs.

In this study, we determined the effects of relaxin and estrogen deficiency and estrogen replacement therapy (ERT) on the cardiac, renal, and pulmonary phenotypes of female relaxin gene knockout (Rln1-/-) and age-matched wild-type (Rln1+/+) mice. One-month-old Rln1+/+ and Rln1-/- mice were bilaterally ovariectomized or sham-operated and aged until 9 or 12 months. A subgroup of ovariectomized mice received ERT from 9 to 12 months of age. At the appropriate time points, heart, kidney, and lung tissues from these mice were collected and analyzed for changes in organ fibrosis, hypertrophy, and airway thickening. Neither ovariectomy nor ERT had any effect on cardiac or renal collagen concentration in all groups studied. In contrast, total lung collagen concentration and airway subepithelial collagen deposition were significantly increased in ovariectomized Rln1+/+ mice (P<0.05 vs. sham) and to a greater extent in ovariectomized Rln1-/- mice (P<0.01 vs. sham). Ovariectomy of Rln1+/+ mice also led to a significant increase in airway smooth muscle (SM) (lung) thickening, which was further exaggerated in Rln1-/- mice. Cardiac hypertrophy, evidenced by increased heart weight and expression of hypertrophy-related genes (all P<0.05 vs. sham) was only observed in Rln1-/- mice. These findings demonstrated an increased pathology in mice that were deficient of both relaxin and estrogen. ERT significantly decreased airway fibrosis, airway SM thickening, and cardiac hypertrophy when administered to ovariectomized Rln1-/- mice (all P<0.05 vs. ovariectomy alone). These findings suggest that relaxin and estrogen appear to play protective roles against airway fibrosis, airway SM thickening, and cardiac hypertrophy in female mice.

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo.

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.

The relaxin gene knockout mouse: a model of progressive scleroderma.

Relaxin is a peptide hormone with anti-fibrotic properties. To investigate the long-term effects of relaxin deficiency on the ageing skin, we compared structural changes in the skin of ageing relaxin-deficient (RLX-/-) and normal (RLX+/+) mice, by biochemical, histological, and magnetic resonance imaging analyses. Skin biopsies from RLX+/+ and RLX-/- mice were obtained at different ages and analyzed for changes in collagen expression and distribution. We demonstrated an age-related progression of dermal fibrosis and thickening in male and female RLX-/- mice, associated with marked increases in types I and III collagen. The increased collagen was observed primarily in the dermis of RLX-/- mice by 1 mo of age, and eventually superseded the hypodermal layer. Additionally, fibroblasts from the dermis of RLX-/- mice were shown to produce increased collagen in vitro. Recombinant human gene-2 (H2) relaxin treatment of RLX-/- mice resulted in the complete reversal of dermal fibrosis, when applied to the early onset of disease, but was ineffective when applied to more established stages of dermal scarring. These combined findings demonstrate that relaxin provides a means to regulate excessive collagen deposition in disease states characterized by dermal fibrosis and with our previously published work demonstrate the relaxin-null mouse as a model of progressive scleroderma.

Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats.

The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and alpha-smooth muscle actin (alpha-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25+/-1%, P<0.01 using biochemical analysis and 3-fold; P<0.01 using quantitative histology), enhanced expression of PCNA (by 70+/-8%; P<0.01), alpha-SMA (by 32+/-2%; P<0.05), and the collagen-degrading enzyme MMP-9 (by 70+/-6%; P<0.05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25+/-2%, P<0.05 using biochemical analysis and 2.4-fold; P<0.01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV (P<0.01) and kidney (P<0.05), completely inhibited cell proliferation (P<0.01) and fibroblast differentiation (P<0.05) in the LV, and increased MMP-2 expression (by 25+/-1%; P<0.05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.

The relaxin gene-knockout mouse: a model of progressive fibrosis.

Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P < .05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.

Detection, localization, and action of the INSL3 receptor, LGR8, in rat kidney.

Recent molecular and pharmacologic studies have identified LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family, as a cognate receptor for insulin-like peptide-3 (INSL3). LGR8 mRNA has been detected in various tissues, but the precise roles of these INSL3-LGR8 systems are unknown. In this study we first investigated the presence and cellular localization of LGR8 mRNA in both adult and developing rat kidney and subsequently examined the possible role of INSL3-LGR8 signaling in cultured mesangial cells. LGR8 mRNA was detected in the kidney by polymerase chain reaction and localized by in situ hybridization in mature glomerular mesangial cells within the renal cortex, with highest levels detected at embryonic day 18 and lowest levels in adult kidney. Synthetic INSL3 inhibited the proliferation of mesangial cells in primary culture, indicating the presence of functional LGR8 on these cells. These findings suggest that INSL3/LGR8 signaling may be involved in the genesis and/or developmental maturation of renal glomeruli and in the regulation of mesangial cell density in the adult kidney.

Relaxin-1-deficient mice develop an age-related progression of renal fibrosis.

BACKGROUND: Relaxin (RLX) is a peptide hormone that stimulates the breakdown of collagen in preparation for parturition and when administered to various models of induced fibrosis. However, its significance in the aging kidney is yet to be established. In this study, we compared structural and functional changes in the kidney of aging relaxin-1 (RLX-/-) deficient mice and normal (RLX+/+) mice. METHODS: The kidney cortex and medulla of male and female RLX+/+ and RLX-/- mice at various ages were analyzed for collagen content, concentration, and types. Histologic analysis, reverse transcription-polymerase chain reaction (RT-PCR) of relaxin and relaxin receptor mRNA expression, receptor autoradiography, glomerular isolation/analysis, and serum/urine analysis were also employed. Relaxin treatment of RLX-/- mice was used to confirm the antifibrotic effects of the peptide. RESULTS: We demonstrate an age-related progression of renal fibrosis in male, but not female, RLX-/- mice with significantly (P < 0.05) increased tissue dry weight, collagen (type I) content and concentration. The increased collagen expression in the kidney was associated with increased glomerular matrix and to a lesser extent, interstitial fibrosis in RLX-/- mice, which also had significantly increased serum creatinine (P < 0.05) and urinary protein (P < 0.05). Treatment of RLX-/- mice with relaxin in established stages of renal fibrosis resulted in the reversal of collagen deposition. CONCLUSION: This study supports the concept that relaxin may provide a means to regulate excessive collagen deposition during kidney development and in diseased states characterized by renal fibrosis.

Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis.

Relaxin (RLX) is a peptide hormone with known antifibrotic properties. However, its significance in the lung and its role as a therapeutic agent against diseases characterized by pulmonary fibrosis are yet to be established. In this study, we examined age-related structural and functional changes in the lung of relaxin-deficient mice. Lung tissues of male and female RLX knockout (-/-) and RLX wild-type (+/+) mice at various ages were analyzed for changes in collagen expression and content. We demonstrate an age-related progression of lung fibrosis in RLX -/- mice with significantly increased tissue wet weight, collagen content and concentration, alveolar congestion, and bronchiole epithelium thickening. The increased fibrosis was associated with significantly altered peak expiratory flow and lung recoil (lung function) in RLX -/- mice. Treatment of RLX -/- mice with relaxin in early and developed stages of fibrosis resulted in the reversal of collagen deposition. Organ bath studies showed that precontracted lung strips relaxed in the presence of relaxin. Together, these data indicate that relaxin may provide a means to regulate excessive collagen deposition in diseased states characterized by pulmonary fibrosis.

Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family.

We have identified a novel human relaxin gene, designated H3 relaxin, and an equivalent relaxin gene in the mouse from the Celera Genomics data base. Both genes encode a putative prohormone sequence incorporating the classic two-chain, three cysteine-bonded structure of the relaxin/insulin family and, importantly, contain the RXXXRXX(I/V) motif in the B-chain that is essential for relaxin receptor binding. A peptide derived from the likely proteolytic processing of the H3 relaxin prohormone sequence was synthesized and found to possess relaxin activity in bioassays utilizing the human monocytic cell line, THP-1, that expresses the relaxin receptor. The expression of this novel relaxin gene was studied in mouse tissues using RT-PCR, where transcripts were identified with a pattern of expression distinct from that of the previously characterized mouse relaxin. The highest levels of expression were found in the brain, whereas significant expression was also observed in the spleen, thymus, lung, and ovary. Northern blotting demonstrated an approximately 1.2-kb transcript present in mouse brain poly(A) RNA but not in other tissues. These data, together with the localization of transcripts in the pars ventromedialis of the dorsal tegmental nucleus of C57BLK6J mouse brain by in situ hybridization histochemistry, suggest a new role for relaxin in neuropeptide signaling processes. Together, these studies describe a third member of the human relaxin family and its equivalent in the mouse.