Diaz et al. BMC Urology (2020) 20:44 https://doi.org/10.1186/s12894-020-00607-4 RESEARCH ARTICLE Open Access Characterizing relaxin receptor expression and exploring relaxin’s effect on tissue remodeling/fibrosis in the human bladder Edward C. Diaz1,2*, Mason Briggs1,3, Yan Wen3, Guobing Zhuang3, Shannon L. Wallace3, Amy D. Dobberfuhl1, Chia-Sui Kao4 and Bertha C. Chen3 Abstract Background: Relaxin is an endogenous protein that has been shown to have antifibrotic properties in various organ systems. There has been no characterization of relaxin’s role in the human bladder. Our objective was to characterize relaxin receptor expression in the human bladder and assess relaxin’s effect on tissue remodeling/ fibrosis pathways in bladder smooth muscle cells. Methods: Relaxin family peptide receptor 1 (RXFP1) and RXFP2 expression was assessed using quantitative reverse transcriptase-PCR (qRT-PCR) and immunohistochemistry (IHC) on primary bladder tissue. Primary human smooth muscle bladder cells were cultured and stimulated with various concentrations of relaxin. Western blot, qRTPCR, ELISA, and zymogram assays were used to analyze fibrosis/tissue remodeling pathway proteins. Results: There was universal mRNA transcript detection and protein expression of relaxin receptors in primary bladder specimens. Immunohistochemistry demonstrated RXFP1 and RXFP2 localizing to both urothelial and smooth muscle cell layers of the bladder. 24 h of in vitro relaxin stimulation did not affect mRNA expression of selected proteins in human bladder smooth muscle cells. However, 48 h of in vitro relaxin stimulation resulted in upregulation of active (p = 0.004) and latent (p = 0.027) MMP-2 in cell lysate, and upregulation of active MMP-2 in supernatant (p = 0.04). There was a dose dependent relationship with increasing expression of MMP-2 with increasing relaxin concentration. Relaxin stimulation resulted in decreased levels of active and total TGF-β1in supernatant and extracellular matrix (p < 0.005 with 100 ng/mL relaxin stimulation). Conclusions: In the human bladder, relaxin receptors are expressed at the dome and trigone and localize to the urothelium and smooth muscle cell layers. Stimulation of human bladder SMCs with relaxin in vitro affects expression of MMP-2 and TGF-β1. Keywords: Relaxin, Fibrosis, Bladder, RXFP1, RXFP2 Background exstrophy without osteotomy in the first 48 h of life. It Relaxin 2, commonly referred to as “relaxin”, is known binds to relaxin family peptide receptor 1 (RXFP1) and in urology for its ability to facilitate closure of bladder RXFP2 [1]. In addition to its ability to increase pelvic ligament and bone laxity in pregnancy, relaxin can in- * Correspondence: [email protected] hibit fibrotic pathways [2] and modulate the extracellular 1 Department of Urology, Stanford University Medical Center, 300 Pasteur matrix [3]. This antifibrotic effect has led to clinical tri- Drive, Grant S-287, Stanford, CA 94305, USA 2Present Address: Division of Pediatric Urology, Advocate Children’s Hospital, als looking at relaxin therapy for fibrotic medical 8901 West Golf Road, Suite 301, Des Plaines, IL 60016, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Diaz et al. BMC Urology (2020) 20:44 Page 2 of 9 conditions such as heart failure [4], scleroderma [5], and Invitrogen), Alexa Fluor 594-conjugated donkey anti- pulmonary fibrosis [6]. rabbit IgG (1:200, Invitrogen). The poorly compliant bladder demonstrates a histo- logic phenotype of fibrosis. There is an evolution of ini- Quantitative reverse transcriptase-PCR (qRT-PCR) tial hypertrophy and then loss of detrusor muscle [7], RNA was extracted with RNA Stat-60 reagent (Tel-Test muscle replacement with collagen [8], and decreased Inc.). Primers for target proteins were created using pre- elastin in the extracellular matrix [9]. Molecular path- viously described protocols [12–16]. Samples were per- ways that contribute to this process are multifactorial formed in duplicate. Brilliant SYBR Green qPCR Master [10]. Preliminary in-vivo data suggests a relationship be- Mix (Stratagene) was used. A 10 min hot start at 95 °C tween relaxin and bladder fibrosis. Using a radiated mur- with forty cycles of 30 s of denaturation (94 °C), 1 min of ine model, a group demonstrated that relaxin therapy annealing (60 °C), and extension at 72 °C for 30 s was reverses radiation-induced fibrosis and restores bladder used. Glyceraldehyde 3-phosphate dehydrogenase function [11]. This evidence supports further investiga- (GAPDH) was used as the endogenous reference. Hu- tion as to whether relaxin-related mechanisms also exist man vaginal cuff tissue was used as a positive control for in the human bladder which, to our knowledge, has not RXFP1 and RXFP2 expression. been documented. The objective of this study is to characterize relaxin receptor expression in the human Bladder smooth muscle cell culture and Relaxin treatment bladder and assess the in-vitro effect of relaxin on ex- Primary human bladder smooth muscle cell (bSMC) line pression of proteins involved in tissue remodeling and fi- was obtained commercially from Lonza. Cells were brosis in human bladder smooth muscle cells. grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% charcoal-stripped Fetal Bovine Serum (FBS) at Methods 37 °C and 5% CO2 incubator. Cells (passage 4) were – Procurement of primary bladder specimens plated in triplicate. At 80 90% confluency, cells were IRB exemption and external approval from Donor Net- then starved in serum-free DMEM with 0.2% lactalbu- work West’s (federally designated organ procurement min enzymatic hydrolysate for 24 h, and then treated – organization) Internal Research Council and Medical with various concentrations of human relaxin-2 (0 100 Advisory Board Research Subcommittee was obtained ng/mL, PeproTech) for 24 or 48 h as described previ- for collection of primary bladder tissue from brain dead ously by our group [17]. cadaveric organ transplant donors. Brain dead donation Supernatant was concentrated 100-fold using Vivaspin was selected to eliminate warm ischemia time. Bladder filtration tubes (Sartorius AG). To acquire proteins, cell tissue (Dome and trigone) was procured by a board cer- layers were washed with (PBS) and lysed by 3 successive tified urologist after removal of organs allocated for hu- 10- min treatments with Complete-Mini Protease Inhibi- man transplant and immediately cryopreserved, placed tor Cocktail (1 tab/10 mL, Sigma- Aldrich) in radioim- in formalin, and processed into cell culture. munoprecipitation assay (RIPA) buffer at 4 °C. Cell debris was removed by centrifugation. Extracellular matrix (ECM) was prepared according to Immunohistochemistry of primary tissue Pedrozo et al. [18] Cells were removed with RIPA buffer, Primary tissue was processed into paraffin blocks. 5 um the remaining cell-free, non-solubilized ECM was sections were made and hematoxylin and eosin stain was washed 3 times with PBS and plates dried overnight at performed. Slides were reviewed by a genitourinary room temperature. Isolated ECM samples were digested pathologist. with plasmin (MP Biomedicals) in DMEM for 3 h at 37° to release TGFβ-118 [19]., Reaction was stopped by the Immunofluorescence for RXFP1 and RXFP2 in primary addition of aprotinin (5 μg/mL, Sigma- Aldrich). tissue Plasmin-digested ECM samples were collected and con- 5 um cryostat sections were fixed in 4% paraformalde- centrated 200-fold with Vivaspin filtration tubes. hyde, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS), and blocked in 1% bo- ELISA measurement of active and latent TGF-β1 vine serum albumin. Primary antibodies: mouse anti- An ELISA kit (Promega Corp) was used. Nunc MaxiSorp human cytokeratin (AE1/AE3) IgG (5μg/ml, Chemicon 96-well plates were coated with TGF-β1 monoclonal International), mouse anti-human desmin IgG (1:50, antibody. Plates were washed and blocked and 100 μLof Sigma-Aldrich), rabbit anti-human RXFP1 IgG (10μg/ acid-treated and non-acid treated samples were then ml, Abcam), rabbit anti-human RXFP2 IgG (10μg/ml, added to the wells in duplicate. After washing, anti- Lifespan Biosciences). Secondary antibody used: Alexa TGF-β1 polyclonal antibody was applied. Horseradish Fluor 488-conjugated goat anti-mouse IgG (1:300, peroxidase HRP–conjugated antibody was used for Diaz et al. BMC Urology (2020) 20:44 Page 3 of 9 detection. Absorbance at 450 nm was measured on a SPSS Statistics 21.0 software