(227) Analysis of Gene Expression of Vitamin D in Rats in Experimental Model of Peyronie's Disease and Human Penile Tunica Albuginea

Abstract

Abstract Introduction Peyronie's disease (PD) is a penile connective tissue disorder that results in an inflammatory process, fibrotic plaque in the tunica albuginea (TA) and penile curvature. The molecular changes of fibrotic plaque formation are complex and include different cellular/protein mechanisms. Vitamin D (vitD) is a steroid hormone that has unconventional pathways of action, and among its effects it has already been described that it plays a role in extracellular matrix remodeling, in the immune response and in fibrosis. The literature regarding the action of vitD in the male genital system is scarce, and the gene expression of its receptors has not yet been demonstrated in penile TA. Objective To identify the gene expression of the vitD receptor in penile TA of rats submitted to the experimental model of PD and human penile TA. Methods The experimental model in rats divided into 2 groups (case and control) described before by Cohen et al. Human penile TA was achieved from Peyronie´s surgery cases and cadaveric controls. The antibody anti vitD (VDR D-6: sc-13133, Santa Cruz Biotechnology®, CA, USA) was used. Immunolabeling was carried out using the avidin-biotin-peroxidase complex method, and 3,3′-diaminobenzidine as liquid chromogen. In each case, the immunolabeling was quantified by computer-assisted digital image analysis following the methodology described by Matos et al. PCR real-time analyses followed the MIQE guideline10. One of the fractions of the tissue samples of the TA was stored in RNAholder. The extraction of total RNA from the samples was carried out using the reagent TRIzol®. The reverse transcription was performed using the reverse transcriptase enzyme ImPromII™ to obtain complementary DNA (cDNA). Messenger RNA expression of vitD was obtained by RT-PCR. Quantitative RT-PCR was carried out using pair of oligonucleotides endogenous RPL13a (human samples) and GAPDH (rat samples), known commercially as forward and reverse primers. It was performed statistical analysis with SPSS® version 17.0 (SPSS® Inc; Illinois, USA). Values were expressed as a mean and standard error with a significance level of 95% (p ≤ 0.05). The tests applied were One-Way ANOVA and unpaired t-test with Welch's correction, confirming the similarity between the variances. Sensitivity and specificity analysis was calculated following the Youden index. Ethical approval local committee number 06/2016. Results The vitD mRNA amplifications demonstrated the presence of its receptors in the penile TA in both the control and the case groups, and the expression of the control group was statistically higher (p < 0,0001) when compared to the case group in rats. In human samples, mRNA amplifications demonstrated the presence of its receptors in the penile TA in both the control and the case groups, and the expression of the control group was statistically lower (p < 0,0001) when compared to the case group. Conclusions The presence of vitD receptors (genic and proteic expression) in penile TA of rats Peyronies´ models and human TA was demonstrated and allows future studies regarding its regulation and modulation according to the evolution and phase of PD and vitD serum levels. Disclosure No