Expression of bone morphogenetic proteins in normal kidney and renal cell carcinoma

Abstract

CILJ ISTRAŽIVANJA. Cilj doktorskog rada bio je prikazati izražaj koštanih morfogenetskih proteina (BMP), njihovih receptora (BMPR) i proteina njihovog signalnog puta (pSmad1/5/8) u zdravom tkivu bubrega, tkivu karcinoma bubrega (RCC) te tkivu na granici između karcinoma i normalnog bubrega. Nadalje, željeli smo prikazati promjenu u njihovoj ekspresiji između zdravog tkiva i RCC u usporedbi s kliničkim i patološkim značajkama ispitanika. ISPITANICI I METODE. Istraživanje je bilo prospektivno, a istražili smo tkivo bubrega u 64 ispitanika u kojih je, na Klinici za urologiju Kliničkog bolničkog centra Rijeka, učinjena nefrektomija zbog nemetastatskog karcinoma bubrega. Analizirali smo zdravo tkivo bubrega, tkivo karcinoma bubrega te granično tkivo, a analiza tkiva je provedena na Zavodu za anatomiju, Medicinskog fakulteta u Rijeci. Zabilježili smo ekspresiju proteina BMP-6, BMP-7, BMPR-IA, BMPR-IB, BMPR-II i pSmad1/5/8 (imunohistokemija i Western Blot: WB) odnosno njihove mRNA: mRNA BMP-2, mRNA BMP-4, mRNA BMP-6, mRNA BMP-7, mRNA BMPR-IA, mRNA BMPR-IB, BMP-RII (lančana reakcija polimeraze nakon reverzne transkripcije: RT-PCR). Razliku u ekspresiji BMP između različitih tkiva utvrdili smo korištenjem Student t testa (WB) i Mann-Whitney testa (RT-PCR). REZULTATI. U normalnom bubregu proteini BMP-7, BMPR-IA, BMPR-IB, BMPR-II i pSmad1/5/8 dominantno su bili prisutni u proksimalnim tubulima. U tkivu RCC pronašli smo ekspresiju proteina BMP-7, BMPR-IA, BMPR-IB, BMPR-II te pSmad 1/5/8. Ekspresiju BMP-6 u normalnom tkivu i u RCC nismo dokazali imunohistokemijskom metodom. Granično tkivo je pokazivalo obrazac ekspresije normalnog tkiva bubrega odnosno karcinoma bubrega u ovisnosti o kojem dijelu graničnog tkiva se radilo. Western blot metodom dokazali smo ekspresiju proteina BMP-6 i BMP-7, BMPR-IB, BMPR-II i pSmad1/5/8 u tkivu zdravog bubrega i u RCC. Ekspresija BMPR-IB i pSmad1/5/8 je bila povećana, a ekspresija BMPR-II, BMP-6 i BMP-7 je bila snižena u bolesnika s RCC. BMPR-IA se, ovom metodom, nije prikazao. mRNA svih BMP i njihovih receptora je pronađena u oba tkiva i bila je viša u RCC. Najsnažniji izražaj pokazao je mRNA BMP-2. Pronašli smo snažniju ekspresiju mRNA BMP-2 u bolesnika s višim tumorskim stadijem i višu ekspresiju mRNA BMP-6 u bolesnika starijih od 60 godina. Također je izražaj mRNA BMP-7 veći u žena. U uznapredovalom tumorskom stadiju (T3 i T4) bolesnika s RCC došlo je do gubitka ekspresije mRNA BMP-6, BMP-7 i BMPR-IB. ZAKLJUČAK. BMP i njihovi receptori su prisutni u zdravom tkivu bubrega te u tkivu karcinoma bubrega. BMP proteini, u ovoj studiji, su podjednakog izražaja u normalnom tkivu i karcinomu bubrega ali je ekspresija njihove mRNA znatno veća u RCC. Spoznaja kako je mRNA BMP-2 snažno eksprimiran u RCC te kako njegova ekspresija ovisi o tumorskom stadiju govori u prilog njegove važnosti u tumorigenezi karcinoma bubrega koju bi trebalo daljnjim istraživanjima ispitati.OBJECTIVES. Aim of our study was to present expression of different bone morphogenetic proteins (BMPs), their receptors (BMPRs) and protein of their signaling pathway (pSmad1/5/8) in healthy kidney tissue, renal cell carcinoma (RCC) and borderline tissue. Also, we compared changes in expression between healthy kidney tissue and RCC and correlate their expression with clinicopathological parameters. PATIENTS AND METHODS. We prospectively examined kidney tissue samples of 64 patients who underwent nephrectomy for localized kidney cancer. All patients were operated in Department of Urology, University Hospital Rijeka. Analysis was performed on healthy kidney tissue, RCC and borderline tissue on Institute of Anatomy, School of Medicine Rijeka. Protein expression of BMP-6, BMP-7, BMPR-IA, BMPR-IB, BMPR-II and pSmad 1/5/8 was determinated with immunohistochemistry and Western blot:WB) and expression of mRNA BMP-2, mRNA BMP-4, mRNA BMP-6, mRNA BMP-7, mRNA BMPR-IA, mRNA BMPR-IB, BMP-RII with reverse transcriptase polymerase chain reaction (RT-PCR). Difference in expression between tissue was estimated by Student t test (WB) and Mann-Whitney test (RT-PCR). RESULTS. In healthy kidney proteins BMP-7, BMPR-IA, BMPR-IB, BMPR-II and pSmad1/5/8 were dominantly expressed in proximal tubule. In RCC expression of proteins BMP-7, BMPR-IA, BMPR-IB, BMPR-II and pSmad 1/5/8 was found. Expression of BMP-6 was not noticed in any tissue with immunohistochemistry. Borderline tissue showed expression like RCC or healthy tissue in dependence which part of borderline tissue was examined. Proteins BMP-6, BMP-7, BMPR-IB, BMPR-II and pSmad1/5/8 were detected with WB in RCC and normal kidney. Expression of BMPR-IB and pSmad1/5/8 was increased and expression of BMPR-II, BMP-6 and BMP-7 was decreased in RCC. BMPR-IA was not detected with WB. mRNA of all BMPs and BMPRs was detected in both tissue with higher expression in RCC, specially for BMP-2. Expression of mRNA BMP-2 was increased in patients with higher tumor stage, mRNA BMP-6 was increased in elderly (> 60 years) and mRNA BMP-7 in female. We also found loss of mRNA BMP-6, BMP-7 i BMPR-IB expression in tumor stage T3 and T4. CONCLUSION. BMPs and their receptors was expressed in healthy kidney tissue and RCC. BMP proteins, in our study, have similar level of expression in both tissue but expression of their mRNA was higher in RCC. High level of mRNA BMP-2 expression in RCC and their increased expression with higher tumor stage are arguments in favour of their role in RCC. Futher investigation is needed to elucidate role of BMP-2 in RCC