Study On The Pathogenic Role And Associated Mechanism Of BMP4-ID2Axis On Congenital Ureteral Pelvic Junction Obstruction

Backgroud: Congenital ureteropelvic junction obstruction (UPJO) isa common casue of hydronephrosis in children, which will precipitate therenal been compressed and decrease renal function progressively.Especially, some patinets may lose the renal function completely due topersisting obstruction. However, the underlying etiopathogenesis of UPJOis still elusive. Recent studies had revealed the histopathological change ofhuman UPJO, the major characteristics of which include atrophy of smoothmuscle, collagen deposition, reduction of nerve fibers and decrement ofICC-like cells (interstitial cells of Cajal-like cells). Among thesecharacteristics, atrophy of smooth muscle may be the primary change. Sothe experiments, which are designed to study the development of smoothmuscle cells in embryonic ureters, may be significant to clarify theetiopathogenesis of UPJO. The development of ureteric bud is importantfor the urinary system. Bmp4was intensely expressed in mesenchyme cellssurrounding the nascent ureter, when ureteric bud branching from the wolffian duct. Besides, Bmp4had been postulated to have two importantfunctions during this period: one was to inhibit ectopic budding from thewolffian duct; the other was to promote the elongation of the branchingureter in the metanephros. Antagonizing Bmp4signaling in vitro orexcising Bmp4gene in vivo would induce a dose dependent loss of ureteralsmooth muscle, which implied that Bmp4regulated the formation ofureteral smooth muscle. The mice lacking Id2(a target gene of Bmp4)exhibited hydronephrosis mimicking the characteristics of human UPJO.Bmp4belongs to the bone morphogenetic proteins family, which have thefunctions of regulating cell differentiation, proliferation and apoptosis.Usually, these functions are mediated through Smads pathway. Id2is one ofthe most important targets of Bmp4, which expresses in various types ofcells and has the basic functions of promoting cell proliferation andinhibiting cell differentiation. Otherwise, Id2is also mediated by Myc,cAMP, and IRS1/PI3-K signals. Bmp4-Id2axis had been demonstrated tohave the functions of regulating the developments of skeletal muscle cellsand vascular smooth muscle cells. Therefore, to clarify the associationbetween BMP4/ID2and human UPJO and study the role of Bmp4-Id2axisin the development of ureteral smooth muscle cells are very important.Part1Expressions of BMP4and ID2in the ureteropelvic junctionof patients with congenital UPJOObjective: To examine the expressions of BMP4and ID2in the ureteropelvic junction (UPJ) of patients with congenital UPJO and clarifythe association between these two factors and UPJO.Methods:1. The UPJ samples were collected in patients with congenital UPJOand Wilm’s tumor, which were arranged as patients group and controlgroup, respectively.2. Masson staining was used to analyze the histopathologicalcharacteristics of UPJ. Then, the expressions of BMP4and ID2weredetected using the methods of Real-time Quantitative PCR (Q-PCR),immunohistochemistry (IHC) and western blot (WB).Results:1. The major changes of UPJ in patients group were included thinningof muscle bundles, disarrangement of muscle layers and collagendeposition, which were in accord with previous studies.2. BMP4was expressed in the mucous and smooth muscle layers. Theexpression levels of BMP4and ID2were significantly lower in the patientsgroup, which indicated these two factors were associated with humanUPJO.Part2Mutation screening of BMP4and ID2genes in patients withcongenital UPJOObjective: To detect whether BMP4and ID2mutations existed in theUPJO patients Methods:1. The samples of peripheral venous blood were collected in UPJOpatients and children undertaking health examination, which were used forgenomic DNA extraction. Then the coding sequences (CDS) of BMP4andId2genes were acquired by PCR amplification.2. Sequencing of the PCR products was accomplished on an ABI3100DNA analyzer. When mutation was detected, repeated sequencing wasperformed to ascertain the result. Then SIFT program and Align-GVGDmethod were used to predict the function of the mutations.Results:1. Three different heterozygous nucleotide sequence variations,including one novel missense variation and two synonymous mutations,were detected in patients. None was found in the controls. These mutationsare c.485G﹥A (p.R162Q), c.1167T﹥C (p.Y389Y) in BMP4and c.108A﹥G (p.L36L) in ID2.2. The missense mutation R162Q is predicted to affect the function ofBMP4by in silico analysis.Part3Construction of murine Id2overexpression plamids andexamination its expression in C2C12cellsObjective: To construct murine Id2overexpression plamids anddetect whether the mRNA and protein could be expressed after the plasmidtranfected into C2C12cells. Methods:1. The sequence of Murine Id2CDS was acquired using a chemicalsynthesis method, which was connected into the multiple clone site (MCS)of GV230vector. The recombinant plasmid Id2/GV230was identified byenzyme digestion and direct sequencing methods.2. The transfection efficiencies were detected at24h and48h afterId2/GV230plasmid tranfected. Id2mRNA and protein in each group wereexamined at48h after the transfection.Results:1. There was a product with a size of406bp in the products ofId2/GV230plasmid digested by XhoI and KpnI enzymes. The sequence ofthis product was identical to that of murine Id2CDS.2. The transfection efficiencies were (18.8±2.5)%and (19.2±2.7)%at24h and48h after the transfection, respectively. The expressions of Id2mRNA and protein in transfected group were significantly higher than thatof control groups at48h.Part4Effects of Bmp4antagonism and Id2overexpression on thedevelopment of smooth muscle cells in embryonic uretersObjective: To identify whether Bmp4, Id2paticipate in the regulativeprocess of ureteral smooth muscle cells (SMC) development.Methods:1. To dissect embryonic kidneys associated with ureters at E12.5d and culture the organs in vitro. The cultured organs were divided to threegroups: normal group, Noggin antagonism group, Noggin antagonism andId2overexpression group.2. After24h of the embryonic kidneys cultured,5μg Noggin wasadded in the culture medium of Noggin antagonism group and Id2overexpression group. Besides, the mixture of Id2/GV230plasmid andPolyJet DNA transfection regent was added into the Id2overexpressiongroup after3days. At last, α-SMA was detected by immunofluorescence ineach group after another48h.Results:1. The organs were maintained in a viable state after cultured for6days in vitro. The expression of α-SMA decreased in Noggin antagonismgroup, which indicated that Bmp4was necessary for ureteric SMCformation.2. After Bmp4antagonized by Noggin, Id2overexpression couldincrease α-SMA level, which indicated that Id2was also indispensable forureteric SMC development. It might also be presumed that the regulativefunction of Bmp4was (at least partly) mediated by Id2.Conclusions:1. The BMP4-ID2axis is closely relative to the pathogenesis of humanUPJO. The abnormality of UPJ smooth muscle may be resulted by thedecreased expression of Bmp4and ID2. 2. There is one missense of BMP4in UPJO patients, and the c.485G﹥A (p.R162Q) mutation may be one of the mechanisms leading to the lowexpressions. Howerver, the mutation rate is low (close to1%), furtherstudies are urgently needed to clarigy whether epigenetic changes existingin these two genes, which could also induce the low expressions.3. Both Bmp4and Id2participate in the regulative process of ureteralsmooth muscle formation. The regulative function of Id2in thedevelopment of ureteral SMC is mediated by Bmp4. And the majormechanism is probably to promote SMC proliferation.