| The main function of kidney is separation the urine,mineral salt, toxins and other discharge metabolites from blood, body produce various waste in metabolism process,most of them are through the glomerulus filtration, renal tubular secretion, and finally discharged to vitro with urine.What’s more, through the function of glomerulus filtration,renal tubular reabsorption and secretion,it can discharged redundant water outof vivo,adjust acid and alkali balance,maintain the stability of the internal environment,thus maintain the normal function of our’s body.Kidney disease can lead to kidney injury, gradually lose it function,and lead to the end stage renal failure (ESRF) in the end.Polycystic kidney disease (PKD) is a renal disease characterized by the growth of numerous cysts filled with fluid in the kidneys,this will affect the normal function The whole process of the disease is the function and structure and micro environmental change of renal tubular, which lead to the formation of kidney cystic lesion and expand gradually(NOEL S,1999).The appearance of interstitial fibrosis in polycystic kidneys is emblematic of progressive disease. Renal fibrosis is a complication of kidney injury and can contribute to organ failure. Currently, there are no effective drugs for the treatment of PKD and renal fibrosis. Yet, the mechanisms that promote PKD and interstitial fibrosis remain largely unknown.G protein-coupled receptors (GPCRs) are a large family of transmembrane proteins that are involved in a wide variety of physiological processes. GPCRs can recognize their ligands and transduce extracellular stimuli into intracellular signals by ligand-receptor interactions. There are thress subfamily of G protein-coupled receptors(GPCRs):Glycoprotein hormone receptor family(FSH,LH,TSH); Orphan receptor family(LGR4,5,6); Relaxation peptide receptor family(LGR7,8),here we research Gpr48,also called LGR4,which is belong to the orphans receptors with7LRRs in their putative hormone binding domains,and widely expressed in multiple tissues(Hsu SY,1998). It has been demonstrated, Gpr48deficient mice showed marked intrauterine growth retardation coupled with embryonic and perinatal lethality (Mazerbourg S,2004).The development of several organs, including eye,erythropoiesis, renal, reproductive, hair, bone and gall bladder, had been reported to be impaired severely when Gpr48is deficient in the current (Song HP,2008)(Jin C,2008)(Mendive F,2006)(Mohri Y,2008)(Kato S,2006).Therefore, GPR48is very crucial for maintaining normal development and physiological functions.The aim of this study was to clarify the role and molecular mechanism of Gpr48in the formation of kidney disease.by establishing Gpr48-/-and Gpr48+/+two genotypes to detect whether Gpr48influence the structure and function of kidney. Our result showed,The incidence of renal cystic lesion of Gpr48-/-genotype mice were as high as60%,while no cysts were observed in the kidneys of all Gpr48+/+genotype mice.Gpr48knockout easy to lead kidney cystic lesion,renal fibrosis is the terminal phase of all chronic kidney disease,so renal cystic lesion eventually caused kidney fibrosis occurs,and we draw a conclusion:Gpr48is required to maintain the normal renal function in the mice. Furthermore, Wnt/β-catenin signal pathway has been demonstrated to regulate renal development and diseases.We presumed that Gpr48might play its role by altering the activity of Wnt pathway. We subsequently demonstrated that Wnt pathway is involved in the regulation of Gpr48on preserving normal renal function. Detailed study is listed as follow:1.Reproduction and genotying of Gpr48Knock-Out mice:First we get two pairs of adult Gpr48+/-mice provided by Mr.Liu’s lab,raised in one cage and according to Mendel law of inheritance,we can get three genotype: Gpr48+/+,Gpr48+/-,Gpr48-/-respectively.By using phenotype observation (Gpr48-/genotype mice development retarded,look smaller than others),X-gal staining and RT-PCR, we identified the genotype of offsprings comprehensive by this three methods.To prevent individual differences,we selected littermate Gpr48-/-and Gpr48+/mice in the follow-up experiment,Gpr48+/-can be kept for reproduction.2.Gpr48deletion mice have an abnormal renal phenotype:Choose3pairs of different ages(1wk,3wk,5wk,8wk) littermate Gpr48+/+and Gpr48-/-mice respective,weighing original weight before experiment,weighing kidney after death and ratio calculation with original weight,take the average value of kidney/body as statistical data.Our result indicate that:Lgr4deletion will lead to renal hypoplasia,reduce kidney/body weight ratios and multiple fluid-filled cystic lesions on surface.3.Gpr48deletion caused renal poly cystic lesions and renal fibrosis:Take one kidney of our experiment littermate Gpr48+/+and Gpr48-/-mice,HE staining after fixed,comparison the internal morphological structure differences of two genotype mice.Extract RNA and protein of the other kidney,detect the transcription and translation level changes of cystic lesion related factors(PKD1).As renal fibrosis is the ultimate results of PKD,so we detect the transcription and translation level changes of fibrosis related factors(α-SMA), and for semiquantitative and location observation with immunohistochemical.Our result indicate that:HE staining demonstrate not only in the surface,but also intenal has the obvious cystic lesion,vacuolus,but only after5weeks,that means in adult Gpr48-/-genotype mice can we detect the cystic lesion. According to statistics,there are as high as60%incidence of knockout mice,while no cysts were observed in the kidneys of all Gpr48+/+genotype mice.We choose adult mice for next research and find out that coding poly cystic protein gene PKD1and PKD2were down-regulated in Gpr48-/-genotype mice,and MMP2were up-regulated which was used for remove the extracellular matrix, thus help to cystic expansion.ColI,ColⅢ and ColIV are all significantly higher expression.As a matrix metalloproteinases,MMP-1was down-regulated,and matrix metalloproteinases inhibitors TIMP1and TIMP2were up-regulated,we get consistent results in protein level and immunohistochemical staining. Thus deduce that degradation reduce synthesis increase of collagen,lead to the collagen accumulation in extracellular matrix.Next,we selected some important fibrosis markers(CTGF, FN and alpha-SMA)for detect,and result showed that all of this factors were up-regulated both in transcription and in protein expression levels.Compared with the wild type mice, FN showed a nearly5-fold increase in their mRNA expression and alpha-SMA expression was increased by almost3times in both the mRNA and protein levels in the Gpr48knockout mice. So we can get a conclusion:Gpr48deletion lean to caused renal polycystic lesions and ultimately lead to renal fibrosis.4. The correlation mechanism of Gpr48gene and kidney signaling pathways According to the literature,PKD and fibrosis involve the change of Tgf-β,Akt,Wnt signal pathway,so we detect related signal factors of Tgf-β,Akt and Wnt signal pathway by RT-PCR and Western-blot,trying to find out Gpr48related signal pathway.The experiment results indicate that:β-catenin as a molecular switch in Wnt signal pathway,was significantly up-regulated in Gpr48-/-genotype mice on translation,while in Gpr48+/+genotype mice, nearly no P-catenin band were detected because of ubiquitination degradation. So Gpr48knockout can result in nuclear accumulation of β-catenin,and thus activate Wnt signal pathway. Akt as a Wnt signal inhibitor,deletion in Gpr48-/-genotype mice,which is more advantageous to up-regulated Wnt protein,While there were no obvious differences of Tgf-β signal factors between Gpr48-/-and Gpr48+/+genotype mice,so we deduced that renal cystic lesion and fibrosis caused by Gpr48deletion may not involve Tgf-β signal pathway. |
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