TIN-ag Expression In Chronic Kidney Disease And It’s Clinical Significance

Background:Chronic kidney disease is kidney damage or glomerular filtration rate less than60ml/min/1.73m2for more than3months, including kidney damage, pathological changes, blood, and urine composition abnormality or imaging exam abnormality. The biomarker of chronic kidney disease includes urine, urinary sediment,24-hour urinary protein, serum creatinine, blood urea nitrogen, eGFR, alpha1-microglobulin, beta2-microglobulin and so on. A Chinese city cross-sectional study of the prevalence of CKD in Zhuhai shows in2013the CKD prevalence was12.5%, the prevalence rate of renal function decline was2.7%and protein urine prevalence rate was10.3%, in Beijing The prevalence of CKD was13.0%, the prevalence of CKD in Shanghai was11.8%, in China’s southwest prevalence of CKD was18.3%. A simple classification of renal disease is classified as diabetic nephropathy, non-diabetic kidney disease, renal transplantation kidney disease. The main type of kidney disease can be divided into type1diabetes and type2diabetic nephropathy; non-diabetic glomerular diseases (autoimmune disease, systemic infection, drugs, tumors); vascular disease (macro vascular disease, high blood pressure, micro vascular lesions); tubulointerstitial disease (urinary tract infection, stones, obstruction, drug toxicity),Polycystic Kidney Disease (ADPKD); kidney chronic rejection; transplant drug toxicity (cyclosporine or tacrolimus);Recipients (glomerular diseases recurrent disease); allograft nephropathy. In the United States, the progression to end-stage renal disease (ESRD) patients with a mortality rate of approximately20%. CKD complications include high blood pressure, anemia, malnutrition, bone disease, calcium and phosphorus metabolism disease, neurology, complications including cardiovascular disease and diabetes.The tubular features include selective reabsorption and excretion molecules as well as the production and release of hormones. Thus, the tubulointerstitial demanding more energy and easy to be injuried. Tubular participate in a variety of diseases, including urinary tract obstruction, reflux nephropathy, pyelonephritis, metabolic diseases, genetic diseases, and poisoning. In view of the importance of ubulointerstitial damage sensitivity, many physiological functions are inappropriate to enlarge clinical manifestations of chronic kidney disease (CKD). Tubulointerstitial damage caused by factors as below:changes in glomerular function, the filter to be misleading, the filtrate flow is blocked, proteinuria, renal tubular epithelial cell activation, the plasma molecular filtration load increases, blood lipids and complement generated. Glomerular filtration dysfunction, inflammation, fibrosis, hypoxia, common in the pathophysiology of the various kidney diseases, can cause tubulointerstitial injury and activation. In turn, the tubulointerstitial injury may cause progressive chronic kidney disease. Tubulointerstitial injury led to the accumulation of inflammatory cells in the interstitium. Tubular damage will lead to the shedding of tubules and glomerular tubules, decrease in the number and function of the nephron. Together, these events create a continuously injury, cell activation, the cycle bug fixes; it is variety of CKD common reasons. Further study of these tubular interstitial damage mechanisms will enable us to better clarify the nature of CKD and identify possible therapeutic intervention pathway Tubulointerstitial nephritis antigen (TIN-ag) is a human anti-tubular basement membrane (TBM) renal interstitial nephritis antibody-mediated antigen. The immunofluorescence staining find TIN-ag is positioned at the proximal of the tubular basement membrane, another small portion is located in the distal tubule and Bowman’s capsule and the basement membrane of the intestinal mucosa. TIN-ag is divided into40-58kD and40-50kD glycoprotein subtypes. This molecule has an affinity for type IV collagen and laminin. It specific expressed in fetal kidney development, lack of TIN-ag can lead to juvenile nephronophthisis (JNPHP) and some other genetic cause tubule interstitial defects. TIN-AG physicochemical characteristics:1) TIN-AG is a high molecular weight glycoprotein, which may be spliced into the molecules of different size;2) it can be combined with other basement membrane components via a covalent bond;3) TBM component can reacts with the human or animal anti-TBM antibody;4) TIN-ag antigen-antibody reaction may exist in multiple TBM assembly. The above analysis suggests that TIN-ag have great significance in renal physiology and pathologyTIN-ag can facilitate the interaction between the cells and matrix in kidney development and interaction with other basement membrane proteins, such as type IV collagen, laminin and proteoglycans. The juvenile nephronophthisis (JNPHP) characteristic by early-onset chronic renal failure, tubular interstitial scale, and is accompanied by autosomal genetic disease in other organs, such as retinitis pigmentosa. TIN-ag is an glycoprotein which expression in the extracellular matrix of basement membrane of the renal tubule, while certain extracellular matrix proteins can regulate the cell cycle and survival in vitro experiments.In vitro experiment it also found that Cisplatin can induced HK-2cells apoptosis, increase the activity of the Caspase7, enhance mitochondrial DNA fragmentation and decrease cell survival. After disposed with TIN-ag, the change was reduced. Coimmunoprecipitation analysis also found that: TIN-ag can combined with vitronectin and integrin alpha V beta3.Afer integration av(33siRNA transfected HK-2cells, cisplatin-induced apoptosis increased, cytochrome C and Bax proteins abnormal translocation, cell survival decreased. In addition, it is found that the avβ3siRNA treated HK-2cells express less focal adhesion kinase (FAK) and phosphorylated FAK and AKT and express more p53and phosphorylation P53,while Integrin-linked kinase (ILK) expression does not change. The above changes significantly improved after treated by TIN-ag protein. Indicating that TIN-ag plays an important regulatory role in acute kidney injury tubule cell apoptosis.The mechanism related to the TIN-ag effectively combined with integrin alpha V beta3and adjust its downstream signaling molecules level.We assumed:TIN-ag defects may lead to CKD. In obstructive kidney disease, type IV collagen protein expression was up-regulated but laminin and TIN-ag was down-regulated.The expression trend of TIN-ag in the renal tissue of patients with CKD is not yet fully understood, there are still many of the research spaces in renal physiology and pathology, The purpose of this study is to explore the TIN-Ag expression in the kidneys of patients with CKD and its clinical significance.The relationship between TIN-Ag and CKD and whether it may be a biomarker of CKD diagnosis to provide experimental evidence Chapter1. Expression of TIN-ag in patients with chronic kidney diseaseObjective:To observe TIN-AG expression in the patients with chronic kidney disease kidney tissue, preliminary study with the relationship between TIN-ag and chronic kidney renal interstitial fibrosis.Method:Randomly selected clinical and renal pathological diagnosis of chronic kidney disease and minimal change nephritis patients77cases. According to the pathological types are divided into five groups:minimal change glomerulonephritis9cases, none minimal change glomerulonephritis68cases, including focal segmental glomerulosclerosis16cases, IgA nephropathy23cases, membranous nephropathy14cases, lupus nephritis15cases. HE, Masson staining to observe pathological changes of the kidney; immunofluorescence to detect kidney tissue TIN-ag expression.Results:kidney tissue for HE staining showed that with microscopy, no obvious abnormalities in glomerular and tubulo-interstitial compartment in the renal tissues of minimal change diseases (MCD) patients, and podocyte effusion was seen with eletronicmicroscope. None minimal change glomerulonephritis (NMCD) patients’renal tissue show various focal tubular atrophy and interstitial fibrosis besides glumerular morphology abnormalities. The immunofluorescence with anti TIN-ag antibody showed that in MCD group, TIN-ag expressed clearly in renal tissue, especially in tubule. In patients with NMCD renal tissue, TIN-ag expression became weaker, especially in the area which renal tubular epithelial atrophy and interstitial fibrosis. Quantity analysis for TIN-ag immunofluorescence staining intensity demonstrated that TIN-ag expression inNMCD significantly decreased, as compared to MCD group (p<0.01). Further analysis found that TIN-ag expression in the renal tissues of primary FSGS, IgA nephropathy, MN and LN was much lower than in that of MCD patients (p<0.05).But there is no difference of TIN-ag expression between the four groups. TIN-ag expression level in patients of collagen area score0is higher than expression in the area of collagen score1-3(p<0.05). TIN-ag expression in Collagen area score1patients is higher than the area of collagen score2-3expression patients (p<0.05).Conclusion:TIN-AG expression in non-minimal change renal tissue is reduced; TIN-ag expression in focal segmental glomerular nephritis group was the lowest. TIN-ag expression is inversely correlated to the severity of renal interstitial fibrosis. Chapter2. Expression of TIN-ag in renal tissue of patients with chronic kidney disease and its relationship with clinical phenotypeObjective:Explore TIN-ag expression changes in chronic kidney disease, as well as the clinical phenotypes.Method:Randomly selected a total of77cases patients diagnosed by clinical and renal pathology with chronic kidney disease. Immunofluorescence detect kidney tissue TIN-ag expression, detection of serum creatinine, urea nitrogen, eGFR,24-hour urine output,24-hour urine protein, al-Microglobulin,(32-Microglobulin, pathological casts, NAG enzyme specific gravity and other clinical phenotypes.Results:1.TIN-ag expression in the renal tubule of CKD patients was negatively correlated with blood al-Microglobulin concentration, the correlation coefficient is-0.244(p<0.05);2. TIN-ag expression in the renal tubule of CKD patients was negatively correlated with pathological casts in the urine, the coefficient is-0.264(p <0.05).3. TIN-ag expression in the renal tubule of CKD patients was negatively associated with total protein content in24hour urine, the coefficient is-0.184(p<0.05).4. TIN-ag expression in the renal tubule of CKD patients was negatively associated with the level of urine NAG expression.5. TIN-ag expression was less in low urine gravity group, as compared to that in normal urine gravity (p<0.05).6. TIN-ag expression was not significant correlated with eGFR and serum creatinine, blood urea nitrogen, P2-Microglobulin concentration.Conclusion:1TIN-Ag expression in chronic nephropathy group was inversely proportional with blood alphal-microglobulin concentration, pathological casts, total protein content in24hour urine, urine NAG expression and urine gravity.2TIN-Ag expression was not significant correlation with serum creatinine concentration, blood urea nitrogen concentration and eGFR.3. TIN-ag is closely related to tubule injury and may have a protective effect or as a biomarker. Chapter3. Secretion and Expression of TIN-ag in Urine of CKD PatientsObjective:To observe whether the TIN-ag expression can take as a biomarker of CKD.Methods:randomly selected chronic kidney disease group (n=8) and healthy control group (n=8), detect blood and urine TIN-ag expression by ELISA. Randomly selected chronic kidney disease group (n=9) and healthy control group (n=16), detect the urine TIN-ag mRNA expression by RT-PCR.Results:in TIN-ag protein expression in the healthy control group was higher than the expression of chronic kidney disease serum (p>0.05). TIN-ag protein expression was higher than the expression levels in the urine of chronic kidney disease group (p<0.05). TIN-ag mRNA expression in the urine of healthy control group was higher than the expression level of the chronic kidney disease group (p<0.05).Conclusion:The expression of TIN-ag protein and TIN-Ag mRNA in the urine of chronic kidney disease group were both decreased.