The Relationship Between Decoy Receptor2Expression And P16Expression, Tubular Interstitial Lesion In IgA Nephropathy

Background and ObjectiveIgA nephropathy is one of the most common chronic kidney disease in Asian. About25to30%patientswith IgA nephropathy ultimately run into the end-stage renal diseaseafter10to20years.The mechanism underlying theprogression of IgA nephropathyis notfully understood yet,and thus effective treatments are still lacking.Stress induced premature senescence(SIPS)can be observed in patients with chronickidney disease, especially in IgA nephropathy. Present study suggests that SIPS is animportant cellular events in disease progression, it plays a very important role in thedevelopment of cancer, diabetes and its complications, cardiovascular and cerebrovasculardiseases, tissue and organ fibrosis and other diseases.Cellular senescence can be inducedrapidly in response to various physiologic stressors independent of the number of celldivisions, so-called SIPS. The feature of SIPS is irreversible cell cycle arrest which inducespermanent loss of cell proliferation, damages the tissue, reduces regenerative capacity andrepair capacity of tissue, makes organ dysfunction. Senescent cells secrete proinflammatorycytokines, chemokines, growth factors and extracellular matrix termed thesenescence-associated secretory phenotype (SASP), which may damage surrounding cellsand cause local inflammation and tissue fibrosis. Our previous study found that p16Ink4a、SA-β-gal positive RTEC icreased with the progression of the IgA nephropathy, at the sametime, type III collagen and fibronectin expression increased, this would result inglomerulosclerosis and tubulointerstitial fibrosis. This study found SIPS was the pathologyfonudation of IgA nephropathy progression. Transl Res published a commentary whichconsider that this word is a step forward in our understanding of tubular senescence in thesetting of glomerulonephritis. Due to the limitation of the biomarker of cell senescence, cellsenscence cann’t be identified by a sigle marker, two or more than two markers was used toconfirm cell senscence. So, it is very important to verify SIPS in IgA nephropathy and to find more specificity SIPS marker for RTEC.Decoy receptor2(DcR2), which is located in the cell membrane,is one of theTNF-related apoptosis-inducing ligand (TRAIL) receptors. It participates in the regulationof cell apoptosis. Recently, it is found that DcR2is the reason of cell apoptosis resistan.Apoptosis resistance is one of the characters of senescent cells, and this is the reason whysenescent cells elimination delayed and gathering in local, so that result in diseaseprolonged and progression. Recently, it is found that DcR2expression increased insenescence premalignant and fibroblast cells, it can be a marker of cell senescence, and it isalso probably the reason of senescence cell resistent to apoptosis. DcR2can be expressed inmost tissue, but mainly in kidney and testis. DcR2can be detected in HK2cells and inRTEC of diabetes mouse. But the study of DcR2in kidney is rare, only a study reports thatDcR2was highly expressed and this would inhibit RTEC apoptosis in diabets mouse. Thereis no study about the relathionship between DcR2expression and disease progression inIgA nephropathy and whether it can be used as a marker of cell senscence in RTEC. Thisstudy explored the relathionship between DcR2and SIPS in IgA nephropathy, DcR2expression changing and the extent of kidney lesion, to proving DcR2is a biomarker of theprogress of IgA nephropathy.Methods1. Clinical data.116primary IgA nephropathy patients aged18-50years withouttreatment were enrolled. All those patients were hospitalized in department of nephrologyin our hosipital between January2013and Spetember2013. Patients who had secondaryIgAN diseases, acute kidney injury, suffering from other diseases(such as cancer, diabetes,tuberculosis, viral hepatitis, severe cardiovascular disease), pregnancy or lactation andthose on ACEI and/or ARB, glucocorticoid and/or nephrotoxic drugs before the study wereexcluded. All the patients were devided into T0, T1and T2groups by tubularatrophy/interstitial fibrosis score for Oxford scoring system.13renal hamartomaaged18-50yearswere recruited as controls, The renal biopsy sampleswere obtained from resectionoperations of renaltumors, located far from tumor sites and pathologicallyconfirmed asnontumor, normal tissues.All patients’ data, including sex, age, BM, SBP,DBP,MAP,ALB,Scr,BUN,UA,CHOL,TG,HDL, LDL, urine protein to creatinine ratio andurinary NAGwere collected. Estimated glomerular filtration rate (eGFR) wascalculated by the CKD-EPIformula as following:eGFR=141×min(SCr/K,1)α×max(SCr/K,1)-1.209×0.993age×[1.018if Female]×[1.159if Black].2. Renal tissue lesion scoring.Oxford IgA nephropathy scoring system was used forthe renal tissue lesion scoring, the criteria as follows:（1） Glomerulus. Mesangial scoreless than or equal to0.5(M0) or>0.5(M1),Segmental glomerulosclerosis absent (S0) or present (S1), Endocapillary hypercellularityabsent (E0) or present (E1)（2） Tubular and interstitial. Tubular atrophy/interstitial fibrosisless than or equalto25%(T0),26–50%(T1), or>50%(T2)（3） Artery. Arcuate arteriosclerosis/Interlobular arteriosclerosisnormal (AA/IA0),intimal thickened to more or less than the thickness of the media(AA/IA2or AA/IA1,respectively). Arteriolar hyalinosis normal (AH0),1-25%affected arterioles(AH1),26-50%affected arterioles (AH2) and>50%affected arterioles (AH3).3. Indicators and methods.（1） Renal tissue SA-β-gal expression detection.In accordance with SA-B-gal staining kit steps to detecte frozen sections of renaltissue SA-β-gal expression.（2） Immunohistochemical detect renal tissue p16Ink4a, DcR2expression.Two-step immunohistochemical staining(EnVision) was used. Continus paraffin-embedded tissue sections, dewaxing, hydration, high-pressure repair antigen, anti-DcR(1:500) or anti-p16Ink4a (1:400) was added, overnight incubation, dropping horseradishperoxidase-labeled anti-mouse or anti-rabbit IgG, incubated at room temperature for30min,DAB microscopic color, hematoxylin, dehydrated, mounted.（3） Immunofluorescence detect renal tissuep16Ink4aand DcR2co-expression anddistribution.Paraffin-embedded tissue sections, dewaxing, hydration, high-pressure repair antigen,anti-DcR(1:400) and anti-p16Ink4a(1:400) mixture was added, overnightincubation,dropping FITC-labeled goat anti-mouse IgG(1:50) andCy3-labeled goatanti-rabbit IgG(1:50) mixture, incubated at room temperature for40min, mounted, viewedby confocal microscope.4. Scoring method. Tissue sections results were performed blinded by two unrelated persons of the study as following:Ten continus fields were selected randomly using a light microscope. RTECs showingpositive (brown) staining for p16Ink4aor DcR2were counted and expressed as thepercentage of total number of RTECs.Ten continus fields were selected randomly using a light microscope. Tubules showingpositive (blue) staining for SA-β-gal were counted and expressed as the percentage of totalnumber of tubules.p16Ink4aand DcR2double positive RTECs were counted and expressed as thepercentage of total number of RTECs under a confocal microscope.5. Statistical analysis. The data were analyzed using SPSS20.0software. Significancelevels were set at P<0.05.Results1. Clinical feartures. IgA nephropathy patients were devided into T0group of79patients, T1group of24patients, T2group of13patients. Age, sex and BMI had nosignificant difference compared with the control group. Compared with the control group,cholesterol, high density lipoprotein, LDL, urinary NAG, urine protein to creatinine ratiowas significantly higher in T0group (p <0.05). Cholesterol, low density lipoprotein, serumcreatinine, blood urea nitrogen, uric acid, urine NAG, urine protein to creatinine ratio wassignificantly higher in T1group(p <0.05); albumin, eGFR was significantly lower than thecontrol group (p <0.05). Systolic blood pressure, diastolic blood pressure, mean arterialpressure, total cholesterol, LDL, serum creatinine, blood urea nitrogen, uric acid, urineNAG, urine protein to creatinine ratio was significantly higher in T2group (p <0.05);albumin, eGFR significantly lower than control group (p <0.05). Compared with T0group,triglycerides, serum creatinine, blood urea nitrogen, urea group was significantly higher inT1group, albumin, eGFR was significantly lower in T1group (p <0.05). Compared withT1group, systolic blood pressure, diastolic blood pressure, mean arterial pressure, serumcreatinine, blood urea nitrogen, uric acid was significantly higher in T2group (p <0.05),eGFR was significantly lower in T2group (p <0.05). Urine protein to creatinine ratio hadno statistically significant difference among IgA nephropathy groups.2. Cell senescence markers were significant increased in IgA nephropathy patients.(1) SA-β-Gal positive cells were mainly tubular epithelial cells, with low expression in normal kidney tissue, increased in IgA nephropathy tissue. Percentageof SA-β-Galpositive renal tubular was significantly higher in IgA nephropathy than the normal controlgroup, and increased with tubular atrophy/interstitial fibrosis score.(2) p16Ink4aexpressed in renal tubular epithelial cells, glomerular cells, renal interstitialcells. In normal renal tissue, p16Ink4aexpression was low,in IgA nephropathy p16Ink4aexpression increased. Compared with normal control group, percentage of p16Ink4apositivetubular epithelial cells, glomerular cells and renal interstitial cells was significantly higherin the IgA nepropathy group, and increased with tubular atrophy/interstitial fibrosis score.(3) Immunohistochemical staining show that DcR2only expressed in renal tubularepithelial cells, DcR2expression in tubular epithelial cells of normal kidney waslow, andincreased in IgA nephropathy renal tubular epithelial cells.The tendency of the DcR2expression was consistent with SA-β-Gal and p16Ink4a: low expression in the normal kidneytissue, increased with tubular atrophy/interstitial fibrosis score. Furthermore, continuousrenal tissue sections of each group show that DcR2positive RTECs distribution were withp16Ink4apositive RTECs.(4) DcR2、p16Ink4aco-expression in RETCs.Immunofluorescence staining show thatDcR2and p16Ink4awere co-expressed in renal tubular epithelial cells, DcR2expressionlevel was lower than p16Ink4a, no positive p16Ink4aexpression withnegative DcR2expressiontubular epithelial cells were found. The percentage of co-expression RTECs increased withtubular atrophy/interstitial fibrosis score.3. The correlation between SA-β-Gal,p16Ink4a, DcR2and clinical feature, kidneylesion score in IgAnepropathy(1) Correlation analysis showed the percentage of p16Ink4a, SA-β-Gal, DcR2positiveRTECshave a positive correlation with and blood pressure, urine NAG, uric acid, ureanitrogen, serum creatinine, urine protein to creatinine ratio;and have a negative correlationwith eGFR in IgA nephropathy patients. Age, BMI, albumin and blood lipid levels were notcorrelated with the percentage of p16Ink4a, SA-β-Gal, DcR2positive RTECs.(2) The percentage of p16Ink4a, SA-β-Gal, DcR2positive RTECs were positivelycorrelated withsegmental sclerosis/adhesion, interstitial fibrosis, arcuatearteriosclerosis/interlobular arteriosclerosis, arteriolar hyalinosis. There were no correlationwith mesangial scoreless, endocapillary hypercellularity and the percentage of p16Ink4a, SA-β-Gal, DcR2positive RTECs.ConclusionsOur study confirms the presence of cell senescence in IgA nephropathy renal tubularepithelial cells, renal tubular epithelial cell senescence may lead the progression of IgAnephropathy.DcR2as a marker of renal tubular epithelial cell senescence is initially proved,the expression levels of DcR2is an important indicator for judging IgA nephropathyprogression.