Evaluating Canine Neutrophil Gelatinase-associated Lipocalin(NGAL) As A Sensitive Biomarker For Early Diagnosis Of Acute Renal Injury

Acute kidney injury(AKI)is one of the most common canine diseases,with high morbidity and mortality due to the lack of early diagnostic biomarkers.Neutrophil gelatinase-associated lipoprotein(NGAL)is an early diagnostic biomarker for AKI in human and other animals.However,canine AKI have different causes and thus whether NGAL could be used as an early diagnostic biomarker remains to be defined.In this study,we established canine renal injury model of ischemia-reperfusion(I/R).NGAL was proved to be a sensitive marker for early diagnosis of canine AKI by comparing with other diagnostic biomarkers using the canine I/R AKI model.Six monoclonal antibodies were prepared by constructing NGAL expressiong vectors and double antibody-sandwiched ELISA was established.By using clinical samples from healthy and AKI-diseased dogs,the sandwich ELISA was proved useful for early diagnosis of canine AKI.1.Establish canine I/R-AKI model and comparative study of diagnostic markersTwelve beagles were randomly divided into sham group(n=6)and I/R group(n=6).In the I/R group,the right kidney was removed surgically,and both artery and vein of left kidney were cross-clamped for 60 min followed by reperfusion.Dogs in the sham group were only removed the right kidney.The renal tissues of left kidneys of both groups were collected at 12 h and 72 h after operation.Histopathological analysis showed that the I/R group had typical AKI lesions,including loss of brush edge of renal tubules,degeneration and necrosis of epithelial cells,protein congestion or lumen stenosis in the tubules.The blood and urine samples were collected at different time points after reperfusion.The blood urea nitrogen(BUN),serum creatinine(sCR)and urinary creatinine(uCr)were detected by automatic biochemical analyzer.Serum NGAL(sNGAL)and urinary NGAL(uNGAL)were detected by ELISA kit.The results showed that,compared with the sham group,the level of sCr in I/R group was significantly increased from 24 h after operation,reaching the upper limit of the reference value at 36 h and the peak value at 60 h.The ratio of uNGAL/uCr(UNCR)rose rapidly from 2 h after I/R and maintained at high levels from 6 to 60 h.The levels of sNGAL and uNGAL increased rapidly from 2 h after I/R,reaching to the peak values at 12 h and then declining gradully.ROC analysis showed that detection sensitivities of uNGAL and sNGAL were significantly higher(P<0.0001)than that of sCr.Both quantitative RT-PCR(qRT-PCR)and immunohistochemistry analyses of dog tissues confirmed that the NGAL could be used as a sensitive biomarker for early diagnosis of canine AKI.2.Preparation and identification of monoclonal antibodies against canine NGALTo develop a canine NGAL detection method with independent intellectual property,the primers were designed according to the published sequence of canine NGAL.The total RNA was extracted from beagle kidneys and used to amplify the mature peptide of canine NGAL cDNA by RT-PCR.The amplicon was cloned into pET-28a(+)vector and transformed into E.coli competent cells.The recombinant NGAL expression was induced with IPTG and purified by nickel affinity column.The BALB/c mice were immunized with the recombinant protein and 6 hybridoma cell lines were generated.The sequence analysis showed that the cloned NGAL sequence was identical to the published sequence.The E.coli-expressed canine NGAL had the expected molecular weight of 25 kDa and was purified to more than 90%.After three rounds of subcloning,6 positive hybridoma cell lines were obtained and the cell culture supernatants had ELISA antibody titers of 1:10240,1:64000,1:32000,1:10240,1:32000 and 1:64000,respectively.The ascites antibody titers of hybridoma-injected mice were 1:64000,1:128000,1:128000,1:64000,1:128000 and 1:128000,respectively.The purified antibodies had an ELISA antibody titer of 1:1024000.The six monoclonal antibodies reacted specifically with canine NGAL.These results suggested that the prepared monoclonal antibodies could be used for canine NGAL detection.3.Development of double antibody-sandwiched ELISA kit for canine NGAL detectionThe antigen binding sites of 6 monoclonal antibodies were analyzed by additive ELISA using recombinant NGAL and the best matching antibody pair was determined by sandwich ELISA for establishing double antibody-sandwiched ELISA.The best reaction conditions for the sandwich ELISA were determined using single factor test.The coefficient of variation and recovery rate between the intra-batch and inter-batch were determined by clinical sample detection.The detection performance of double antibody-sandwiched ELISA was compared with commercial ELISA kits using the serum and urine samples from healthy and AKI dogs(n=36).The results showed that,among 6 monoclonal antibodies tested,NC-1 and NC-3 shared the same antigen binding site,while NC-2,NC-4,NC-5 and NC-6 shared another antigen binding site.Finally,NC-5 and NC-3 were selected as the capture antibody and detection antibody of the sandwich ELISA since their high P/N ratio.The single factor test showed that the optimal conditions for NC-5(capture antibody)coating were 2 ?g/mL antibody in 50 mM Tris-HCl(pH 8.0).The best conditions for the ELISA plate blocking were incubation with 1%casein in 0.1M PBST for 3 h at 37?.The sample was incubated for 45 min at 37? and the optimal dilution(1:8000)of detection antibody was added.After 45-min incubation at 37?,the substrate solution(0.1mg/mL,TMB)was added and incubated for 10 min at room temperature in dark.By using purified recombinant canine NGAL,the standard curve was generated with a detection limit of 0.292 ng/mL.For the serum samples with low or high NGAL concentration,the intra-batch coefficient of variation was 8.63%or 6.70%,and the inter-batch coefficient of variation was 10.34%or 7.48%.For the urine samples with low or high NGAL concentration,the intra-batch coefficient of variation was 8.33%or 4.54%,and the inter-batch coefficient of variation was 10.01%or 4.62%.The averaged recovery ratio of NGAL in serum and urine samples were 91.9%and 96.7%,respectively.Compared with commercial NGAL kit,the correlation coefficients of the home-made sandwich ELISA for urine and blood sample detection were 97.18%and 99.06%,respectively.These results suggested the usefulness of home-made sandwich ELISA for canine NGAL detection.4.Validation of NGAL sandwich ELISA for early diagnosis of canine AKIA total of 256 blood samples and 106 urine samples were collected from 27 breeds of dogs.According to clinical examination and BUN,sCr detection,256 dogs were divided into non-kidney disease group including healthy(n=40),parvovirus-infected(n=52)and unexplained diarrhea dogs(n=50),and kidney disease group including dogs with acute kidney injury(n=60)and chronic kidney disease(n=54).Both sNGAL and uNGAL were detected with the home-made sandwich ELISA and UNCR was calculated.For 11 dogs without renal disease but with uNGAL level above the reference,concentrations of BUN,sCr,uCr,sNGAL and uNGAL in blood and/or urine samples were detected using the sandwich ELISA and UNCR was calculated.The results showed that concentrations of sNGAL,uNGAL and UNCR in dogs without kidney disease were significantly(P<0.0001)lower than that in kidney disease dogs.In addition,the average sNGAL level in chronic kidney disease(CKD)dogs was significantly(P<0.0001)lower than that in AKI dogs.Compared to that in dogs without kidney disease,the levels of sNGAL,uNGAL and UNCR in AKI dogs were significantly(P<0.0001)different.For the non-kidney disease dogs with BUN and sCr concentrations above the reference values,8/11 dogs had significantly increased levels of sNGAL and uNGAL on day 5 after primary detection,which were diagnosed as AKI dogs by combining with the clinical symptoms.These data suggested that the home-made NGAL ELISA could be used for early diagnosis of canine AKI.