Ya-Jane Lee

Urine neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute canine kidney injury

BackgroundBiomarkers for the early prediction of canine acute kidney injury (AKI) are clinically important. Recently, neutrophil gelatinase-associated lipocalin (NGAL) was found to be a sensitive biomarker for the prediction of human AKI at a very early stage and the development of AKI after surgery. However, NGAL has not yet been studied with respect to dog kidney diseases. The application of NGAL canine AKI was investigated in this study.ResultsThe canine NGAL gene was successfully cloned and expressed. Polyclonal antibodies against canine NGAL were generated and used to develop an ELISA for measuring NGAL protein in serum and urine samples that were collected from 39 dogs at different time points after surgery.AKI was defined by the standard method, namely a serum creatinine increase of greater than or equal to 26.5 μmol/L from baseline within 48 h. At 12 h after surgery, compared to the group without AKI (12 dogs), the NGAL level in the urine of seven dogs with AKI was significantly increased (median 178.4 pg/mL vs. 88.0 pg/mL), and this difference was sustained to 72 h.ConclusionAs the increase in NGAL occurred much earlier than the increase in serum creatinine, urine NGAL seems to be able to serve as a sensitive and specific biomarker for the prediction of AKI in dogs.

Prognosis of acute kidney injury in dogs using RIFLE (Risk, Injury, Failure, Loss and End-stage renal failure)-like criteria

A retrospective case-series study evaluated the prognosis of 853 dogs with acute kidney injury (AKI) based on the RIFLE (Risk, Injury, Failure, Loss and End-stage renal failure) criteria, derived from human medicine. The 30-day mortality of dogs with AKI in each class was found to be 23.8 per cent (40 of 168) dogs for Risk, 41.0 per cent (107 of 261) dogs for Injury and 78.5 per cent (333 of 424) dogs for Failure. Using the dogs in the Risk class as the reference, the mortality of dogs in either the Injury or Failure class was significantly higher than that of dogs in the Risk class (P<0.05). The longest median survival time was observed in the Risk class (nine days) and the shortest median survival time was observed in the Failure class (three days). Using a multiple logistic regression model, a new score that simultaneously considered RIFLE class, diarrhoea status and serum phosphorus level was calculated to predict prognosis. Evaluation using the area under the receiver-operating characteristic curve (AUROC) indicated that the new scoring method (AUROC 0.80) was a better prognostic indicator than using RIFLE criteria alone (AUROC 0.73).

Prognostic Factors and a Prognostic Index for Cats with Acute Kidney Injury

BACKGROUND The clinical manifestations of acute kidney injury (AKI) range from mild to fatal in cats; however, prognosis factors have been rarely studied. HYPOTHESIS/OBJECTIVES To find the clinical factors significantly correlated with the outcome among cats with AKI and to develop a simple prognostic index. ANIMALS Seventy cats with AKI were recruited. METHODS Demographic and clinicopathological data obtained from 70 cats with AKI were retrospectively collected. Students t-test or Mann-Whitney U-test and Pearson chi-square test or Fishers exact were applied to determine the factors associated with survival in cats with AKI. Using logistic regression, the statistically significant factors associated with prognosis were identified and a new prediction model was generated. RESULTS The overall case fatality rate was 64% (45/70). The results showed that nonsurviving cats had significantly lower levels of PCV, WBC, RBC, LDH and albumin, a lower albumin/globulin ratio, lower blood glucose, and a reduced body temperature, as well as being older. Serum urea and creatinine concentrations were not statistically significant as prognostic factors, but a decrease in these 2 variables in 3 days was significantly related to a reduction in death. A summary prognostic index including body temperature and LDH and albumin concentrations had area under the receiver-operating characteristic curve (AUROC) for predicting death of 0.86 (P < .05) and a cut-off value of 0.82, a sensitivity of 77% and a specificity of 90%. CONCLUSIONS The prognosis in cats with AKI is quite different from that found for human and dogs.

Neutrophil Gelatinase–Associated Lipocalin in Dogs with Naturally Occurring Renal Diseases

Background Neutrophil gelatinase–associated lipocalin (NGAL) is released from renal tubular cells after injury and serves in humans as a real‐time indicator of active kidney damage, including acute kidney injury (AKI) and chronic kidney disease (CKD). However, NGAL concentrations in dogs with naturally occurring AKI or CKD rarely have been explored in detail. Hypothesis/Objectives The goal of this study was to evaluate whether NGAL can serve as a useful biomarker in dogs with naturally occurring renal disease. Animals Client‐owned dogs with renal disease (57) and control dogs without any disease (12) were examined. Methods Serum NGAL (sNGAL) and urine NGAL (uNGAL) concentrations were measured in each animal by a newly developed ELISA system. Demographic, hematologic, and serum biochemical data were recorded. Survival attributable to AKI and CKD was evaluated at 30 days and 90 days, respectively. Results Serum and urine NGAL concentrations in azotemic dogs were significantly higher than in nonazotemic dogs and were highly correlated with serum creatinine concentration (P < .05). Among CKD dogs, death was associated with significantly higher sNGAL and uNGAL concentrations compared with survivors. Receiver‐operating characteristic curve (ROC) analysis showed that sNGAL was better than serum creatinine concentration when predicting clinical outcomes for CKD dogs (P < .05). The best cutoff point for sNGAL was 50.6 ng/mL, which gave a sensitivity and a specificity of 76.9 and 100%, respectively. Furthermore, dogs that had higher concentrations of sNGAL survived for a significantly shorter time. Conclusion sNGAL is a useful prognostic marker when evaluating dogs with CKD.

Diagnosis of feline polycystic kidney disease by a combination of ultrasonographic examination and PKD1 gene analysis

Persian-related and non-Persian-related cats were examined by ultrasonography and/or molecular testing to determine the prevalence of feline polycystic kidney disease (PKD) and the presence of a PKD1 gene mutation. PCR was used to amplify exon 29 of the PKD1 gene using genomic DNA extracted from blood samples, and the PCR products were analysed by direct DNA sequencing. Among the 111 cats included in the study, 54 were examined by both ultrasonography and gene testing for a point mutation in exon 29 of the PKD1 gene. The prevalence of PKD diagnosed by ultrasonography was 25.9 per cent in all the cats and 24.2 per cent in Persian-related cats. The prevalence of the transversion mutation in exon 29 of the PKD1 gene was 13.5 per cent in all cats and 15.7 per cent in Persian-related cats. Three cats that were diagnosed with PKD by ultrasonography did not have the mutation within exon 29. Nucleotide analysis of exon 29 indicated that male cats had a higher point mutation rate than female cats.

The different molecular forms of urine neutrophil gelatinase-associated lipocalin present in dogs with urinary diseases.

BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a useful biomarker for the early prediction of renal diseases. NGAL may exist as monomer, dimer and/or NGAL/MMP-9 complex forms in humans. In this study, the existence of various forms of NGAL in urine (uNGAL) was determined and whether these forms are related to the different urinary diseases found in dogs is further discussed.ResultsEighty-one urine samples from dogs with different forms of renal disease (41), pyuria (19) and a number of non-renal related diseases (10), as well as healthy dogs (11), were collected. uNGAL concentrations and their molecular forms in dogs were measured by ELISA and Western blot analysis, respectively. The uNGAL concentrations of dogs with pyuria (median: 15.35 ng/mL) were significantly higher than those of the healthy control animals (median: 3.92 ng/mL) (p < 0.01), but lower than those of dogs with renal diseases (median: 23.77 ng/mL). Each NGAL molecular form could be detected in dog urine. In particular, monomer was detected more frequently in patients with renal disease than those with non-renal diseases; while the dimer form appeared in a significantly higher percentage of cases with pyuria compared to those without pyuria. The NGAL/MMP-9 complex was found to exist not only in the patients with cystitis, but also in the cases with renal injury.ConclusionDifferent molecular forms of uNGAL can indicate different origins of the urinary abnormalities. Determining the molecular forms of uNGAL present in diseased dogs may provide clinical workers with a tool that will help the early and more precise detection of different urinary diseases.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

Detection of indoxyl sulfate levels in dogs and cats suffering from naturally occurring kidney diseases.

Indoxyl sulfate (IS), a protein-bound uraemic toxin, has been found to accumulate in the serum of people with renal diseases and is associated with free radical induction, nephrotoxicity cardiovascular toxicity, and osteoblast cytotoxicity. Although IS has been studied in humans and in experimental models, the role of IS in dogs and cats with kidney disease has not been investigated. A high performance liquid chromatography system was applied to detect plasma IS concentrations in non-azotaemic animals (63 dogs, 16 cats) and in animals with renal azotaemia (66 dogs, 69 cats). The IS levels of azotaemic animals were significantly higher (P <0.01) than those of non-azotaemic animals (median [IQR] 20.4 (9.5) mg/L vs. 7.2 (8.8) mg/L for dogs; median [IQR] 21 (18.9) mg/L vs. 14.8 (12.3) mg/L for cats). The IS level was significantly correlated with blood urea nitrogen, serum creatinine and phosphate concentrations. Dogs with acute kidney injury had significantly higher IS levels (P <0.01) than those with chronic kidney diseases (CKD) (median [IQR] 57.7 (40.8) mg/L vs. 17.7 (25.1) mg/L). When CKD was graded using the International Renal Interest Society (IRIS) staging system, IS levels were correlated with CKD severity in both dogs and cats. The IS concentration is directly related to loss of renal function. Further studies are necessary to determine whether measurement of IS provides any additional diagnostic or prognostic information in dogs and cats with kidney disease.

Molecular Detection of Autosomal-Dominant Feline Polycystic Kidney Disease by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C→A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

Neutrophil Gelatinase-Associated Lipocalin in Cats with Naturally Occurring Chronic Kidney Disease

Background Neutrophil gelatinase‐associated lipocalin (NGAL) is a biomarker for the early prediction of renal damage and the progression of chronic kidney disease (CKD) in humans and dogs. Hypothesis Neutrophil gelatinase‐associated lipocalin also may play a role in the progression of CKD in cats. Animals Eighty CKD and 18 control cats. Methods Cats were categorized into different stages according to the International Renal Interest Society (IRIS) staging system. Urine and plasma samples were collected and tested for NGAL concentrations using an in‐house sandwich ELISA system and urinary NGAL (uNGAL)‐to‐creatinine ratio (UNCR) was determined. Cats in which serum creatinine concentration increased by >0.5 mg/dL from baseline within 30 days were defined as exhibiting progression. Results The urinary NGAL and UNCR of CKD cats were significantly higher than those of healthy cats (P < .05) and were highly correlated with serum creatinine concentration. The area under the receiver operating characteristic curve (AUROC) for uNGAL, when predicting the progression of CKD, was 0.71 and the best cutoff value was 2.06 ng/mL with a sensitivity of 76.9% and a specificity of 75%. The AUROC for UNCR when predicting the progression of CKD was 0.79 and the best cutoff value was 4.08 × 10−6 with a sensitivity of 76.9% and specificity of 79.2%. Cats with UNCR values higher than their cutoffs experienced significantly faster deterioration with a median of 19 days. Conclusions Both urinary NGAL and UNCR are useful markers for the prediction of CKD progression in cats.