A specific immunoassay for detection of feline kidney injury molecule 1

Abstract

Objectives The aim of this study was to design and carry out a preliminary evaluation of a urine point-of-care test for kidney injury molecule 1 (KIM-1) in healthy and diseased cats. Methods Part of the feline KIM-1 gene was amplified, ligated into a plasmid with a signal peptide and monomeric human IgGFc, and transfected into a mammalian cell line. Supernatant was purified and tested for the fusion protein by gel electrophoresis and Western blot. Mice were immunized three times with purified proteins, and hybridomas were generated from splenocytes. Antibodies were tested by ELISA for detection of recombinant KIM-1 and naturally occurring KIM-1 in disease-state urine. Next, a lateral flow assay (LFA) with capture and detection antibodies was constructed, and tested with 34 urine samples from healthy and diseased cats. Antibodies were also tested for reactivity with formalin-fixed paraffin-embedded kidney tissue. Results Three antibodies were assessed. Antibodies detected between 0.4 and 60 ng/ml feline KIM-1 fusion protein in the LFA. Urine samples from healthy cats yielded faint bands in the LFA corresponding to optical density (OD) values of 4.8–8.8. Samples from cats with suspected or confirmed acute kidney injury (AKI) had OD values ranging from 1.6–20.5. Urine KIM-1 varied over multiple days in cats with sepsis or urethral obstruction despite normalizing serum creatinine concentration. In tissue sections, KIM-1 antibodies labeled tubular cells with morphological features of injury. Conclusions and relevance A practical patient-side assay for detection of KIM-1 in feline urine has been developed. Preliminary results show marked though transient increases in cats with sepsis and urethral obstruction-associated AKI, and expression in injured tubules. Although initial data indicating that the LFA is sensitive and specific for KIM-1 in cats with AKI are promising, values associated with different types of injury, urine collection, urine storage and specific gravity need to be investigated.

Evaluation of symmetric dimethylarginine in cats with acute kidney injury and chronic kidney disease

“…Serum SDMA concentrations were within reference intervals in 4/22 cats with novel AKI and 5/19 cats with CKD, and were elevated in 3/15 control cats. When considering the novel AKI group only (Figure 1), serum SDMA concentrations were significantly higher in cats with IRIS grade IV AKI (45 [13‐69] μg/dL; P = .015) and IRIS grade V AKI (70 [40‐170] μg/dL; P = .003) compared with cats with IRIS grade II AKI (16 11‐32 μg/dL). There were only 2 cats with IRIS grade III AKI, which had SDMA concentrations of 9 μg/dL and 18 μg/dL.…”

Renal biomarkers in cats: A review of the current status in chronic kidney disease

“…Some cats (31%) with conditions associated with AKI had a substantial but transient increase in uKIM‐1. Only 1 CKD cat expressed uKIM‐1 at lower levels than healthy cats 96 . Overall, uKIM‐1 is highly expressed after early severe tubular injury but further tubular loss may result in negative results.…”

“… 167 More recently, the same research group also developed a specific lateral flow assay for the detection of feline uKIM‐1. 96 In mice, uKIM‐1 is stable for 5 days at room temperature and at least for 1 year at −80°C, and multiple freeze–thaw cycles do not significantly alter uKIM‐1 concentrations. 168 The effects of storage time and temperature on uKIM‐1 concentrations in cat samples remain unknown.…”

“…Only 1 CKD cat expressed uKIM‐1 at lower levels than healthy cats. 96 Overall, uKIM‐1 is highly expressed after early severe tubular injury but further tubular loss may result in negative results. Therefore, kinetic monitoring of this biomarker using serial sample analysis might be important in practice.…”

“…Feline KIM‐1 has been detected in urine using the commercially available anti‐rat KIM‐1 lateral flow immunoassay 167 . More recently, the same research group also developed a specific lateral flow assay for the detection of feline uKIM‐1 96 . In mice, uKIM‐1 is stable for 5 days at room temperature and at least for 1 year at −80°C, and multiple freeze–thaw cycles do not significantly alter uKIM‐1 concentrations 168 .…”