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A specific immunoassay for detection of feline kidney injury molecule 1
S Karlyn Bland1, Mary Ellen Clark1, Olivier Côté2
1Department of Pathobiology, University of Guelph, Guelph, ON, Canada.
Insights
A new point-of-care test for feline kidney injury molecule 1 (KIM-1) shows promise for diagnosing acute kidney injury (AKI) in cats. This urine test detected increased KIM-1 levels in cats with AKI, indicating kidney damage.
Area of Science:
- Veterinary Medicine
- Biomarker Discovery
- Diagnostic Assay Development
Background:
- Acute kidney injury (AKI) is a significant concern in feline health, often diagnosed using serum creatinine, which may not reflect early or transient damage.
- Kidney injury molecule 1 (KIM-1) is a promising biomarker for renal tubule injury, but a validated point-of-care test for cats is lacking.
Purpose of the Study:
- To design and conduct a preliminary evaluation of a urine point-of-care test for feline KIM-1.
- To assess the test's ability to detect KIM-1 in healthy versus diseased cats, particularly those with AKI.
Main Methods:
- Developed a lateral flow assay (LFA) using antibodies against feline KIM-1.
- Purified recombinant feline KIM-1 fusion protein for antibody testing.
- Tested the LFA with 34 urine samples from healthy and diseased cats and evaluated antibody reactivity with kidney tissue.
Main Results:
- The developed LFA detected feline KIM-1 fusion protein at concentrations between 0.4 and 60 ng/ml.
- Urine samples from cats with suspected or confirmed AKI showed significantly higher optical density (OD) values (1.6-20.5) compared to healthy cats (4.8-8.8).
- KIM-1 antibodies labeled injured tubular cells in kidney tissue, and urine KIM-1 levels varied in cats with sepsis or urethral obstruction.
Conclusions:
- A practical, patient-side assay for detecting KIM-1 in feline urine has been developed.
- Preliminary findings suggest transient increases in KIM-1 in cats with sepsis and obstruction-associated AKI, correlating with injured tubules.
- Further investigation is needed to refine the assay and understand KIM-1 values across different injury types and sample conditions.
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.
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