We report here the relative performance of four biomarkers to accurately assess kidney injury in thirteen structurally and mechanistically different models of renal tubular injury in rats. Irrespective of whether the kidney injury was induced by well established kidney toxicants or ischemia, urinary Kim-1 not only outperformed BUN, SCr and urinary NAG, which are conventional markers for assessing renal injury, but did so achieving an ROC-AUC of 0.91 to 0.99. The AUC for Kim-1 remained greater than 0.9 whether the entire histopathology grade of 0 to 5 were included or whether the analyzed group was restricted to histopathology grade scores of 0 and 1, demonstrating that urinary Kim-1 measurements are highly sensitive, specific and accurate in diagnosing either drug-induced kidney tubular necrosis, degeneration, and/or dilatation or regenerative basophilia when lesions are either subtle with little organ involvement, or very severe with disturbed renal function. We further show by exclusion analysis that a threshold increase of 1.87-fold of urinary Kim-1 concentration for 95 % specificity derived from one laboratory was similarly and independently defined in other laboratories using other study designs for kidney injury.
In this set of 17 studies, the increase in urinary Kim-1 was compared to histopathology, which is considered the “gold standard” for assessing preclinical renal injury. The AUC and the sensitivity of Kim-1 was nearly 1, irrespective of the mechanism of kidney injury and correlated highly with histopathologic changes even for low-grade injuries (grade 1). This is the first report demonstrating that current markers of assessing nephrotoxicity, BUN and SCr, are effective only with more severe histopathological grades (> grade 2) in preclinical studies. For example, the sensitivity of SCr was remarkably low at 0.20 for histology grades 0 to 1 and increased to only 0.56 with severity grades of 0 to 3 in the Merck studies while urinary Kim-1 was sensitive and specific for assessing subtle forms of proximal tubular damage (histology grade 0 to 1). These AUC-ROC numbers represent the exclusion data analysis approach. Exclusion analysis sets aside data values that show discordance between histopathology and early biomarker values in nephrotoxicant treated animals since further investigations would be required to unequivocally show which approach indicates truth. Exclusion analysis gives the most conservative threshold of elevation and is most appropriate for preclinical investigations since histopathology data are available. Inclusion analysis was similarly performed in parallel (Supplementary Table 2, 3 and 5
) and is most similar to a clinical setting where histopathology observations are not available for biomarker correlation. An inclusion approach assumes that discordance between early biomarker values and true injuries may be attributed to false positive biomarker values. The inclusion analysis approximately doubles the Kim-1 threshold indicative of injury to 3.9-fold. Direct comparison of AUC shows a lower value for inclusion analysis compared to exclusion analysis across the curve (Supplementary Table 2
Urinary Kim-1 has been shown in additional studies to be a sensitive and early diagnostic indicator of renal injury in a variety of acute and chronic rodent kidney injury models resulting from drugs8, 9, 16
, environmental toxicants7, 9, 17
, and protein overload18
. Human studies have also yielded promising results for potential utility of urinary KIM-1 as a diagnostic biomarker for AKI. Han et al. demonstrated marked expression of KIM-1 in kidney biopsy specimens from six patients with acute tubular necrosis, and found elevated urinary levels of KIM-1 after an initial ischemic renal insult, prior to the appearance of casts in the urine 10
. Liangos et al.
reported urinary KIM-1 and NAG in 201 patients with established AKI and demonstrated that elevated levels of urinary KIM-1 and NAG were significantly associated with the clinical composite endpoint of death or dialysis requirement, even after adjustment for disease severity or comorbidity11
. Zhang et al.
compared the tissue expression of KIM-1 with histopathological and functional parameters of acute tubular injury (ATI) and acute cellular rejection (ACR) in renal transplant biopsies from 62 patients19
. KIM-1 expression was present in all biopsies from patients with histological changes showing ATI and in 92 % of kidney biopsies from patients with ACR. KIM-1 staining sensitively and specifically identified proximal tubular injury and significantly correlated with declining renal function. A longitudinal prospective study conducted by van Timmeren et al
reported that elevated urinary KIM-1 serves as an independent predictor of long-term graft loss in renal transplant recipients (n=145 patients) independent of donor age, creatinine clearance and proteinuria13
. In our recent clinical cross sectional study comparing nine urinary biomarkers (KIM-1, NGAL, IL-18, NAG, protein, HGF, VEGF, IP-10 and Cystatin C) in 204 patients with or without acute kidney injury urinary KIM-1 had an AUC-ROC of 0.93 and was significantly higher in patients who progressed either to death or to requirement for renal replacement therapy (RRT) when compared to those who survived and did not require RRT12
The opportunity to use the same marker for both the preclinical and clinical setting facilitates clinical monitoring of toxicity that has been demonstrated at higher doses in preclinical development or in a single test species when human relevance is suspected.
To facilitate the biomarker measurement, a sensitive, specific, reproducible and high-throughput assay is critical for its widespread use and applicability. We have previously reported development and evaluation of a sandwich ELISA assay for measurement of Kim-1 using two epitopically distinct monoclonal antibodies raised against rat Kim-1 ectodomain8
. Nevertheless, our ELISA required adjustments of urine samples to fit to the linear range of the standard curve decreasing the throughput of the assay. Therefore, we developed the microbead based assay to measure Kim-1 in rat urine samples, and found that the dynamic range was increased to span from 4 pg/mL to 40,000 pg/mL. Other advantages of this assay include the ability to quantitate Kim-1 using only 30 μL of undiluted urine samples and reducing the assay time from 6 hours to 3.5 hours while maintaining the intra and inter assay variability to less than 10%. The easy transferability of the Kim-1 assay was demonstrated by the Kim-1 measurements of the Novartis studies conducted in a second laboratory. More recently, we have also reported the development of a rapid point of care diagnostic dipstick assay for measuring Kim-1 in rodent and human urine samples within 15 minutes20
In summary, we report that urinary Kim-1 levels correlate with different grades of kidney tubular histopathologies in thirteen mechanistically distinct models of acute kidney injury. Using exclusion and inclusion data analysis, Kim-1 had the highest AUC-ROC (> 0.88) as compared to BUN, SCr and NAG. Especially for low-grade toxicity (grade 1) Kim-1 was the only marker of the four capable of consistently detecting renal tubular injury. Urinary Kim-1 outperformed (p<0.01) serum creatinine, BUN and urinary NAG as biomarkers of renal tubular injury in these mechanistically distinct models of kidney injury performed at three different sites. Binary and ordinal logistic regression models for exclusion and inclusion data analysis showed that addition of Kim-1 represented a statistically significant improvement and increased the concordance probability to histopathology.
Thus, due to the striking evidence of the performance of urinary Kim-1 it was deemed qualified as a highly sensitive and specific marker of drug-induced kidney injury by both FDA and EMEA and is expected to greatly facilitate evaluation of tubular toxicity in certain preclinical and clinical settings.