A rapid urine test for early detection of kidney injury

Abstract

Kidney injury molecule-1 (Kim-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a highly sensitive and specific urinary biomarker to monitor drug-induced kidney injury in preclinical studies and on a case-by-case basis in clinical trials. Here we report the development and evaluation of a rapid direct immunochromatographic lateral flow 15-min assay for detection of urinary Kim-1 (rat) or KIM-1 (human). The urinary Kim-1 band intensity using the rat Kim-1 dipstick significantly correlated with levels of Kim-1 as measured by a microbead-based assay, histopathological damage, and immunohistochemical assessment of renal Kim-1 in a dose- and time-dependent manner. Kim-1 was detected following kidney injury induced in rats by cadmium, gentamicin, or bilateral renal ischemia/reperfusion. In humans, the urinary KIM-1 band intensity was significantly greater in urine from patients with acute kidney injury than in urine from healthy volunteers. The KIM-1 dipstick also enabled temporal evaluation of kidney injury and recovery in two patients who developed postoperative acute kidney injury following cytoreductive surgery for malignant mesothelioma with intraoperative local cisplatin administration. We hope that future, more extensive studies will confirm the utility of these results, which show that the Kim-1/KIM-1 dipsticks can provide a sensitive and accurate detection of Kim-1/KIM-1, thereby providing a rapid diagnostic assay for kidney damage and facilitating the rapid and early detection of kidney injury in preclinical and clinical studies.

Figure 1. Evaluation of rat RenaStick to detect urinary Kim-1 in cadmium induced nephrotoxicity model

Urinary Kim-1 measurements using rat RenaStick (a–e), histological damage (f–i), and Kim-1 immunostaining (j–m) following cadmium-induced kidney injury. Male Sprague Dawley rats (n = 4/group) were injected subcutaneously with varying doses of CdCl2 (Cd dose of 0.6, 1.2, or 2.4 mg/kg) or saline 5 days per week for 4 weeks. (a–d) After 4 weeks, rats were placed in metabolic cages and urine was collected. Seventy microliters of urine sample from each rat was mixed with 70 µl of TRIS-buffered saline solution, added to the RenaStick sample well, and incubated for 15 min. (e) Each RenaStick was scanned using a CAMAG Thin Layer Chromatography scanner and the densitometric units obtained were plotted. *Statistically higher than control/sham (P<0.05). (f–i) At necropsy, samples of both kidneys were isolated from each animal and placed in 10% neutral buffered formalin (NBF) for hematoxylin and eosin (H&E) staining and histology assessment. (j–m) In addition, non-fixed cryosections of the kidney tissue were processed for the immunoflourescent visualization of Kim-1. (f and j) Control, (g and k) 0.6 mg Cd/kg, (h and l) 1.2 mg Cd/kg, (i and m) 2.4mg Cd/kg. All fields were chosen from the outer renal cortex. Arrows in panels g–i indicate retraction and sloughing of cells, tubular congestion, and necrosis. The fluorescent images in panels k–m indicate Kim-1 staining in the apical membrane of the proximal tubules of rats treated with cadmium in similar fields of the outer renal cortex.

Figure 2. Evaluation of rat RenaStick to detect urinary Kim-1 in gentamicin-induced nephrotoxicity model

Urinary Kim-1 measurements using rat RenaStick (a–d), BUN and serum creatinine (e, f), and histological damage (g–i) following gentamicin-induced kidney toxicity. Male Sprague Dawley rats received gentamicin sulfate at 0, 40, or 120 mg/kg/day (n = 5 rats per dose group) for 9 days, and urine samples were collected on day 10 by keeping rats in metabolic cages. (a–c) Urinary Kim-1 was measured from each rat by mixing 70 µl of urine sample with 70 µl of TRIS-buffered saline solution and adding it to the RenaStick sample well. Results were read in 15 min. (d) Each RenaStick was scanned using a CAMAG Thin Layer Chromatography scanner and the densitometric units obtained were plotted. On day 10, blood and kidney tissue were obtained from animals for evaluation of (e) blood urea nitrogen (BUN), (f) serum creatinine, and (g–i) histomorphological evaluation of kidneys (hematoxylin and eosin staining). All fields were chosen from the cortex. Arrows in panels g and i indicate sloughing of cells, tubular dilation, and necrosis. Bar represents 50 µm. *Represents statistically higher than control (P<0.05).

Figure 3. Detection of urinary Kim-1 using rat RenaStick in bilateral renal ischemia/reperfusion model

Urinary Kim-1 measurements using rat RenaStick (a–i), kidney histopathology (j–m), immunohistochemistry for Kim-1 (n–q), and BUN and serum creatinine (r–s) over time following 20min of bilateral renal ischemia/reperfusion injury. Male Wistar rats were subjected to 0 (sham) or 20 min of bilateral ischemia by clamping the renal pedicles for 20 min and then removing the clamps and confirming reperfusion. (a–h) Rats were placed in metabolic cages and urine was collected at 6, 9, 18, 24, 48, 72, 96, and 120 h following reperfusion. Urinary Kim-1 was measured from each rat by mixing 70 µl of urine sample with 70 µl of TRIS-buffered saline solution and adding it to the RenaStick sample well. Results were read in 15 min. (i) Each RenaStick was scanned using a CAMAG Thin Layer Chromatography scanner and the densitometric units obtained were plotted. (j–q) Rats were killed at various times and blood and kidney tissue were collected. Representative photomicrographs of H&E-stained kidney sections and immunohistochemistry for Kim-1 are presented from the following time points: (j and n) 0 h (sham control); (k and o) 9 h; (i and p) 24 h; (m and q) 120 h. All fields were chosen from the outer stripe of outer medulla (OSOM). Arrows in panels from k–m indicate sloughing of cells, tubular dilation, and necrosis. Panels o–q show Kim-1 staining in the apical membrane of the proximal tubules in the OSOM region. Bar represents 50 mm. (r) Blood urea nitrogen (BUN) and (s) serum creatinine were measured over time following reperfusion. *Represents statistically higher than sham-operated animals (P<0.05).

Figure 4. Detection of KIM-1 using human RenaStick in urine from patients with or without kidney injury

Urinary KIM-1 measurements using human RenaStick in (a–c) a cross-sectional study involving 24 patients with or without acute kidney injury and (d–f) prospective study in two patients with cisplatin-induced kidney toxicity over time.