Introduction: 

One of the most frequent causes of acute kidney injury (AKI) is acute tubular necrosis (ATN) of ischemic origin. To achieve the best prognosis there is a need for early diagnostic tools of AKI. The neutrophil gelatinase associated lipocalin (NGAL) is an accepted biomarker of tubular epithelial cell injury and inflammation. The aim of this study was the detailed analysis of the diagnostic role of NGAL in ischemia-reperfusion (IR) injury.

Methods: 

We performed 10, 20 and 30 minutes of ischemia on the left kidneys with right nephrectomy in C57BL/6 mice. After 1, 3, 6, 15, 24, 48, 72 and 96 hours of reperfusion we examined NGAL (1) renal expression (real-time PCR), (2) localization (immunohistochemistry on tissue microarray (TMA) slides), (3) plasma and (4) urine protein levels (ELISA). Furthermore we evaluated (5) tubular histopathology (PAS stain) and (6) renal function by blood urea retention (Reflotron). Systemic inflammation was monitored by plasma (7) IL-6 and (8) p40 (IL-12, IL-23) protein levels (ELISA).

Results: 

Following IR injury NGAL appeared mainly in a punctuate form in juxtamedullary tubular cells at the luminal pole and in the tubular lumen. After 1 day of reperfusion, NGAL mRNA and its 24h urine protein level raised significantly (1.7 fold, p<0.05 and 6.3 fold, p<0.01, respectively) as a consequence of only 10 minutes of ischemia, not inducing any other renal damage marker. Acute tubular necrosis (p<0.01) and increase in blood urea (5.4 fold, p<0.001) and plasma NGAL protein (3.2 fold, p<0.05) was detected only if the ischemia lasted at least 20 minutes. Thirty minutes ischemia lead to anuria and to severe ATN already after 1h (p<0.01), blood urea at 3h (1.8 fold, p<0.05), renal NGAL mRNA at 6h (10 fold, p<0.01), plasma NGAL at 15h of reperfusion (5.2 fold, p<0.01). In the sham operated group NGAL renal mRNA and its plasma protein level also increased (the most at 15h), but this elevation was minor compared to the IR injury (50 and 5 times lower, respectively). Plasma inflammatory cytokines also increased, but only temporarily. In the moderate ischemia group (20 min) renal blood urea and plasma NGAL levels have returned to the baseline, but renal NGAL mRNA remained elevated longer (13.9 fold, p<0.05).

Discussion and conclusion: 

NGAL is upregulated after IR injury and accumulates in vesicles at the apical cytoplasmic compartment of renal tubular epithelial cells. Thus, urine NGAL protein (if obtainable) is the most sensitive biomarker of ischemic injury of tubular cells.