Effects Of Neutrophil Gelatinase Associated Lipocalin In Lipopolysaccharide-induced Acute Kidney Injury And Its Mechanism

Acute kidney injury (AKI; or acute renal failure) is an important issue for patients during critical care, with sepsis being the most common trigger for AKI in the intensive care unit (ICU). Because of the lack of sensitive and specific biomarkers for indicating renal cell injury, the mortality rates for septic AKI have remained high. Recently, genomic, transcriptomic, and proteomic techniques have identified neutrophil gelatinase associated lipocalin (NGAL) as an early marker of AKI. NGAL has been investigated in a range of different clinical settings, such as contrast-induced nephropathy, AKI after cardiac surgery or kidney transplantation and AKI in the critical care setting. Overall, the sensitivity for NGAL to predict AKI is0.815(95%confidence interval,0.732-0.892) and it is a promising biomarker for AKI, similar to troponin for acute myocardial infarction.However, the use of NGAL in detecting or predicting the outcomes of patients with septic AKI is complex and depends on the clinical setting. In previous research of critically ill patients, both serum NGAL (sNGAL) and plasma NGAL (pNGAL) levels have the potential to act as early biomarkers for AKI. While both of them are highly sensitive, they have not been found to be very specific predictors of septic AKI. Shapiro et al assessed the diagnostic accuracy of pNGAL in predicting AKI, wherein it was revealed that pNGAL concentrations of>150ng/mL were96%sensitive but only51%specific for patients with suspected septic AKI. Martensson et al observed that pNGAL is elevated in patients with systemic inflammatory response syndrome, severe sepsis, and septic shock, and it should be used with caution as a marker of AKI in ICU patients with septic shock. In contrast, studies have shown that urinary NGAL (uNGAL) is more useful for predicting AKI, as uNGAL levels are not elevated in septic patients without AKI. Understanding the role of NGAL in impaired renal tissues may help clarify this issue.Furthermore, a biomarker should temporally reflect the pathophysiology initiated by a stimulus leading to injury so as to alert the clinician to a potentially reversible stage of the illness. In the case of septic AKI, the relationship between NGAL and early inflammation mechanisms in the kidney will ought to be clearly established. However, the mechanisms of septic AKI remain undefined. Increasing evidence suggests that intrarenal inflammatory mediators play an important role in the pathogenesis of kidney injury. Specially, tumor necrosis factor a (TNFα) is a pivotal proinflammatory mediator and interleukin (IL)-6is an accessory factor. However, it remains unknown whether NGAL expression is related to changes in renal IL-6and TNFa during the early stage of septic AKI.Based on the available evidence, the use of NGAL as a marker of septic AKI is promising but requires further investigation. In particular, the relationship between NGAL expression, humoral NGAL and renal inflammation requires additional clarification. In addition, the mechanism of NGAL which was expressed in the injured epithelia must be investigated to explain the function of NGAL as acute phase reactive proptein in renal during sepsis.However, few studies have specifically investigated the expression and its mechanism of NGAL in renal tissues during sepsis. Therefore, the purpose of this study was to investigate the pattern and localization of renal NGAL expression, and to explore the relationship between renal inflammation mediators, such as TNFa and IL-6, and humoral NGAL in a rat model of LPS-induced kidney injury, as well as the antiapoptotic mechanism of NGAL was explored in lipopolysaccharide (LPS)-treated HK-2to provide systematic and reliable theoretical base for the application of NGAL in diagnosis and treatment of septic AKI.Part1Preparation of LPS-induced acute kidney injury in ratsObjective:To induce septic AKI by using lipopolysaccharide (LPS,3.5 mg/kg, ip) and evaluate the SCr and kidney pathological changes in order to make clear the acute renal damage.Methods:The rats were divided into six groups according to processing factors and the time difference within24hours. The control group (Con) for salt water treatment which was given0.9%salt water (1ml, ip). The other groups (LPS groups) which were given LPS (3.5mg/kg, ip) were divided into5groups according to treating time (1h,3h,6h,12h,24h). According to the presupposed time points, the animals were anesthetized with pentobarbital sodium to take blood samples to determine SCr and harvest kidney to verify the histological damages under light and electron microscopy.Results:Rats treated with LPS showed significantly elevated levels of SCr, which were significantly increased from3to12hours after LPS administration compared with those in Con group (P<0.01).The kidneys from the Con group showed normal glomeruli and tubules. In contrast, the kidneys of the LPS-treated rats were characterized by transient renal damage. Under a light microscope, there were no intrinsic lesions within the glomeruli, but lesions consisting of epithelial swelling were observed in the3-12-hour groups. Further, intracellular edema was also detected and was more severe in the straight segment of the proximal tubules in the LPS-treated rats. Ultrapathology analysis further demonstrated the presence of intracellular edema, coupled with disarranged microvilli, and/or injury or destruction of the mitochondrial outer membrane in the proximal epithelia.Conclusions:On the basis of changes to serum creatinine levels and renal histology, we were able to confirm LPS-induced AKI, and thus confirm that we had established a suitable animal model. Part2Renal NGAL expression in LPS-induced acute kidney injury in the ratObjective:Neutrophil gelatinase associated lipocalin (NGAL) is a highly predictive biomarker of acute kidney injury. To understand the role of NGAL in renal injury during sepsis, we investigated the temporal changes and biological sources of NGAL in a rat model of acute kidney injury, and explored the relationship between renal inflammation, humoral NGAL and NGAL expression during endotoxemia.Methods:To induce acute renal injury, rats were treated with LPS(3.5mg/kg, ip), and the location of NGAL mRNA was evaluated by in situ hybridization. Quantitative RT-PCR was also used to determine the dynamic changes in NGAL, tumor necrosis factor a (TNFa) and interleukin (IL)-6mRNA’expression1,3,6,12, and24hours following LPS treatment. The correlation among NGAL, TNFa and IL-6was analyzed. Urinary and plasma NGAL (u/pNGAL) levels were measured, and the relationship between humoral NGAL and NGAL expression in the kidney was investigated.Results:Renal function was affected3-12hours after LPS. NGAL mRNA was significantly upregulated in tubular epithelia at the same time (P<0.01). The course of NGAL mRNA upregulation occurred in parallel with renal damage. There was a transient increase in TNFa and IL-6mRNA levels within3hours following LPS administration(P<0.01),and a strong correlation between TNFa and NGAL mRNA (r=0.995, P<0.001) but not with IL-6mRNA. Both pNGAL and uNGAL levels were markedly increased compared with those in the control group (P<0.01); however, only uNGAL levels were correlated with NGAL mRNA (r=0.850, P<0.001).Conclusions:NGAL mRNA upregulation is sensitive to LPS-induced renal TNFa increase and injury, which are observed in the tubular epithelia. Urinary NGAL levels accurately reflect changes in NGAL in the kidney. Part3Exploration of the antiapoptotic mechanism of NGAL in LPS-treated HK-2Objective:NGAL mRNA has been found to be up-regulated in renal epithelia during endotoxemia in our previous study. To understand the role of NGAL in renal epithelium during sepsis, we investigated the temporal changes of NGAL in LPS-treated HK-2, and explored the relationship between NGAL mRNA and Caspase3mRNA. Furthermore, we investigate whether specific inhibition targeting NGAL using small interfering RNA (siRNA) would provide renal protection by reducing apoptosis in LPS-treated HK-2.Methods:An HK-2cell model was used, in which NGAL mRNA and Caspase3mRNA were evaluated by qRT-PCR, and the correlation among them was analyzed as well. In vitro delivery of siRNA was performed to investigate whether NGAL could protect HK-2from LPS-induced injury by inhibiting cell apoptosis.Results:①The expression of NGAL mRNA was upregulated within6hours after LPS50uM treatment in HL-2(LPS1h and LPS3h groups, P<0.001; LPS6h group, P<0.01), and decreased to baseline after12hours (P>0.05). The expression of Caspase3mRNA was upregulated within3hours after LPS (LPS1h group, P<0.001; LPS3h group, P<0.05), and decreased to baseline after6hours (P>0.05). There was a strong correlation between Caspase3and NGAL mRNA (r=0.448, P<0.05).②The transfection of siRNA effectively interfered the expression of NGAL mRNA (P<0.001). The expression of NGAL mRNA decressed to35%level of Con group in siRNA group, and46%level in siRNA+LPS group. The expression of Caspase3mRNA was significantly upregulated in siRNA+LPS relative to Con, LPS and siRNA groups (P<0.001).③We have detected the necrosis and apotosis in HK-2after transfection by flow cytometry.There was1.08%necrosis and10.55%apoptosis in Con group. The necrosis was1.25%, apoptosis was13.71%in siRNA group, and there was no significant difference relative to Con. There were significant increase of necrosis (2.38%) and apoptosis(21.53%) in LPS relative to Con and siRNA groups (P<0.001) There were significant increase of necrosis (2.34%) in siRNA+LPS relative to Con and siRNA groups (P<0.001), but not to LPS group (P>0.05). However, the apoptosis (36.06%) of siRNA+LPS has significantly changes relative to Con, LPS and siRNA groups (P<0.001). There was a transient increase in TNFa and IL-6mRNA levels within3hours following LPS administration, and a strong correlation between TNFa and NGAL mRNA (r=0.995, P<0.001) but not with IL-6mRNA. Both pNGAL and uNGAL levels were markedly increased compared with those in the control group (P<0.001); however, only uNGAL levels were correlated with NGAL mRNA (r=0.850, P<0.001).④We detected the apoptosis in the four groups by TUNEL stain as well. There was seldom apoptosis in Con and siRNA groups, more apoptosis in LPS group and most apoptosis in siRNA+LPS.Conclusions:This study reveals that apoptosis is the main pathological reaction in LPS-treat HK-2. NGAL mRNA upregulation is sensitive to LPS treatment, and NGAL can inhibit the increase of Caspase3mRNA. So, NGAL can protect HK-2from apoptosis which is induced by LPS.From the existing research results, we drew the following conclusions through the experimental observation in LPS-induced septic AKI in rats and LPS-treated HK-2cells.①LPS can induce septic AKI in rats, and the main pathological histology is showed in epithelia including edema, microvilli disarray and apoptosis.②NGAL mRNA upregulation is sensitive to LPS-induced renal TNFa increase and injury, which are observed in the tubular epithelia; Urinary NGAL levels accurately reflect changes in NGAL in the kidney, so it is an ideal biomarker of septic AKI; Plasma NGAL is not yet perfected because it is not correlated with NGAL mRNA in renal. The results can provide a new theory basis of better application for NGAL in septic AKI diagnosis.③Apoptosis is the main pathological reaction in LPS-treat HK-2. NGAL mRNA upregulation is sensitive to LPS treatment, and NGAL can inhibit the increase of Caspase3mRNA. So, NGAL can protect HK-2from apoptosis which is induced by LPS. This study results provide new ideas and targets for improving diagnosis and treatment of septic AKI.