Objective To explore the mechanism of protecting cells from hypoxia/ reoxygenation (H/R) injury by constructing specific small interference RNA (siRNA) to inhibit NALP3 expression in rat renal tubular epithelial cells (NRK-52E).Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition,the cells were cultured with hypoxia for 1 h and then with reoxygenation for 1 h,2 h,4 h,8 h,16 h and 24 h.The activity of lactae dehydrogenase (LDH) in the culture medium,cell count and cell viability,the expression of NALP3 were determined by biochemical method,trypan blue exclusion and Western blotting.(2) The siRNA was transfected into NRK-52E.The irrespective siRNA transfected group wasused as control.NALP3 expression was examined by Western blotting.(3) The cells were divided into 4 groups:control group,H/R group,irrespective siRNA transfected group and NALP3-siRNA transfected group.To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition,the cells were cultured with hypoxia for 1 h and then with reoxygenation for 4 h.And the expression of NALP3 was determined by Western blotting.(4)Cellular apoptosis was examined by Annexin V/PI staining and flow cytometry.NF-κB DNA binding activity,IκB-α,Bcl-2 and Bax expression were examined by EMSA and Western blotting.Results (1)Compared with the control group,the activity of LDH significantly increased,cell count and cell viability significantly decreased (all P＜0.05).The expression of NALP3 significantly increased and peaked at 4 h after H/R.(2)The specific siRNA could efficiently inhibit NALP3 expression in NRK-52E.Compared with the irrespective siRNA transfected group,the protein expression of NALP3 was significantly down-regulated in NALP3 siRNA transfected group (P＜0.05).(3)After hypoxia 1 h and reoxygenation 4 h,the activity of LDH and the expression of NALP3 increased.Compared with the irrespective siRNA transfected group,LDH concentration in media and the expression of NALP3 significantly decreased in NALP3-siRNA transfected group.(4)After hypoxia 1 h and reoxygenation 4 h,NF-κB DNA binding activity was increased,IκB-α phosphorylation and degradation,Bcl-2 and Bax were significantly up-regulated.However,compared with the irrespective siRNA transfected group,NF -κB DNA binding activity,IκB-α degradation and Bax/Bcl-2 were significantly decreased (P＜0.05) in NALP3-siRNA transfected group.At the same time,the ratio of apoptosis was significantly increased in three groups than that in control.Compared with the irrespective siRNA transfected group,the ratio of apoptosis in NALP3-siRNA transfected group was significantly decreased (P＜0.05).Conclusions H/R induces the expression of NALP3 in NRK-52E.The synthesized siRNA can inhibit the expression of NALP3 and protect NRK-52E from hypoxia/reoxygenation injury.The mechanism may be via inhibiting the activation of NF-κB,modulating expression of Bcl-2 and Bax,as well as decreasing cell apoptosis.

Acute kidney injury (AKI), associated with significant morbidity and mortality, is widely known to involve epithelial apoptosis, excessive inflammation, and fibrosis in response to ischemia or reperfusion injury, which results in either chronic pathological changes or death. Therefore, it is imperative that investigations are conducted in order to find effective, early diagnoses, and therapeutic targets needed to help prevent and treat AKI. However, the mechanisms modulating the pathogenesis of AKI still remain largely undetermined. MicroRNAs (miRNAs), small non-coding RNA molecules, play an important role in several fundamental biological and pathological processes by a post transcriptional regulatory function of gene expression. MicroRNA-21 (miR-21) is a recently identified, typical miRNA that is functional as a regulator known to be involved in apoptosis as well as inflammatory and fibrotic signaling pathways in AKI. As a result, miR-21 is now considered a novel biomarker when diagnosing and treating AKI. This article reviews the correlative literature and research progress regarding the roles of miR-21 in AKI.
Acute Kidney Injury ; diagnosis ; drug therapy ; genetics ; pathology ; Animals ; Apoptosis ; Biomarkers ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; Molecular Targeted Therapy