l-NIL prevents the ischemia and reperfusion injury involving TLR-4, GST, clusterin, and NFAT-5 in mice

Abstract

On renal ischemia-reperfusion (I/R) injury, recruitment of neutrophils during the inflammatory process promotes local generation of oxygen and nitrogen reactive species, which, in turn, are likely to exacerbate tissue damage. The mechanism by which inducible nitric oxide synthase (iNOS) is involved in I/R has not been elucidated. In this work, the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine (l-NIL) and the NOS substrate l-arginine were employed to understand the role of NOS activity on the expression of particular target genes and the oxidative stress elicited after a 30-min of bilateral renal ischemia, followed by 48-h reperfusion in Balb/c mice. The main findings of the present study were that pharmacological inhibition of iNOS with l-NIL during an I/R challenge of mice kidney decreased renal injury, prevented tissue loss of integrity, and improved renal function. Several novel findings regarding the molecular mechanism by which iNOS inhibition led to these protective effects are as follows: 1) a prevention of the I/R-related increase in expression of Toll-like receptor 4 (TLR-4), and its downstream target, IL-1β; 2) reduced oxidative stress following the I/R challenge; noteworthy, this study shows the first evidence of glutathione S-transferase (GST) inactivation following kidney I/R, a phenomenon fully prevented by iNOS inhibition; 3) increased expression of clusterin, a survival autophagy component; and 4) increased expression of nuclear factor of activated T cells 5 (NFAT-5) and its target gene aquaporin-1. In conclusion, prevention of renal damage following I/R by the pharmacological inhibition of iNOS with l-NIL was associated with the inactivation of proinflammatory pathway triggered by TLR-4, oxidative stress, renoprotection (autophagy inactivation), and NFAT-5 signaling pathway.

Glutathione S-Transferase and Clusterin, New Players in the Ischemic Preconditioning Renal Protection in a Murine Model of Ischemia and Reperfusion

“…The acute kidney injury is observed after 48 hours of reperfusion. According to our previous results using the murine model of IRI in BALB/c mice [49], the lower GFR values and higher values of injury and acute inflammation biomarkers are present 48 hours after reperfusion. The sham animals were subjected to the same surgery process, but they did not undergo renal pedicle occlusion [50].…”

“…We used a validated model of renal ischemia and reperfusion injury using the BALB/c mice that were also used in our previous study [49]. Adult mice (20-25g) were housed in a 12h light/ dark cycle.…”

“…The kidneys were fixed in 10% buffered formalin, embedded in paraffin, sectioned, dewaxed, rehydrated, rinsed in water, and stained with hematoxylin-eosin (HE) and periodic acid Schiff (PAS). The morphologic analysis was carried out in a blinded manner as detailed previously by Dr. Luis Contreras, pathologist [49]. The cortex and medulla were evaluated for epithelial necrosis, loss of brush border, tubular dilation, and tubular congestion, among other kidney alterations observed in AKI response.…”

Intravenous Arginine Administration Downregulates NLRP3 Inflammasome Activity and Attenuates Acute Kidney Injury in Mice with Polymicrobial Sepsis

“…To investigate the role of NO, L-N (6)-iminoethyl-lysine (L-NIL) (Sigma, St. Louis, MO, USA), an inducible (i) NOS inhibitor, was administered to mice in the septic saline (SSL, n = 15) and septic Arg (SAL, n = 15) groups. L-NIL (3 mg/kg BW) was given intraperitoneally at the end of CLP and at 6 h after sepsis induction [20]. Mice in the SSL and SAL groups were sacrificed at 6, 12, and 24 h to collect blood and kidney samples.…”

Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells

“…Another study provided evidence that NFAT5 is essential for the expression of iNOS in TLR-stimulated macrophages (4,15). Recently, we have described in a mouse model of renal ischemia and reperfusion that pharmacological inhibition of iNOS [N 6 -(1iminoethyl)lysine, hydrochloride] increased the NFAT5 signaling pathway in the kidney (32). Although this evidence points toward functional reciprocity between these two signaling pathways, the interaction of NFAT5 and iNOS signaling in the setting of hypoxia remains completely controversial.…”