4-benzyl-2-methyl-1-2-4-thiadiazolidine-3-5-dione and Acute-Kidney-Injury

Topics: Acute Kidney Injury; Animals; Aquaporin 1; Autophagy; Cells, Cultured; Enzyme Inhibitors; Glycogen Synthase Kinase 3 beta; Male; Mice; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiadiazoles

Clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac (DCLF) is limited by multiple adverse effects, including renal toxicity leading to acute kidney injury. In mice with DCLF-induced nephrotoxicity, TDZD-8, a selective glycogen synthase kinase (GSK)3β inhibitor, improved acute kidney dysfunction and ameliorated tubular necrosis and apoptosis associated with induced cortical expression of cyclooxygenase-2 (COX-2) and prostaglandin E2. This renoprotective effect was blunted but still largely preserved in COX-2-null mice, suggesting that other GSK3β targets beyond COX-2 functioned in renal protection. Indeed, TDZD-8 diminished the mitochondrial permeability transition in DCLF-injured kidneys. In vitro, GSK3β inhibition reinstated viability and suppressed necrosis and apoptosis in DCLF-stimulated tubular epithelial cells. DCLF elicited oxidative stress, enhanced the activity of the redox-sensitive GSK3β, and promoted a mitochondrial permeability transition by interacting with cyclophilin D, a key component of the mitochondrial permeability transition pore. TDZD-8 blocked GSK3β activity and prevented GSK3β-mediated cyclophilin D phosphorylation and the ensuing mitochondrial permeability transition, concomitant with normalization of intracellular ATP. Conversely, ectopic expression of a constitutively active GSK3β abolished the effects of TDZD-8. Hence, inhibition of GSK3β ameliorates NSAID-induced acute kidney injury by induction of renal cortical COX-2 and direct inhibition of the mitochondrial permeability transition.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Survival; Cyclooxygenase 2; Cyclophilins; Diclofenac; Epithelial Cells; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Kidney Tubular Necrosis, Acute; Kidney Tubules; Mice; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Knockout; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Oxidation-Reduction; Peptidyl-Prolyl Isomerase F; Phosphorylation; Protein Kinase Inhibitors; Thiadiazoles

Renal proximal tubular damage and repair are hallmarks of acute kidney injury. As glycogen synthase kinase-3β (GSK3β) is an important cellular regulator of survival and proliferation, we determined its role during injury and recovery of proximal tubules in a mercuric chloride-induced nephrotoxic model of acute kidney injury. Renal proximal tubule-specific GSK3β knockout mice exposed to mercuric chloride had improved survival and renal function compared to wild-type mice. Apoptosis, measured by TUNEL staining, Bax activation, and caspase 3 cleavage, was reduced in the knockout mice. The restoration of renal structure, function, and cell proliferation was also accelerated in the GSK3β knockout mice. This enhanced repair, evidenced by increased Ki-67 and BRDU staining, along with increased cyclin D1 and c-myc levels, was recapitulated by treatment of wild-type mice with the small-molecule GSK3 inhibitor TDZD-8 following injury. This confirmed that hastened repair in the knockout mice was not merely due to lower initial injury levels. Thus, inhibition of GSK3β prior to nephrotoxic insult protects from renal injury. Such treatment after acute kidney injury may accelerate repair and regeneration.

Topics: Acute Disease; Acute Kidney Injury; Animals; Anti-Infective Agents, Local; Apoptosis; Cell Proliferation; Disease Models, Animal; Female; Gene Deletion; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Kaplan-Meier Estimate; Kidney Tubules, Proximal; Mercuric Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; Thiadiazoles

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Disease Models, Animal; Epithelial Cells; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kidney Tubules; Mice; NF-kappa B; Rats; Thiadiazoles

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3beta (GSK3beta) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3beta promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3beta, Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3beta mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference-mediated knockdown of GSK3beta promoted cell survival. Furthermore, RNA interference-mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3beta. In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine(163) site targeted by stress-activated GSK3beta. In rats, TDZD-8 inhibited ischemia-induced activation of GSK3beta, Bax, and caspase 3; ameliorated tubular and epithelial cell damage; and significantly protected renal function. Taken together, GSK3beta-mediated Bax activation induces apoptosis and tubular damage that contribute to acute ischemic kidney injury.

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line; Epithelial Cells; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Kidney Tubules, Proximal; Male; Mice; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA Interference; Thiadiazoles

Excessive inflammation and apoptosis are pathological features of endotoxaemic acute renal failure. Activation of glycogen synthase kinase-3 (GSK-3) is involved in inflammation and apoptosis. We investigated the effects of inhibiting GSK-3 on lipopolysaccharide (LPS)-induced acute renal failure, nuclear factor-kappaB (NF-kappaB), inflammation and apoptosis.. The effects of inhibiting GSK-3 with inhibitors, including lithium chloride (LiCl) and 6-bromo-indirubin-3'-oxime (BIO), on LPS-treated (15 mg x kg(-1)) C3H/HeN mice (LiCl, 40 mg x kg(-1) and BIO, 2 mg x kg(-1)) and LPS-treated (1 microg x mL(-1)) renal epithelial cells (LiCl, 20 mM and BIO, 5 microM) were studied. Mouse survival was monitored and renal function was analysed by histological and serological examination. Cytokine and chemokine production, and cell apoptosis were measured by enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling staining, respectively. Activation of NF-kappaB and GSK-3 was determined by immunostaining and Western blotting, respectively.. Mice treated with GSK-3 inhibitors showed decreased mortality, renal tubular dilatation, vacuolization and sloughing, blood urea nitrogen, creatinine and renal cell apoptosis in response to endotoxaemia. Inhibiting GSK-3 reduced LPS-induced tumour necrosis factor-alpha (TNF-alpha) and CCL5/RANTES (released upon activation of normal T-cells) in vivo in mice and in vitro in murine kidney cortical collecting duct epithelial M1 cells. Inhibiting GSK-3 did not block TNF-alpha-induced cytotoxicity in rat kidney proximal tubular epithelial NRK52E or in M1 cells.. These results suggest that GSK-3 inhibition protects against endotoxaemic acute renal failure mainly by down-regulating pro-inflammatory TNF-alpha and RANTES.

Topics: Acute Kidney Injury; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cells, Cultured; Down-Regulation; Endotoxins; Glycogen Synthase Kinase 3; Inflammation Mediators; Lipopolysaccharides; Male; Mice; Mice, Inbred C3H; Protein Kinase Inhibitors; Rats; Thiadiazoles