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Delayed Administration of a Single Dose of Lithium Promotes Recovery from AKI

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Delayed Administration of a Single Dose of Lithium Promotes Recovery from AKI BASIC RESEARCH www.jasn.org Delayed Administration of a Single Dose of Lithium Promotes Recovery from AKI † † † † † Hui Bao,* Yan Ge, Zhen Wang,* Shougang Zhuang, Lance Dworkin, Ai Peng,* and † Rujun Gong *Department of Nephrology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China; and †Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island ABSTRACT Evidence suggests that glycogen synthase kinase 3b (GSK3b) contributes to AKI; however, its role in post- AKI kidney repair remains uncertain. Here, delayed treatment with a single dose of lithium, a selective inhibitor of GSK3b and a US Food and Drug Administration–approved mood stabilizer, accelerated re- covery of renal function, promoted repopulation of renal tubular epithelia, and improved kidney repair in murine models of cisplatin- and ischemia/reperfusion-induced AKI. These effects associated with reduced GSK3b activity and elevated expression of proproliferative molecules, including cyclin D1, c-Myc, and hypoxia-inducible factor 1a (HIF-1a), in renal tubular epithelia. In cultured renal tubular cells, cisplatin exposure led to transient repression of GSK3b activity followed by a prolonged upregulation of activity. Rescue treatment with lithium inhibited GSK3b activity, enhanced nuclear expression of cyclin D1, c-Myc, and HIF-1a, and boosted cellular proliferation. Similarly, ectopic expression of a kinase-dead mutant of GSK3b enhanced the expression of cyclin D1, c-Myc, and HIF-1a and amplified cellular proliferation after cisplatin injury, whereas forced expression of a constitutively active mutant of GSK3b abrogated the effects of lithium. Mechanistically, GSK3b colocalized and physically interacted with cyclin D1, c-Myc, and HIF-1a in tubular cells. In silico analysis revealed that cyclin D1, c-Myc, and HIF-1a harbor putative GSK3b consensus phosphorylation motifs, implying GSK3b-directed phosphorylation and subsequent degradation of these molecules. Notably, cotreatment with lithium enhanced the proapoptotic effects of cisplatin in cultured colon cancer cells. Collectively, our findings suggest that pharmacologic targeting of GSK3b by lithium may be a novel therapeutic strategy to improve renal salvage after AKI. J Am Soc Nephrol 25: 488–500, 2014. doi: 10.1681/ASN.2013040350 The current clinical management of AKI is largely epithelial cells seems to be mainly responsible for limited to symptomatic treatments and general nephron repair and renal recovery.3,4 Impaired kid- supportive measures, including fluid resuscitation ney repair or incomplete renal recovery after AKI and renal replacement therapy.1 Specific therapeu- has been recognized as an independent risk factor tic interventions that either rescue kidney injury or for AKI to CKD transition, which has lately been improve survival are still unavailable in clinical considered to be an important and underestimated practice. After acute injury, the kidney has a limited cause of CKD.5 potential of self-repair as marked by a spontaneous recovery of kidney function and the repopulated Received April 5, 2013. Accepted August 3, 2013. renal tubular epithelia on renal histology in patients Published online ahead of print. Publication date available at and experimental animal models. A burgeoning www.jasn.org. body of evidence suggests that direct engraftment Correspondence: Dr. Rujun Gong, Division of Kidney Disease of hematopoietic mesenchymal stem cells is less and Hypertension, Department of Medicine, Rhode Island Hos- likely to be a major mechanism involved in tubular pital, Brown University School of Medicine, 593 Eddy Street, cell regeneration during kidney repair.2 Instead, Providence, RI 02903. Email: [email protected] self-duplication of nonlethally injured, surviving Copyright © 2014 by the American Society of Nephrology 488 ISSN : 1046-6673/2503-488 J Am Soc Nephrol 25: 488–500, 2014 www.jasn.org BASIC RESEARCH The development, progression, and recovery of AKI is a 5 and 7, respectively; treatment with a higher dose of lithium complex and highly orchestrated pathophysiologic process (80 mg/kg) yielded a further reduction of serum creatinine levels that is regulated by a myriad of signaling pathways. Of many of by 46% and 66% on days 5 and 7, respectively, signifying an these pathways, glycogen synthase kinase 3b (GSK3b)has accelerated renal recovery from AKI. The lithium-promoted re- emerged as the integration point and plays a crucial role in covery in renal function was accompanied by enhanced kidney the pathogenesis of AKI. GSK3b is a well conserved, ubiqui- repair, as demonstrated by fewer renal tubular injury lesions and tously expressed serine/threonine protein kinase originally augmented repopulation of tubular epithelia on histology (Fig- characterized as one that regulates glucose metabolism.6 In- ure 1C). Semiquantitative morphometric analysis indicated a terest in GSK3b expanded greatly with the realization that it markedly accelerated regression of tubular injury scores on day is a key regulator of multiple pivotal pathophysiologic processes 7 after lithium treatment (Figure 1D). Collectively, these data extending well beyond glycogen metabolism to inflammation, suggest that lithium possesses a potent proreparative effect that immunomodulation, embryo development, tissue injury, repair, promotes renal function recovery and kidney repair after AKI. and regeneration.7 More recently, a growing body of evidence suggests that GSK3b plays a detrimental role in AKI.8,9 Never- Lithium Treatment after Cisplatin Injury Inhibits GSK3b theless, the role of GSK3b in renal recovery and kidney repair Activity and Reinforces Tubular Proliferation and after AKI remains elusive. Repair in the Kidney Lithium, a US Food and Drug Administration (FDA)–approved In most organ systems, a number of proproliferative molecules first-line drug commonly used for the past 50 years to treat bi- that are active in embryo development are re-expressed after polar affective disorders,10 is a selective inhibitor for GSK3b.11 acute injury, resulting in reinforced cell cycle progression. Lithium has been well known to have a potent promotional effect These proproliferative molecules include cell cycle regulators on tissue repair and regeneration after injury in multiple organ like cyclin D1 and transducers of mitogenic signals like c-Myc, systems, including the central nervous system and hematopoietic which are proproliferative molecules that govern cellular system,12–15 whereas the effect of lithium on kidney repair and mitosis and proliferation. Furthermore, renal recovery from renal recovery after AKI is unknown. This study examined the AKI is inevitably confounded by ischemia or hypoxia that is effect of delayed administration of a single low dose of lithium secondary to endothelial injury and vascular constriction even on a murine model of cisplatin or ischemia/reperfusion-induced if the original etiology is nonischemic.17–19 Recent evidence AKI. The potential role and implication of GSK3b,thetargetof suggests that hypoxic preconditioning of cisplatin-injured lithium, in kidney repair was delineated. proximal tubular cells increases cell proliferation and that this effect is largely mediated by the induced activation of hypoxia-inducible factor-1a (HIF-1a), which controls sur- RESULTS vival and proliferation of renal epithelial cells.20 To determine whether lithium inhibited GSK3b activity in vivo in the kidney Rescue Treatment with Lithium Accelerates Renal and to decipher whether cell cycle progression and tubular Recovery in a Murine Model of Cisplatin-Induced AKI proliferation was responsible for the lithium-accelerated renal Cisplatin-based chemotherapy for cancer has been greatly recovery after AKI, kidney homogenates were subjected to limited by multiple adverse effects, including AKI.16 Asingle immunoblot analysis followed by densitometric analysis (Fig- intraperitoneal injection of cisplatin (20 mg/kg) in mice eli- ure 2A). After cisplatin injury, inhibitory phosphorylation of cited severe injury to the kidney. By day 3, cisplatin elicited a GSK3b waselevatedondays5and7duringtherecovery typical pattern of acute tubular necrosis, characterized by ep- phase, suggesting that GSK3b inactivation is involved in kid- ithelial simplification, vacuolization of proximal tubular epi- ney repair. This was associated with mild induction of the thelium, luminal ectasia, epithelial necrosis, sloughing of expression of cyclin D1, c-Myc, and HIF-1a (Figure 2A). De- tubular cell into lumen, and loss of brush border (Figure layed lithium treatment accentuated the inhibitory phosphor- 1A),whereashistologyofkidneysfrommicetreatedalone ylation of GSK3b in a dose-dependent manner, concomitant with saline or lithium chloride (80 mg/kg) remained normal. with an amplified expression of cyclin D1, c-Myc, and HIF-1a Consistent with the morphologic changes, cisplatin injury re- (Figure 2A). To validate the immunoblot data, fluorescence sulted in a remarkable elevation in serum creatinine levels that immunohistochemistry staining was carried out on frozen peaked on postinjury day 3 (Figure 1B), congruent with the kidney specimens that were obtained on postinjury day 7. As injury phase of AKI. After day 3, serum creatinine levels in shown in Figure 2B, cisplatin injury elicited very weak and cisplatin-injured mice started to regress gradually but were sporadic expression of cyclin D1, c-Myc, and HIF-1a mainly still
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