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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

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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 significantly higher than those observed in the saline-trea- in the injured tubules. Low-dose lithium treatment amplified ted group or the lithium-treated group until postinjury day 7, to a moderate extent, while higher-dose lithium treatment suggesting a spontaneous but incomplete renal recovery after amplified to a greater extent, the expression of cyclin D1, AKI (Figure 1B). Rescue treatment with a low dose of lithium c-Myc, and HIF-1a that was predominantly observed in the chloride (40 mg/kg) significantly reduced serum creatinine renal tubular epithelia. It was noted that the staining of cyclin levels in cisplatin-injured animals by 30% and 47% on days D1, c-Myc, and HIF-1a in renal tubular cells mainly colocalized

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Figure 1. Delayed lithium therapy accelerates kidney repair and renal recovery in a murine model of cisplatin-induced AKI. On day 0, mice receive cisplatin (20 mg/kg) or an equal volume of saline or LiCl (80 mg/kg) treatment via a single intraperitoneal injection. On day 3, cisplatin-injured mice are treated with saline, low-dose (L) LiCl (40 mg/kg), or high-dose (H) LiCl (80 mg/kg). (A) Representative micrographs of hematoxylin and eosin–stained renal cortex of mice treated with saline (a), lithium (b), or cisplatin (c) on day 3. (B) Serum creatinine levels decrease at a more rapid rate in cisplatin + LiCl–treated mice compared with cisplatin + saline–treated mice (n=5 per group). (C) Representative micrographs of hematoxylin and eosin–stained renal cortex of cisplatin + saline–treated mice (d and g), cisplatin + LiCl (L)–treated mice (e and h), and cisplatin + LiCl (H)–treated mice (f and i) on days 5 and 7. (D) Tubular injury score for mice treated with saline, LiCl (H), cisplatin + saline, cisplatin + LiCl (L), or cisplatin + LiCl (H) on day 7 (n=5 per group). *P,0.05; **P,0.001 versus cisplatin + saline-treated mice. ns, not significant; Scr, serum creatinine. Original magnification, 3200; 3400 in insets. with 49,6-diamidino-2-phenylindole (DAPI) staining and ap- bilateral kidney ischemia reperfusion injury (IRI) was utilized. peared in nuclei in addition to cytoplasm (Figure 2B). During the Eight hours after IRI, mice were given 40 mg/kg or 80 mg/kg of recovery phase of AKI, even though kidney repair is the domi- lithium chloride or saline via intraperitoneal injection. Renal nant histologic event, kidney injury characterized by tubular cell histology was examined 24 and 48 hours after ischemia and death, especially apoptosis, also occurs continuously for several exhibited a typical pattern of IRI-induced AKI shown by tu- days after injury. Indeed, apoptotic cells were evidently detected bular dilation, swelling, necrosis, sloughing of tubular cells, in cisplatin-injured kidneys even on postinjury day 5 and were loss of brush border, luminal congestion, and interstitial substantially diminished by lithium in a dose-dependent fashion hemorrhage. These changes were prominently attenuated (Figure 2, C and D), as revealed by the terminal deoxynucleotidyl in animals treated with low- or high-dose lithium (Figure transferase–mediated digoxigenin-deoxyuridine nick-end label- 3A). Serum creatinine levels were consistently precipitously ing (TUNEL) assay, suggesting that delayed lithium therapy im- elevated 24 and 48 hours after renal IRI but were sig- proves the late phase kidney injury. nificantly corrected by delayed lithium treatment in a dose- dependent fashion, denoting a promoted renal recovery Lithium Rescue Therapy Accelerates Renal Recovery (Figure 3A). In parallel with renal function and histologic in a Murine Model of Renal Ischemia Reperfusion Injury findings, mild expression of cyclin D1, c-Myc, and HIF-1a To determine whether this proreparative activity of lithium is was induced in the kidney 24 and 48 hours after IRI; this was also effective in other forms of AKI, another murine model of substantially enhanced after rescue treatment with lithium

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Figure 2. Lithium rescue treatment promotes renal tubular expression of proproliferative molecules in the recovery phase of cisplatin- induced renal injury. (A) Western immunoblot analysis and densitometric analysis demonstrate higher expression of cyclin D1, c-Myc, and HIF-1a in renal cortical homogenates from cisplatin + LiCl–treated mice (n=5 per group). (B) Immunofluorescence staining shows enhanced expression of cyclin D1, c-Myc, and HIF-1a by proximal tubules in kidney specimens from lithium-treated mice on day 7 after cisplatin injury. Note that the staining of cyclin D1, c-Myc, and HIF-1a in renal tubular cells mainly colocalizes with DAPI staining and appears in nuclei in addition to cytoplasm. (C) GSK3b inhibition protects mice from renal cell apoptosis in cisplatin-injured kidneys. Representative micrographs of TUNEL staining of kidney specimens on day 5. TUNEL-labeled nuclei are revealed as bright green spots in the renal cortex in kidney specimens counterstained with PI. Lithium significantly attenuates apoptosis in cisplatin + LiCl–treated mouse kidneys. (D) Absolute counting of the numbers of TUNEL-positive cells as the means of 20 random high-power fields. *P,0.05; **P,0.001 versus cisplatin + saline–treated mice (n=5 per group). ns, not significant. Original magnification, 3200 in A; 3400inB.

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Figure 3. Delayed lithium therapy accelerates kidney repair and renal recovery in mice with renal IRI. Mice are subjected to 22 minutes of bilateral renal ischemia and then allowed to recover for 24 or 48 hours. Lithium or saline is given 8 hours after ischemia. (A) Representative micrographs of hematoxylin and eosin–stained renal cortex. (B) Serum creatinine levels are drastically elevated 24 and 48 hours after renal IRI but are significantly corrected by delayed lithium treatment in a dose-dependent fashion (n=5 per group). (C) Western immunoblot analysis of kidney homogenates demonstrates enhanced expression of cyclin D1, c-Myc, and HIF-1a in kidneys with IRI after lithium treatment (n=5 per group). *P,0.05; **P,0.001 versus IRI + saline–treated group. ns, not significant; Scr, serum creatinine. Original magnification, 3200. as revealed by immunoblot analysis of kidney homogenates least 24 hours, denoting upregulated kinase activity of GSK3b. (Figure 3C). Lithium treatment initiated 12 hours after cisplatin injury markedly enhanced cell proliferation in a dose-dependent fash- Delayed Lithium Treatment Inhibits GSK3b and ion at both 24 hours (Figure 4B) and 48 hours (Figure 4G) as Promotes Proliferation in Cultured Renal Tubular Cells assessed by the diphenyltetrazolium bromide (MTT) cell pro- Injured with Cisplatin liferation assay. This was concomitant with dose-dependent To determine whether lithium promotes renal recovery via a suppression of GSK3b activity and induction of the expression potential proreparative effect directly on renal tubular cells, of cyclin D1, c-Myc, and HIF-1a (Figure 4, C and H). Den- cultured murine tubular epithelial (TKPT) cells were exposed sitometric analysis confirmed that the lithium-induced inhib- to cisplatin and then treated with lithium. As shown in Figure itory phosphorylation of GSK3b positively correlated with 4A, despite a transient induction of inhibitory phosphoryla- the expression of cyclin D1, c-Myc, and HIF-1a, respectively tion of GSK3b, cisplatin injury alone resulted in a prolonged (Figure 4, D–FandI–K), implying that GSK3b inhibition is repression of the inhibitory phosphorylation of GSK3b for at associated with enhanced expression of proproliferative

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Figure 4. Inhibition of GSK3b by lithium after cisplatin injury promotes proliferation of cultured renal tubular cells, associated with amplified expression of cyclin D1, c-myc, and HIF-1a. (A) Western immunoblot analysis for phosphorylated GSK3b in renal tubular (TKPT) cells at different time after cisplatin (100 mM) injured; immunoblots from three separate experiments are analyzed by densi- tometry. (B) MTT assay of TKPT cells injured with cisplatin for 12 hours and then treated with NaCl (10 mM) or different doses of LiCl (1, 5, and 10 mM) for 24 hours (n=6). (C) Western immunoblot analysis for cyclin D1, c-Myc, and HIF-1a in TKPT cells that are subjected to different treatments as described in B. Immunoblots from three separate experiments are subjected to densitometric analysis. (D–F) Densitometric analysis of immunoblot results demonstrates that lithium-induced inhibitory pGSK3b markedly correlates with increased expression of cyclin D1, c-Myc, and HIF-1a at 24 hours (P,0.05). (G) MTT assay of TKPT cells injured with cisplatin for 12 hours and then treated with NaCl (10 mM) or different doses of LiCl (1, 5, and 10 mM) for 48 hours (n=6). (H) Western immunoblot analysis for cyclin D1, c-Myc, and HIF-1a in TKPT cells that are subjected to different treatments as described in G. Immunoblots from three separate ex- periments were subjected to densitometric analysis. (I–K) Densitometric analysis of immunoblot results demonstrates that lithium-in- duced inhibitory pGSK3b markedly correlates with increased expression of cyclin D1, c-Myc, and HIF-1a at 48 hours (P,0.05). *P,0.05 versus cisplatin + NaCl–treated group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant.

J Am Soc Nephrol 25: 488–500, 2014 Delayed Lithium Therapy Promotes Recovery after AKI 493 BASIC RESEARCH www.jasn.org molecules that drives cell cycle progression and cellular pro- Cisplatin injury drastically diminished the nuclear expression of liferation. cyclin D1 but barely affected the basal weak expression of c-Myc and HIF-1a. Delayed lithium treatment reinstated the nuclear Lithium Induces Nuclear Accumulation of expression of cyclin D1 and considerably induced nuclear accu- Proproliferative Molecules in Cisplatin-Injured Renal mulation of c-Myc and HIF-1a in a dose-dependent manner. Tubular Cells Absolute counting of the nuclei positive for these proproliferative Fluorescence immunocytochemistry was performed to define the molecules corroborated the morphologic observation (Figure 5, subcellular localization of lithium-induced expression of propro- B–D). liferative molecules, including cyclin D1, c-Myc, and HIF-1a, (Figure 5A). Cells were stimulated with or without cisplatin for GSK3b Inhibition Is Necessary and Sufficient for 12 hours and then treated with lithium at different concentrations Lithium-Promoted Cellular Proliferation and Expression for 24 hours. In exponentially growing renal tubular epithelial of Proproliferative Molecules cells under basal conditions, cyclin D1 was evidently expressed Toexamine the possible causal relationship between lithium- and mainly localized in the nuclei, whereas c-Myc and HIF-1a induced inhibition of GSK3b and lithium-promoted cell were faintly and sporadically expressed in the nuclei of few cells. proliferation after cisplatin injury, the activity of GSK3b

Figure 5. Lithium induces nuclear accumulation of proproliferative molecules in cisplatin-injured renal tubular cells. TKPT cells are treated with cisplatin (100 mM) for 12 hours and then treated with NaCl (10 mM) or different doses of LiCl (1, 5, and 10 mM) for 24 hours. (A) Immunofluorescence staining for cyclin D1, c-Myc, and HIF-1a shows more nuclei positive cells in LiCl-treated TKPT cells after cisplatin injury. (B–D) Absolute counting of cells that are nuclear positive for cyclin D1, c-Myc, or HIF-1a as the means of 20 random high-power fields. *P,0.05; **P,0.001 versus cisplatin + NaCl–treated cells. Original magnification, 3200.

494 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 488–500, 2014 www.jasn.org BASIC RESEARCH was selectively manipulated by forced expression of vectors Lithium Suppresses Growth and Promotes Apoptosis encoding the hemagglutinin (HA)–conjugated wild-type of Colon Cancer Cells (WT), kinase-dead mutant (KD), or constitutively active mu- To assess whether lithium treatment affects cancer cell growth tant (S9A) of GSK3b. Cells were transfected with these vectors or the tumor killing efficacy of cisplatin, cultured SW480 human after cisplatin injury for 12 hours. Immunofluorescence colon cancer cells were injured with cisplatin in the presence staining for HA revealed a satisfactory transfection efficiency or absence of lithium. Under basal conditions, the addition of (.75%) (Figure 6C). Ectopic expression of KD after cisplatin lithium to the culture significantly suppressed colon cancer cell injury substantially boosted cell proliferation as measured by growth by .20% after 24 or 48 hours, as assessed by the MTT the MTT assay, reminiscent of the effect of lithium in WT cell growth assay (Figure 7A). Colon cancer cell growth was expressing cells, inferring that GSK3b inhibition is sufficient much more prominently suppressed by cisplatin. Concomitant for lithium-promoted cellular proliferation. In contrast, treatment with lithium did not blunt the effect of cisplatin, but forced expression of S9A prominently repressed cell prolifer- instead displayed a trend of potentiated anticancer activity, re- ation after cisplatin injury and abrogated the promotional sulting in a further slight suppression of cell growth (Figure 7A). effect of lithium, suggesting that GSK3b inhibition is required Mechanistically, lithium elicited the activation of proapoptotic for lithium-enhanced tubular cell proliferation (Figure 6A). caspase-3 in colon cancer cells under basal conditions and Consistent with the findings in MTT cell proliferation assays, slightly promoted its activation in cisplatin-injured cells, as de- the expressions of the proproliferative molecules, including termined by immunoblot analysis (Figure 7B) as well as TUNEL cyclin D1, c-Myc and HIF-1a, were all markedly upregulated assays (Figure 7, C and D). in cells expressing KD, but were evidently abolished in S9A expressing cells as determined by immunoblot analysis (Figure 6B), suggesting that GSK3b inhibition is necessary DISCUSSION and sufficient for lithium-enhanced expression of the propro- liferative molecules. Emerging evidence suggests that the number of patients with incomplete renal recovery from AKI is underestimated and Proproliferative Molecules Colocalize and Physically these individuals are at increased risk for CKD and ESRD.1,24 Interact with GSK3b as its Putative Substrates in Renal Renal repair after AKI is influenced by multiple pathobiologic Tubular Cells factors, including aging, preexisting kidney diseases, and the To understand the molecular essence of GSK3b-regulated nature and the degree of the original injury per se.25,26 It is expression of the proproliferative molecules including cyclin imperative to develop novel therapeutic modalities to pro- D1, c-Myc, and HIF-1a, we examined the physical relation- mote kidney repair and maximize the potential of complete ship between GSK3b and these molecules. Fluorescence renal recovery. This study demonstrated for the first time that immunocytochemistry staining indicated that GSK3b and rescue therapy with lithium promotes kidney repair and accel- HIF-1a were expressed in both cytoplasm and nucleus in erates renal recovery after cisplatin- or IRI-induced AKI in mice. the cultured tubular epithelial cells, whereas cyclin D1 and GSK3b is a key transducer involved in a large number of c-Myc mainly exhibited a nuclear distribution. Of note, a sig- cellular signaling pathways7 and has been implicated in the nificant amount of GSK3b staining perfectly colocalized with regulation of cell proliferation, at least partly through its con- each of the three proproliferative molecules and this colocal- trol of the stability of cell cycle proteins such as cyclin D1, ization was predominantly situated in nuclei (Figure 6D). To c-Myc, and HIF-1a.27–30 GSK3b is able to render phosphor- validate the cytologic findings, immunoprecipitation was per- ylation of cyclin D1 and c-Myc and initiate their ubiquitination formed and revealed that GSK3b evidently coprecipitated and proteasomal degradation. In NIH-3T3 cells, phosphory- with each of the three molecules, indicating that GSK3b lation of cyclin D1 by GSK3b led to cyclin D1 exclusion from physically interacts with cyclin D1, c-Myc, and HIF-1a,re- the nucleus to initiate its proteasomal degradation.27 Simi- spectively (Figure 6E). Evidence suggests that upon phos- larly, mitogen signaling inhibits the GSK3b-mediated degra- phorylation, cyclin D1, c-Myc, and HIF-1a will be subjected dation of c-Myc, resulting in the activation of its target genes, to nuclear exit and subsequent proteasomal degradation.21–23 including cyclin D1 and other cell cycle mediators.28,30 More- To further explore the mechanism by which GSK3b regulates over, Flügel et al.29 demonstrated that both insulin and lith- the expression of cyclin D1, c-Myc, and HIF-1a,theamino ium stabilized HIF-1a in hepatic cells through inhibitory acid sequences of the three molecules were subjected to com- phosphorylation of GSK3b. In our study, inhibition of putational active site analysis for putative GSK3b consen- GSK3b by lithium or forced expression of KD consistently sus motifs (Figure 6F). In silico analysis revealed that residues elevated the expression of these proproliferative molecules in T286 of cyclin D1, residue T58 of c-Myc, and residues T505, renal tubular cells. S524, and S598 of HIF-1a reside in consensus motifs GSK3b is also a core element of the canonical Wnt/b-cat- for phosphorylation by GSK3b, suggesting that cyclin D1, enin pathway. Recent evidence indicates that activation of c-Myc, and HIF-1a are putative cognate substrates for b-catenin signaling might be responsible for impaired wound GSK3b. healing and fibrotic kidney repair after AKI.31 For example,

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Figure 6. GSK3b inhibition is necessary and sufficient for lithium-promoted cellular proliferation. TKPT cells are injured with cisplatin (100 mM) for 6 hours, and cells are then subjected to liposome-mediated transient transfection with vectors encoding the HA-conju- gated WT, KD mutant, or constitutively active (S9A) mutant of GSK3b. Some cells are treated with LiCl (10 mM) 6 hours after the transfection. (A) An MTT assay is carried out 24 hours after cisplatin injury. Forced expression of KD enhances cellular proliferation, whereas S9A shows an opposing effect (n=6). (B) After cisplatin injury and transient transfection as described in A, cells are subjected to different treatments as indicated. Whole cell lysates are harvested and analyzed for cyclin D1, c-Myc, and HIF-1a by Western im- munoblot analysis, with three replicates. (C) Fluorescence immunocytochemistry staining of HA demonstrates that the transfection efficiency is .75%. (D) Representative immunofluorescence staining micrographs show colocalization of GSK3b with cyclin D1, c-Myc, or HIF-1a in TKPT cells. (E) Whole cell lysates are immunoprecipitated with anti-GSK3b antibody and immunoprecipitates are probed for cyclin D1, c-Myc, and HIF-1a by immunoblot analysis (n=3). (F) In silico analysis of the amino acid sequences of cyclin D1, c-Myc, and HIF-1a shows consensus motifs for phosphorylation by GSK3b.*P,0.05; **P,0.001 versus cisplatin-treated WT cells. EV, empty vector; ns, not significant. Original magnification, 3400. in a scratch wound healing study in cultured renal tubular isoforms of GSK3a and b is required to show an appreciable epithelial cells, inhibitors of GSK3b including lithium at change in b-catenin levels,33 suggesting that there may be a high concentrations mildly induced b-catenin accumulation therapeutic window and dose for chemical inhibitors of and thereby retarded wound healing.32 A drastic inhibition of GSK3b in treating diseases without elevating b-catenin lev- GSK3, however, seems to be essential to activate the b-catenin els33 and thus a risk of b-catenin–mediated adverse effects like pathway. In support of this, Doble et al. revealed a functional renal fibrosis. Regular or low doses of lithium seem to satisfy redundancy of GSK3a and GSK3b in Wnt/b-catenin signaling this concern. Indeed, kidney diseases, like glomerular or in- by using compound knockouts of GSK3a and b.33 They found terstitial nephropathy, are uncommon for psychiatric patients that gene deletion of at least three of the four alleles of both who received lithium therapy for a long period of time (usually

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Figure 7. Lithium suppresses cell growth and sensitizes apoptosis in colon cancer cells. SW480 colon cancer cells are treated with cisplatin (20 mM) in the presence or absence of lithium chloride (10 mM) or sodium chloride (10 mM) for 24 or 48 hours. (A) An MTT cell growth assay demonstrates that lithium suppresses growth of SW480 colon cancer cells and slightly potentiates the antitumor activity of cisplatin (n=6). (B) Western immunoblot analysis of cell lysates for cleaved (activated) caspase-3 in SW480 cells that are subjected to different treatments for 24 hours. (C) Representative micrographs of TUNEL staining of SW480 cells after different treatments for 24 hours. (D) Quantitative analysis of the percentages of TUNEL-positive cells after different treatments. *P,0.05 versus cisplatin + NaCl– treated group; **P,0.001 versus vehicle + NaCl–treated group.

.10 years).34 Of note, both doses of lithium used in our study Due to the powerful proreparative effect of GSK3b inhibi- (40 and 80 mg/kg) are much lower than the standard dose of tion exhibited in the kidney and other organs, one of the major lithium (120 mg/kg) that has been safely and routinely used concerns of clinical application of lithium for cisplatin-in- for neurobiology research in rodents.35 Consistent with pre- duced AKI is its possible effect on cancer cell growth and the vious work,36 renal expression of b-catenin in our animals was tumor killing efficacy of cisplatin, which thus requires height- barely affected (data not shown). Nevertheless, more clinical ened awareness of safety considerations. To date, GSK3b has studies are merited to determine the appropriate dose of lith- been found to play distinct roles in tumor development and ium for humans to inhibit GSK3b without elevating b-catenin growth in different types of tumors, likely depending on the levels in the kidney. status of GSK3b overexpression.41 For instance, GSK3b seems Lithium, a first-generation GSK3b inhibitor, has histori- to serve as a “tumor suppressor” in skin42 and mammary tu- cally been used safely for decades as a mood stabilizer.10 Lith- morigenesis.43 By contrast, GSK3b promotes tumorigenesis ium is well known for its proreparative activities in multiple and development of ovarian, colon, liver, and pancreatic car- systems, as well as its beneficial effect on organic neurologic cinomas.44–46 In support of this, inhibition of GSK3b activity degenerative diseases, including Alzheimer’sdisease.37 In by pharmacologic inhibitors suppressed proliferation of ovar- human patients with psychiatric disorders, lithium treatment ian cancer cells in vitro and prevented the formation of ovarian was found to induce an increase in brain gray matter.38 In xenograft tumors in nude mice.44 Similarly, inhibitors of addition, lithium is one of the treatment choices for GSK3b significantly inhibited cell growth and proliferation neutropenia39 and may also assist hematopoietic stem cell of subcutaneous xenografts of human colon cancer cells in mobilization in bone marrow transplant donors.40 Our study athymic mice.45 Our study consistently demonstrated that consistently demonstrated that rescue therapy with lithium lithium suppressed cell growth, induced apoptosis, and promotes kidney repair after AKI. Thus, lithium possesses a slightly potentiated the proapoptotic effect of cisplatin in co- proreparative activity that is exhibited in many organ systems lon cancer cells, inferring that lithium may actually favor the after acute or chronic injury. efficacy of cisplatin-based chemotherapy for certain types of

J Am Soc Nephrol 25: 488–500, 2014 Delayed Lithium Therapy Promotes Recovery after AKI 497 BASIC RESEARCH www.jasn.org cancers such as colon cancer. Moreover, in patients subjected lithium chloride (40 or 80 mg/kg) was administered to mice (single to systemic chemotherapy for malignant tumors, lithium intraperitoneal injection). Five mice were randomized to each of the carbonate has been safely used to accelerate hematopoietic following groups for each observed time point: (1) sham + saline, recovery and shorten the period of leukopenia,12,47,48 again mice with sham injury were treated with saline; (2) sham + LiCl, mice suggesting the safety of lithium therapy in chemotherapy pa- with sham injury treated with lithium (80 mg/kg); (3) IRI + saline, tients. mice with IRI treated with saline; (4) IRI + LiCl (L), mice with IRI In summary, our study demonstrated that rescue therapy treated with lithium (40 mg/kg); and (5) IRI + LiCl (H), mice with IRI with low-dose lithium promoted renal tubular epithelia treated with a higher dose of lithium (80 mg/kg). Blood and organs repopulation, improved kidney repair, and accelerated renal were collected at 24 and 48 hours after IRI for further investigation. function recovery after cisplatin or IRI-induced AKI. This proreparative effect of lithium is likely to be attributable to Renal Pathology inhibition of GSK3b and subsequent stabilization of propro- Formalin-fixed kidneys were embedded in paraffin and prepared in liferative molecules, including cyclin D1, c-Myc, and HIF-1a. 3-mm–thick sections. Sections were stained with hematoxylin and Our findings suggest that therapeutic targeting of GSK3b by eosin to estimate gross histologic kidney injury. One observer per- using FDA-approved drugs with GSK3b inhibitory activities formed semiquantitative morphometric analysis in a blinded such as lithium might represent a novel therapeutic strategy for manner. Severity of tubulointerstitial injury and tubular dilation/ rescue treatment of AKI to promote renal recovery after AKI. sloughing was semiquantitatively scored on a scale from 0 to 5 and reported as the mean of 20 random high-power (3200) fields per hematoxylin and eosin–stained section as follows: 0, none; 1, ,10%; CONCISE METHODS 2, 11%–25%; 3, 26%–45%; 4, 46%–75%; and 5, .76%. Indirect immunofluorescence staining of GSK3b,p-GSK3b,HIF-1a, Animal Experimental Design c-Myc, and cyclin D1 was carried out on methanol/acetone-fixed Animal studies were approved by the institution’s animal care and use (1:1) frozen cryostat sections using specific antibodies from Santa committee and they conformed to US Department of Agriculture Cruz Biotechnology (Santa Cruz, CA), followed by applying Alexa regulations and the National Institutes of Health guidelines for hu- Fluor–conjugated secondary antibodies (Invitrogen, Carlsbad, CA). mane care and use of laboratory animals. As a negative control, the primary antibodies were replaced by pre- immune IgG from the same species; no staining occurred. Murine Models of Cisplatin-Induced AKI Five FVB/NJ mice aged 8 weeks were randomly assigned to each of the Creatinine Measurements following groups for each observed time point: (1) group control, mice Serum creatinine levels were measured using a creatinine assay kit received saline alone; (2) group LiCl, mice were only treated with LiCl (Biovision, Mountain View, CA) according to the manufacturer’s (80 mg/kg, single intraperitoneal injection); (3) group cisplatin + saline, instructions.9,50 mice were subjected to cisplatin (20 mg/kg, single intraperitoneal in- jection) injury and saline was given on day 3 after cisplatin injury; (4) Cell Culture and Plasmid Transfection group cisplatin + LiCl (L), mice were subjected to cisplatin (20 mg/kg, TKPT cells were grown in DMEM/F12 that contained 5% FBS. The single intraperitoneal injection) injury and low-dose LiCl (40 mg/kg, eukaryotic expression vectors encoding EV, WT, S9A, and KD were single intraperitoneal injection) was given on day 3 after cisplatin injury; transfected to TKPT cells was as previously described51 by using Lip- and (5) group cisplatin + LiCl (H), mice were subjected to cisplatin ofectamine 2000 according to the manufacturer’s instructions (Invi- (20 mg/kg, single intraperitoneal injection) injury and a higher dose of trogen). After transfection with equal amounts of vectors, cells were lithium (80 mg/kg, single intraperitoneal injection) was given on day 3 then subjected to different treatments and assessed by MTT viability after cisplatin injury. Animals were euthanized on the indicated days and assay as previously described.52 SW480 colon cancer cells were cul- blood and organs were collected for further investigation. tured in DMEM/F12 that contained 10% FCS. Cells were seeded into a six-well plate at a density of 13105 cells per well, followed by Murine Models of Renal IRI different treatments. After 24 and 48 hours of treatment, cell growth Male FVB/NJ mice aged 8 weeks were subjected to bilateral renal IRI to was assessed by the MTT cell growth assay.52 induce AKI.49 Briefly, mice were anesthetized with pentobarbital (60 mg/kg, intraperitoneal injection) and placed on a sterile dispos- Western Immunoblot Analyses able towel over a warming pad. A midline incision was made, and the Cultured cells were lysed, mice kidneys were homogenized in radio- renal artery and vein were isolated from surrounding tissue and then immunoprecipitation assay buffer supplemented with protease in- occluded with a nontraumatic vascular clamp (85 g pressure; Roboz hibitors, and samples were processed for immunoblot as previously Surgical Instrument Company, Gaithersburg, MD) for 22 minutes. In described.51 The antibodies against GSK3b, p-GSK3b, HA, HIF-1a, the sham group, renal pedicles were isolated but no clamps were c-Myc, and cyclin D1 were purchased from Santa Cruz Biotechnol- applied.Attheendoftheischemic period, the vascular clamps ogy. For detection of the physical interactions between GSK3b and were removed and the kidneys were observed for 5 minutes to doc- HIF-1a, c-Myc, or cyclin D1, GSK3b antibody (Santa Cruz Biotech- ument reflow. Eight hours after ischemia or sham injury, saline or nology) was used as the immunoprecipitation antibody and the

498 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 488–500, 2014 www.jasn.org BASIC RESEARCH antibodies against HIF-1a, c-Myc, or cyclin D1 (Santa Cruz Biotech- among nephron segments, and a large pool of mitotically active and nology) were used to probe the immunoprecipitation products by dedifferentiated cells. JClinInvest93: 2175–2188, 1994 immunoblot analysis. 5. Yang L, Humphreys BD, Bonventre JV: Pathophysiology of acute kidney injury to chronic kidney disease: Maladaptive repair. Contrib Nephrol 174: 149–155, 2011 Fluorescence Immunocytochemistry 6. Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A: Glycogen syn- Cultured cells were fixed with 4% paraformaldehyde and then stained thasekinase3:Morethananamesake.Br J Pharmacol 156: 885– with primary antibodies against cyclin D1, c-Myc, HIF-1a,GSK3b, 898, 2009 7. Cohen P, Frame S: The renaissance of GSK3. Nat Rev Mol Cell Biol 2: HA, or preimmune IgG and then with the Alexa Fluorophore 488– 769–776, 2001 conjugated secondary antibody (Invitrogen). Finally, cells were coun- 8. Nelson PJ, Cantley L: GSK3beta plays dirty in acute kidney injury. JAm terstained with DAPI or propidium iodide, mounted with Vectashield Soc Nephrol 21: 199–200, 2010 mounting medium (Vector Laboratories, Burlingame, CA), and vi- 9. Bao H, Ge Y, Zhuang SG, Dworkin LD, Liu ZH, Gong R: Inhibition of sualized using a fluorescence microscope.51 glycogen synthase kinase-3b prevents NSAID-induced acute kidney injury. Kidney Int 81: 662–673, 2012 10. Baldessarini RJ, Tondo L, Hennen J: Lithium treatment and suicide risk Measurement of Cell Apoptosis in major affective disorders: Update and new findings. J Clin Psychiatry Fixed kidney sections or cell cultures were subjected to TUNEL 64[Suppl 5]: 44–52, 2003 staining by using a cell apoptosis detection kit (Roche Applied Science, 11. Meijer L, Flajolet M, Greengard P: Pharmacological inhibitors of gly- – Indianapolis, IN) according to the manufacturer’s instructions, fol- cogen synthase kinase 3. Trends Pharmacol Sci 25: 471 480, 2004 12. Vieweg WV, Yank GR, Rowe WT, Hovermale LS, Clayton AH: Increase in lowed by counterstaining with DAPI or propidium iodide.9 white blood cell count and serum sodium level following the addition of lithium to carbamazepine treatment among three chronically psychotic Statistical Analyses male patients with disturbed affective states. Psychiatr Q 58: 213–217, For immunoblot analysis, bands were scanned and the integrated pixel 1986-1987 density was determined using a densitometer and the National 13. Moore GJ, Bebchuk JM, Wilds IB, Chen G, Manji HK: Lithium-induced increase in human brain grey matter. Lancet 356: 1241–1242, 2000 Institutes of Health image analysis program. All data are expressed as 14. Yan XB, Wang SS, Hou HL, Ji R, Zhou JN: Lithium improves the be- the mean 6 SD. Statistical analysis of the data from multiple groups havioral disorder in rats subjected to transient global cerebral ischemia. was performed by ANOVA followed by the Newman–Keuls test. Data Behav Brain Res 177: 282–289, 2007 from two groups were compared by the t test. P,0.05 was considered 15. Xu J, Culman J, Blume A, Brecht S, Gohlke P: Chronic treatment with a significant. low dose of lithium protects the brain against ischemic injury by re- ducing apoptotic death. Stroke 34: 1287–1292, 2003 16. Arany I, Safirstein RL: Cisplatin nephrotoxicity. Semin Nephrol 23: 460– 464, 2003 ACKNOWLEDGMENTS 17. Yasuda H, Yuen PST, Hu X, Zhou H, Star RA: Simvastatin improves sepsis-induced mortality and acute kidney injury via renal vascular ef- fects. Kidney Int 69: 1535–1542, 2006 This work was made possible in part by funding from the International 18. 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