Delayed administration of a single dose of lithium promotes recovery from AKI

Abstract

Evidence suggests that glycogen synthase kinase 3β (GSK3β) 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 GSK3β and a US Food and Drug Administration-approved mood stabilizer, accelerated recovery 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 GSK3β activity and elevated expression of proproliferative molecules, including cyclin D1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α), in renal tubular epithelia. In cultured renal tubular cells, cisplatin exposure led to transient repression of GSK3β activity followed by a prolonged upregulation of activity. Rescue treatment with lithium inhibited GSK3β activity, enhanced nuclear expression of cyclin D1, c-Myc, and HIF-1α, and boosted cellular proliferation. Similarly, ectopic expression of a kinase-dead mutant of GSK3β enhanced the expression of cyclin D1, c-Myc, and HIF-1α and amplified cellular proliferation after cisplatin injury, whereas forced expression of a constitutively active mutant of GSK3β abrogated the effects of lithium. Mechanistically, GSK3β colocalized and physically interacted with cyclin D1, c-Myc, and HIF-1α in tubular cells. In silico analysis revealed that cyclin D1, c-Myc, and HIF-1α harbor putative GSK3β consensus phosphorylation motifs, implying GSK3β-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 GSK3β by lithium may be a novel therapeutic strategy to improve renal salvage after AKI.

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, ×200; ×400 in insets.

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-1α 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-1α 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-1α in renal tubular cells mainly colocalizes with DAPI staining and appears in nuclei in addition to cytoplasm. (C) GSK3β 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, ×200 in A; ×400 in B.

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-1α 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, ×200.

Figure 4.

Inhibition of GSK3β by lithium after cisplatin injury promotes proliferation of cultured renal tubular cells, associated with amplified expression of cyclin D1, c-myc, and HIF-1α. (A) Western immunoblot analysis for phosphorylated GSK3β in renal tubular (TKPT) cells at different time after cisplatin (100 μM) injured; immunoblots from three separate experiments are analyzed by densitometry. (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-1α 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 pGSK3β markedly correlates with increased expression of cyclin D1, c-Myc, and HIF-1α 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-1α in TKPT cells that are subjected to different treatments as described in G. Immunoblots from three separate experiments were subjected to densitometric analysis. (I–K) Densitometric analysis of immunoblot results demonstrates that lithium-induced inhibitory pGSK3β markedly correlates with increased expression of cyclin D1, c-Myc, and HIF-1α at 48 hours (P<0.05). *P<0.05 versus cisplatin + NaCl–treated group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant.

Figure 5.

Lithium induces nuclear accumulation of proproliferative molecules in cisplatin-injured renal tubular cells. TKPT cells are treated with cisplatin (100 μM) 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-1α 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-1α as the means of 20 random high-power fields. *P<0.05; **P<0.001 versus cisplatin + NaCl–treated cells. Original magnification, ×200.

Figure 6.

GSK3β inhibition is necessary and sufficient for lithium-promoted cellular proliferation. TKPT cells are injured with cisplatin (100 μM) for 6 hours, and cells are then subjected to liposome-mediated transient transfection with vectors encoding the HA-conjugated WT, KD mutant, or constitutively active (S9A) mutant of GSK3β. 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-1α by Western immunoblot 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 GSK3β with cyclin D1, c-Myc, or HIF-1α in TKPT cells. (E) Whole cell lysates are immunoprecipitated with anti-GSK3β antibody and immunoprecipitates are probed for cyclin D1, c-Myc, and HIF-1α by immunoblot analysis (n=3). (F) In silico analysis of the amino acid sequences of cyclin D1, c-Myc, and HIF-1α shows consensus motifs for phosphorylation by GSK3β. *P<0.05; **P<0.001 versus cisplatin-treated WT cells. EV, empty vector; ns, not significant. Original magnification, ×400.

Figure 7.

Lithium suppresses cell growth and sensitizes apoptosis in colon cancer cells. SW480 colon cancer cells are treated with cisplatin (20 μM) 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.