The American Journal of Pathology, Vol. 184, No. 10, October 2014

ajp.amjpathol.org

EPITHELIAL AND MESENCHYMAL CELL BIOLOGY Glycogen Synthase Kinase 3b Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity Implications for the Protective Effect of Low-Dose Lithium in Podocytopathy

Weiwei Xu,*y Yan Ge,y Zhihong Liu,* and Rujun Gongy

From the National Clinical Research Center of Kidney Disease,* Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; and the Division of Kidney Disease and Hypertension,y Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island

Accepted for publication June 10, 2014. Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell Address correspondence to fi Rujun Gong, M.D., Ph.D., signaling molecules, including glycogen synthase kinase 3b (GSK3b), a multitasking kinase lately identi ed Division of Kidney Disease and as a mediator of kidney injury. However, the role of GSK3b in podocytopathy remains obscure. In doxorubicin Hypertension, Department of (Adriamycin)-injured podocytes, lithium, a GSK3b inhibitor and neuroprotective mood stabilizer, obliterated Medicine, Rhode Island Hospital, the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton Brown University School of integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3b overactivity and the Medicine, 593 Eddy St., hyperphosphorylation andoveractivation of paxillin, a focal adhesioneassociated adaptor protein.Moreover, Providence, RI 02903. E-mail: forced expression of a dominant negative kinase dead mutant of GSK3b highly mimicked, whereas ectopic [email protected]. expression of a constitutively active GSK3b mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3b. Furthermore, paxillin interacted with GSK3b and served as its substrate. In mice with doxorubicin ne- phropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3b overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cyto- skeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3b-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytop- athy. (Am J Pathol 2014, 184: 2742e2756; http://dx.doi.org/10.1016/j.ajpath.2014.06.027)

Glomerular visceral epithelial cells or podocytes are a core principal component of the cytoskeletal machinery of foot structural constituent of the glomerular filtration barrier, with processes and forms a subcortical network of branched fila- elaborate interdigitating foot processes that envelop the ments as well as bundled filaments that run longitudinally capillaries of the glomeruli in the kidney, control glomerular through the processes with contractility.8 Actin exists in foot permselectivity, and prevent protein in the bloodstream from processes in a state of dynamic equilibrium between assem- leaking into the urine.1e4 Converging evidence suggests that bly and disassembly, which is important for maintaining the the podocytic filter barrier is not static but a highly dynamic structure that is regulated via the motility of podocyte foot 5e7 Supported in part by NIH grant R01DK092485 (R.G.), the China processes. The molecular basis of foot process motility 973 program 2012CB517600 (Z.L.), and the International Society of lies in the constant dynamics of the molecular machinery Nephrology Sister Renal Center Trio Program. 5e7 that sustains the foot process architecture. Actin is the Disclosures: None declared.

Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.06.027 GSK3b Regulates Podocyte FA Dynamics homeostasis of the glomerular filtration barrier. In response Seattle, WA).31 The cells between passages 21 to 25 were to various pathogenic mediators, including oxidative stress, used. Podocytes were cultured in RPMI 1640 medium (Life circulating permeability factors, and nephrotoxins such as Technologies, Grand Island, NY) supplemented with 10% doxorubicin (Adriamycin), the parallel actin bundles depo- fetal bovine serum (Life Technologies), 0.075% sodium bi- lymerize, resulting in foot process effacement, a pathologic carbonate (Sigma-Aldrich, St. Louis, MO), 0.075% sodium hallmark of podocyte injury and dysfunction.9e13 pyruvate (Sigma-Aldrich), 100 U/mL of penicillin, and 100 Focal adhesions (FAs), by which cells are anchored to the mg/mL of streptomycin (Life Technologies) in a humidified extracellular matrix, are a crucial determinant of actin cyto- incubator with 5% CO2. The cells were cultured at 33 C with skeleton integrity and cell motility.14,15 Molecules from FA 50 U/mL of recombinant mouse interferon-g (Millipore, structures connect the extracellular matrix to bundles of actin Billerica, MA) on collagen-coated Petri dishes. The cells were filaments, enabling the growing actin network to push the transferred to 37C incubators without interferon-g to induce plasma membrane and the contractile stress fibers to pull the differentiation, which took 14 days, and then were treated cell body, corresponding to protrusive and retractive activ- with doxorubicin (0.25 mg/mL; Sigma-Aldrich) and/or lithium ities.14,16 Dynamic turnover of FAs is indispensable for chloride (10 mmol/L; Sigma-Aldrich). Podocytes were constant motility and reorganization of cell edges that man- cultured under permissive conditions (33C) and were pre- ifest as boundary curvature waves.17 A cycle of cellular pared for immunoblot analysis after sodium chloride (10 boundary motion commences with the formation of nascent mmol/L) treatment for 24 hours. Subcultures of the immor- adhesions, which initiate actin assembly and, thus, allow the talized mouse podocytes were maintained under nonpermissive growing actin network to push the cell protrusion forward. conditions (37C) to induce differentiation for 14 days and then The nascent adhesion or focal complex, a precursor of the were treated with lithium chloride (10 or 20 mmol/L) for 24 or FA, is smaller in size, with weaker adhesive force and rapid 48 hours. As control, cells were treated with sodium chloride turnover.18,19 Subsequently, nascent adhesions will either (10 mmol/L) for 24 hours. Cells were subsequently collected disassemble rapidly or mature to be FAs. The FAs usually and prepared for Western blot analysis and immunohisto- contain multiple structural and regulatory molecules, among chemical staining. The expression vectors encoding the which paxillin acts as a pivotal adaptor protein to provide constitutively active GSK3b mutant (S9A-GSK3b-HA/ docking sites for cytoskeletons and to recruit FA regulators pcDNA3), kinase-dead GSK3b mutant (KD-GSK3b-HA/ that control actin dynamics and FA stability.20 The rate of FA pcDNA3), and wild-type GSK3b (WT-GSK3b-HA/pcDNA3) turnover determines cell motility and governs the podocyte were a gift from Dr. Gail V.W. Johnson (University of foot process dynamics. Consistently, targeted manipulation Alabama at Birmingham, Birmingham, AL).32 The vector of FA turnover in podocytes by enhancing or intercepting the encoding green fluorescent proteinepaxillin was a gift from Dr. activity of FA regulatory molecules incurred foot process Luc Sabourin (Ottawa Hospital Research Institute, Ottawa, ON, effacement and podocyte dysfunction.21e23 Canada).33 Podocytes were transfected by using Lipofectamine Glycogen synthase kinase 3b (GSK3b), a well-conserved 2000 reagent (Life Technologies) as previously described.34 and ubiquitously expressed serine/threonine protein kinase, plays a key role in the regulation of cytoskeleton organization Cell Migration Assay and cellular motility.24,25 Indeed, inhibition of GSK3b has been found to reduce cell motility in multiple cells, including Confluent monolayers of differentiated podocytes were vascular smooth muscle cells,26 glioma cells,27 gastric cancer scraped with a 10-mL pipette after different treatments. cells,28 and renal tubular epithelial cells.29 In the kidney, Images of the same area were acquired at indicated time GSK3b has lately been implicated in acute kidney injury points using an inverted microscope and were analyzed and repair.30 However, its role in podocyte injury and foot usingtheImageJversion1.48(NIH,Bethesda,MD) process cytoskeletal disarrangement remains unknown. image processing program. The percentage of cell This study examined the role of GSK3b in a model of migration area was calculated as hypermotility-associated podocytopathy induced by doxo- ðArea0 hour Areaindicated timeÞ=Area0 hour ð1Þ rubicin injury in vivo and in vitro. The potential intervention effect of GSK3b blockade by a single low dose of lithium, a selective GSK3b inhibitor and US Food and Drug Admin- Time-Lapse Fluorescence Microscopy istrationeapproved mood stabilizer, on podocyte motility and dysfunction was accordingly delineated. Podocytes transected with green fluorescent proteine paxillin were subjected to different treatments and placed in Materials and Methods a heating chamber (37 C) on the stage of a time-lapse fluorescence microscope (Axiovert; Zeiss, Cologne, Ger- Cell Culture and Transient Transfection many). Images were taken at 2-minute intervals. The FA turnover rate was calculated using the Focal Adhesion Conditionally immortalized mouse podocytes in culture were Analysis Server web tool (http://faas.bme.unc.edu, last a gift from Dr. Stuart Shankland (University of Washington, accessed October 7, 2013), as described previously.35

The American Journal of Pathology - ajp.amjpathol.org 2743 Xu et al

Western Immunoblot Analysis >75% of the glomerulus, respectively. A whole-kidney average glomerulosclerosis score was obtained by aver- Cultured cells were lyzed and animal tissues homogenized in aging scores from all glomeruli on one section. radioimmunoprecipitation assay buffer supplemented with protease inhibitors and samples were processed for immu- Immunofluorescence Staining noblot analysis. The antibodies against paxillin, GSK3b, p-GSK3b (S9), synaptopodin, and glyceraldehyde-3- Podocytes or cryosections of kidneys were fixed with phosphate dehydrogenase were purchased from Santa Cruz 4% paraformaldehyde (Sigma-Aldrich), permeabilized, and Biotechnology (Santa Cruz, CA), and those against paxillin, stained with primary antibodies against paxillin, GSK3b, and phosphorylated paxillin (S126), and nephrin were acquired synaptopodin, followed by Alexa fluorophoreeconjugated from Cell Signaling Technology Inc. (Danvers, MA) and Pro- secondary antibody staining (Life Technologies). Filamen- gen Biotechnik GmbH (Heidelberg, Germany), respectively. tous actin (F-actin) was stained by rhodamine phalloidin (Cytoskeleton Inc., Denver, CO). Finally, cells were coun- Animal Experiment Design terstained with DAPI, mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and visu- Animal studies were approved by the Rhode Island Hospital alized using a fluorescence microscope. As a negative con- Animal Care and Use Committee, and they conform to the trol, the primary antibody was replaced by preimmune serum US Department of Agriculture regulations and the NIH’s from the same species. The confocal images were acquired Guide for the Care and Use of Laboratory Animals.36 Male using an LSM 710 Meta confocal microscope (Zeiss). For BALB/c mice weighing 20 to 25 g and aged 8 weeks were dual-color staining, images were acquired sequentially to randomly assigned to the following treatments. A single dose avoid dye interference. ImageJ software was used for post- of lithium chloride (40 mg/kg) or an equal molar amount (1 processing of the images, eg, scaling, merging, and co- mEq/kg) of sodium chloride as saline was given via i.p. in- localization analysis. jection on day 0. Doxorubicin (10 mg/kg) or an equal volume of vehicle was given as a tail vein injection 6 hours later. Glomerular Isolation Groups control, LiCl, NaCl þ ADR, and LiCl þ ADR refer to sodium chloride þ vehicle, lithium chloride þ vehicle, Mice were anesthetized and perfused by infusing the abdom- sodium chloride þ doxorubicin, and lithium chloride þ inal artery with 5 mL of phosphate-buffered saline containing doxorubicin treatments, respectively. Spot urine was col- 8 107 Dynabeads M-450 beads (Dynal Biotech ASA, Oslo, lected on postinjury days 0, 1, 3, 5, 7, 10, and 14. Mice were Norway). After perfusion, the kidneys were removed, minced sacrificed on days 3, 7, and 14. Six mice were randomly into 1-mm3 pieces, and digested in collagenase A, and the assigned to each group for each observed time point. glomeruli-containing Dynabeads were collected using a magnetic particle concentrator as described previously.37 Urine Analyses Transmission Electron Microscopy To discern the protein compositions in urine, equal amounts of urine samples were subjected to SDS-PAGE followed by For transmission electron microscopy, kidney cortical tis- Coomassie Blue (Sigma-Aldrich) staining. Urine albumin sues were cut into small pieces (1 mm3), fixed with 2.5% concentration was measured using a mouse albumin glutaraldehyde, and embedded in Epon 812 (Polysciences enzyme-linked immunosorbent assay quantitation kit Inc., Warrington, PA). Conventional electron micrographs (Bethyl Laboratories Inc., Montgomery, TX). Urine creati- were obtained using an EM-10 microscope (Zeiss) operated nine concentration was measured by a creatinine assay kit at 60 kV. The podocyte foot process density was estimated (BioAssay Systems, Hayward, CA). by dividing the total length of glomerular basement mem- brane by the total number of foot processes present in each Morphologic Studies micrograph.

Formalin-fixed kidneys were embedded in paraffin and pre- Statistical Analysis pared sections (3-mm thick). For general histologic analysis, sections were processed for periodic acideSchiff staining. For immunoblot analysis, bands were scanned and the inte- The morphologic features of all the sections were assessed grated pixel density was determined using a densitometer and by a single observer (W.X.) in a blinded manner. A semi- the ImageJ analysis program. All in vitro studies and quantitative glomerulosclerosis score was used to evaluate immunoblot analyses were performed with triplicate samples the degree of glomerulosclerosis. The severity of sclerosis for and were repeated three to six times. All the data are each glomerulus was graded from 0 to 4 as follows: 0 repre- expressed as means SD or as otherwise indicated. Statis- sents no lesions; 1, sclerosis of <25% of the glomerulus; and tical analysis of the data from multiple groups was performed 2, 3, and 4, sclerosis of 25% to 50%, >50% to 75%, and by analysis of variance followed by Student-NewmaneKeuls

2744 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics tests. Data from two groups were compared by Student’s Results t-test or Wilcoxon rank sum test. Linear regression analysis was applied to examine possible relationships between two Lithium Abrogates Podocyte Hypermotility Induced by parameters. P < 0.05 was considered significant. Doxorubicin Stimulation

The conditionally immortalized differentiated murine podo- cytes in culture exhibited typical arborized morphologic features and were characterized as expressing multiple podocyte markers (Supplemental Figure S1). Evidence sug- gests that podocytes are motile cells with considerable constitutive motility.38 Indeed, as revealed by a traditional cell migration assay for assessing cellular motility, podocytes under basal conditions possessed a substantial migratory ca- pacity that lessened the distances between the leading edges of the migrating podocyte sheets. Cells were pretreated with lithium or sodium for 6 hours, followed by doxorubicin injury or vehicle treatment. The basal migrating activity of podocytes was relatively reduced by lithium treatment alone but was unaffected by sodium treatment. In contrast, doxorubicin- injured podocytes demonstrated strikingly accelerated closure of the gap between the invading fronts of the cells, suggesting enhanced podocyte motility. This effect of doxo- rubicin was markedly abrogated by lithium treatment (Figure 1A). These morphologic findings were further corroborated by quantitative measurements of cell migration area (Figure 1B). In addition to rectifying hypermotility, lithium also seemed to elevate the expression of podocyte markers, such as nephrin, in the conditionally immortalized differentiated murine podocytes (Supplemental Figure S1).

Lithium Preserves FA and Actin Cytoskeleton Integrity in Doxorubicin-Injured Podocytes

FA turnover is a prerequisite for cell spreading and migration; accordingly, FA dynamics has been implicated in the control of cellular motility.15 To understand whether the effects of doxorubicin and lithium on podocyte motility were associated with alterations in FA and the ensuing changes in actin cyto- skeleton, podocytes were subjected to phalloidin labeling for F-actin and to immunofluorescence staining for paxillin, a core structural component of FA. Differentiated podocytes either under normal conditions or after vehicle treatment demon- strated abundant FAs that were located at the cell edges, accompanied by intense phalloidin-labeled ventral stress fibers that were anchored to FAs at both ends (Figure 2A). The nuclear staining of paxillin was seemingly nonspecific because it was also probed in the negative control staining, where the Figure 1 Lithium treatment abrogates Adriamycin (ADR; doxorubicin)- primary antibody was replaced with preimmune IgG elicited podocyte hypermotility as assessed by cell migration assay. A: Differentiated podocytes were stimulated with 0.25 mg/mL of ADR or an equal (Figure 2A). Lithium treatment alone slightly reduced the volume of vehicle 6 hours after pretreatment with 10 mmol/L lithium chloride number of FAs but barely affected the size of FAs, suggesting (LiCl) or 10 mmol/L sodium chloride (NaCl), and subsequently scratch was a stabilized FA. Correspondingly, lithium aloneetreated processed using a 10-mL pipette. Control cells were treated with NaCl and podocytes exhibited a stretched cellular shape and a ventral vehicle. The observation was made immediately after scratch (0:00 hour) and stress fiber with long paralleled cortical stress fibers as major at 24:00 hours. B: Quantification of the cell migration area by computerized morphometric analysis. Data are given as means SD. n Z 20 areas from F-actin that were indistinguishable from the morphologic fea- three independent experiments. *P < 0.05 versus all other groups; yP < 0.05 tures of control podocytes. In contrast, in doxorubicin-injured versus sodium treatment alone. Original magnification, 200. podocytes, the number of FAs was substantially increased,

The American Journal of Pathology - ajp.amjpathol.org 2745 Xu et al

Figure 2 Lithium preserves FAs and actin cytoskeleton integrity in Adriamycin (ADR; doxorubicin)-injured podocytes. Differentiated podocytes were stimulated with 0.25 mg/mL of ADR or an equal volume of vehicle 6 hours after pretreatment with 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl). Eight hours later, cells were fixed and subjected to double staining for cytoskeletal F-actin with rhodamine phalloidin and paxillin, an FA marker. A: Control podocytes displayed evident FAs located at the cell edges associated with intense phalloidin-labeled ventral stress fibers anchored to FAs at both ends. Lithium aloneetreated podocytes demonstrated slightly fewer FAs, a more stretched cellular shape, and ventral stress fibers with long paralleled cortical stress fibers similar to the morphologic features of control podocytes. In contrast, ADR-injured podocytes had an increased number of small FAs and displayed an asterlike cell shape as well as actin cytoskeleton disruption that manifested as increased expression of cortical actin filaments, drastically diminished ventral stress fibers, more transverse arcs, and sporadic short dorsal stress fibers connected to the FA at only one end. Lithium treatment prevented the effect of ADR, restored the number and size of FAs, retained stress fibers, and largely preserveed actin cystoskeleton integrity in podocytes. The staining of paxillin in the nucleus was nonspecific because it was also noted in negative control, where the antipaxillin primary antibody was replaced with preimmune IgG. Boxed regions in the paxillin images are shown at higher magnification. B: Computerized morphometric quantification of FA size. ADR reduced the average size of FAs from approximately 3 mm2 to <1 mm2, and this effect was obliterated by lithium pretreatment. C: Quantification of the number of FAs per podocyte. Lithium treatment alone reduced the number of FAs from 137 to approximately 71 per cell, whereas ADR increased FA number to approximately 275 per cell, and this effect was abrogated by lithium pretreatment. Data are given as means SD. n Z 30 cells from three independent experiments. *P < 0.05 versus control; yP < 0.05 versus LiCl þ ADR. Scale bar Z 10 mm(A). Original magnification: 800 (boxed regions in A). and FAs dramatically shrunk to the size that was likely morphometric measurements of FA sizes and absolute FA tantamount to that of focal complexes or nascent adhesions, counts (Figure 2,BandC). denoting a more dynamic FA. In agreement, doxorubicin- injured podocytes exhibited an asterlike cell shape and actin Lithium Normalizes the Doxorubicin-Accelerated cytoskeleton disruption that manifested as increased expres- Dynamics of FA Turnover in Podocytes sion of cortical filaments, diminished ventral stress fibers, more transverse arcs, and sporadic short dorsal stress fi- Fixed podocytes provide only a brief snapshot of FA bers that were connected to the FA only at one end. This expression. By observing alive podocytes, however, addi- cytopathic effect is reminiscent of the actin changes that tional insights into FA dynamics could be gained to validate are observed in foot process effacement in vivo in doxo- or complement the findings from fixed cells. To further rubicin podocytopathy.1e4 Lithium treatment strikingly define the functional impact of lithium- and doxorubicin- prevented the doxorubicin-induced increase in FA regulated FA numbers and sizes on FA dynamics, live numbers and shrinkage in FA sizes, retained stress fibers, imaging of podocytes was performed. Green fluorescent and largely preserved actin cytoskeleton integrity. These proteineconjugated paxillin was ectopically expressed in morphologic findings were subsequently corroborated by podocytes by transient transfection so that turnover of FAs

2746 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics

Figure 3 Lithium corrects the Adriamycin (ADR; doxorubicin)-accelerated dynamics of FA turnover in podocytes. A: Differentiated podocytes were transiently transfected with a vector encoding green fluorescent protein (GFP)epaxillin and were subjected to time-lapse microscopy for 1 hour, followed by fluorescence immunocytochemical staining for synaptopodin. Representative fluores- cent micrographs of synaptopodin staining showed that podocytes retain the podocyte marker protein synaptopodin. B: Podocytes transfected with GFP- paxillin were injured with ADR for 8 hours after 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) treatment for 6 hours. Subse- quently, live podocytes were subjected to time- lapse fluorescence microscopy for 1 hour with 2 minutes between microscopic image frames. The Detail column represents a series of time-lapse microscopic image frames aligned to show the temporal evolution of individual FAs from assembly to disassembly in differently treated podocytes. Because image frames were captured at a fixed rate (0.5 frames per minute), the number of image frames showing the temporal evolution of an indi- vidual FA accordingly correlated the FA dynamics. Thus, hypodynamics and hyperdynamics of FA turnover were indicated by more and less frames, respectively. ADR-treated podocytes shrank rapidly, as shown by the whole cell image and exhibit an accelerated FA turnover (Detail column). This effect was abrogated by lithium pretreatment. Boxed regions focal adhesions, whose turnover is shown in Detail column. C: Quantification of FA assembly rates. Lithium treatment alone slightly reduced the assembly rate. ADR drastically increased the FA as- sembly rate, which was significantly obliterated by lithium pretreatment. D: Quantification of FA disassembly rates. ADR markedly increased the FA disassembly rate, and lithium pretreatment pre- vented the effect. Horizontal bars indicate the median values and the top and bottom lines of the boxes indicate the 3rd and 1st quartile, respectively (C and D). Data are given as medians ranges (C and D). n Z 30 cells from six experiments (C and D). *P < 0.05 versus all other groups by Wilcoxon rank sum test (C and D). Scale bar Z 10 mm(A and B).

The American Journal of Pathology - ajp.amjpathol.org 2747 Xu et al

Figure 4 Inhibitory phosphorylation of GSK3b is negatively associated with paxillin phosphorylation and activation in podocytes. A: Cell lysates were prepared from treated differentiated podocytes at the indicated time points after Adriamycin (ADR; doxorubicin) injury and were subjected to Western blot analysis. ADR injury reduced inhibitory phosphorylation of GSK3b but enhanced paxillin phosphorylation, whereas lithium chloride (LiCl), as a selective inhibitor of GSK3b, counteracted this effect, potentiated GSK3b phosphorylation, and diminished paxillin phosphorylation at different time points. B: Densitometric analysis of immunoblots quantified the relative levels of phosphorylated paxillin/total paxillin ratios and phosphorylated GSK3b/total GSK3b ratios at different time points. C: Linear regression analysis showed a negative correlation between inhibitory phosphorylation of GSK3b and paxillin phosphorylation and activation in podocytes. The correlation coefficient r was 0.7739. White, gray, and black colors represent 2, 8, and 24 hours, respectively. Data are given as means SD. n Z 6 separate experiments (B); n Z 6 representative experiments (C). *P < 0.05 versus control; yP < 0.05 versus LiCl þ ADR. could be visualized and documented by time-lapse fluores- molecules, such as paxillin. Differentiated podocytes were cence microscopy. After transfection and time-lapse mi- pretreated with lithium or sodium for 6 hours, followed by croscopy, control cells retained podocyte morphology and doxorubicin injury or vehicle treatment. Inhibitory phos- evidently expressed typical podocyte marker molecules, phorylation of GSK3b was evidently increased by lithium, including synaptopodin (Figure 3A), suggesting that podo- in agreement with the role of lithium as a specific inhibitor cytes were maintained in a healthy state. Consistent with a of GSK3b (Figure 4A). Doxorubicin injury prominently constitutive motility, basal FA dynamics were evidently diminished GSK3b phosphorylation at all observed time noted in podocytes under normal conditions and were barely points, denoting GSK3b overactivity. This effect was largely affected by vehicle and sodium treatment (Figure 3B). abrogated by lithium treatment. Paxillin phosphorylation, on Doxorubicin injury substantially accelerated FA turnover, as the contrary, exhibited opposing tendencies in response reflected by a reduced number of microscopic image frames to doxorubicin injury or lithium treatment: doxorubicin showing the temporal evolution of an individual FA enhanced whereas lithium abolished paxillin phosphoryla- (Figure 3B). In contrast, lithium treatment resulted in more tion. Densitometric analysis of immunblots confirmed these stable FA dynamics and prominently counteracted the effect findings and revealed an inverse correlation between the of doxorubicin in the observed podocytes. Computerized changes in GSK3b phosphorylation and the changes in morphometric analysis of FA turnover rates revealed that paxillin phosphorylation (Figure 4, B and C). both assembly and disassembly rates of FA were signifi- cantly elevated in doxorubicin-injured cells. Concomitant GSK3b Is Necessary and Sufficient for Paxillin lithium treatment largely prevented the effect of doxorubicin and normalized the FA assembly and disassembly rates to Overactivation, FA Instability, and Podocyte the levels of normal podocytes (Figure 3, C and D). Hypermotility To examine a possible causal relationship between GSK3b Lithium Obliterates Doxorubicin-Elicited GSK3b and paxillin phosphorylation and activation as well as the Overactivity and Paxillin Hyperphosphorylation ensuing changes in FA dynamics and podocyte motility, the activity of GSK3b was selectively manipulated by forced Next we tested whether lithium counteracted the effect of expression of vectors encoding the hemagglutinin- doxorubicin on FA turnover through a direct action on FA conjugated wild-type GSK3b, a dominant negative kinase

2748 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics

Figure 5 GSK3b regulates paxillin phosphory- lation in podocytes and determines the ensuing FA dynamics. A: Differentiated podocytes were trans- fected with vectors encoding the hemagglutinin (HA)-conjugated wild-type (WT) GSK3b or a domi- nant negative KD or a constitutively active (S9A) mutant of GSK3b. Cells were harvested 48 hours after transfection, and cell lysates were subjected to immunoblot analysis for phosphorylated (p) paxillin, total paxillin, HA, and glyceraldehyde-3- phosphate dehydrogenase (GAPDH). B: Densito- metric analysis of immunoblots quantified the relative levels of p-paxillin/total paxillin ratios. Cells were treated with 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) for 6 hours before injury with vehicle or 0.25 mg/mL of Adriamycin (ADR; doxorubicin). C: Podocytes were fixed 8 hours after injury and were subjected to phalloidin labeling of F-actin (red) and immuno- fluorescence staining for paxillin (green). Forced expression of KD diminished the ADR-elicited podocyte injury, marked by an increased number and reduced size of FAs as well as actin cytoskeleton disorganization that manifests as increased expression of cortical filaments and diminished stress fibers, reminiscent of the protective effect of lithium. In contrast, ectopic expression of S9A prominently increased the number and reduced the size of FAs and disrupted actin cytoskeleton integrity under basal conditions, mimicking the effect of ADR injury. On ADR injury, the protective effect of lithium on FAs and actin cytoskeleton was largely abolished in cells expressing S9A. Arrow- heads indicate representative FAs. D: FA number quantification by computerized morphometric analysis. E: Computerized morphometric analysis of the size of FAs. F: Number and size quantification of FAs in ADR-injured podocytes after lithium treat- ment (Figure 2A) or in ADR-injured KD-expressing podocytes (Figure 5C) by computerized morpho- metric analysis; not statistically significant be- tween the two groups. Data are given as means SD. n Z 6 representative experiments (B); n Z 25 cells, three independent experiments (DeF). *P < 0.05 versus all other groups (B) or versus vehicle treated WT-expressing cells (D and E); yP < 0.05 versus ADR-injured WT-expressing cells (D and E), zP < 0.05 versus ADR-injured and LiCl- treated WT-expressing cells (D and E). Scale bar Z 5 mm(C).

dead mutant (KD) or a constitutively active mutant (S9A) of protective effect of lithium (Figure 5,CeE). In contrast, GSK3b. Immunofluorescence staining for hemagglutinin ectopic expression of S9A prominently elicited hyper- revealed a satisfactory transfection efficiency (>70%). phosphorylation and overactivation of paxillin (Figure 5,A Forced expression of KD reduced paxillin phosphorylation and B) under basal conditions, associated with an increased (Figure 5, A and B) and diminished the doxorubicin-elicited number and reduced size of FAs as well as disruption of actin podocyte injury, marked by an increased number and cytoskeleton integrity, mimicking the effect of doxorubicin reduced size of FAs as well as actin cytoskeleton disorga- (Figure 5,CeE). Moreover, on doxorubicin injury, the pro- nization that manifested as increased expression of cortical tective effect of lithium on FAs and actin cytoskeleton was filaments and diminished stress fibers, reminiscent of the largely abolished in cells expressing S9A (Figure 5,CeE),

The American Journal of Pathology - ajp.amjpathol.org 2749 Xu et al

Figure 6 Modulation of GSK3b activity affects podocyte motility. Differentiated podocytes were transfected with vectors encoding the hemagglutinin (HA)- conjugated wild-type (WT), a dominant negative KD, or a constitutively active (S9A) mutant of GSK3b and then were treated with 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) for 6 hours before injury with vehicle or 0.25 mg/mL of Adriamycin (ADR; doxorubicin). Cells were then subjected to cell migration assay for the indicated time. Cell migration assay of podocytes transfected with WT or KD (A) or with WT or S9A (B) after the indicated treatments. Quantification of the cell migration area of podocytes transfected with WT or KD (C) or with WT or S9A (D) after the indicated treatments by computerized morphometric analysis. E: Quantification of the cell migration assay of ADR-injured podocytes after lithium treatment (Figure 1A) or ADR-injured KD-expressing podocytes from A by computerized morphometric analysis; not statistically significant between the two groups. Data are given as means SD. n Z 20 areas from three independent experiments (C and E); n Z 6 areas, three independent experiments (D). *P < 0.05 versus vehicle-treated WT-expressing cells (C and D); yP < 0.05 versus ADR-injured KD-expressing cells (C) or versus ADR- and LiCl-treated S9A-expressing cells (D). Original magnification, 200 (A and B). suggesting that inhibitory phosphorylation of GSK3b is, at migrating podocyte sheets as assessed by the cell migration least in part, responsible for the protection conferred by assay, thus inferring enhanced and impeded podocyte motility, lithium. Consistently, on doxorubicin injury, lithium-treated respectively (Figure 6,AeD). Moreover, the suppressive ef- podocytes and KD-overexpressing podocytes displayed fect of lithium on doxorubicin-elicited cell migration was comparable numbers and sizes of FAs (Figure 5F). Consistent substantially abrogated in S9A-overexpressing podocytes with the role of FA and actin cytoskeleton in cellular motility, injured with doxorubicin (Figure 6, B and D), again suggesting forced expression of S9A reinforced, whereas ectopic that GSK3b inhibition is an indispensable and key mechanism expression of KD mitigated, the doxorubicin-accelerated accounting for the effect of lithium on podocyte motility. closure of the gap between the leading edges of the Consistently, lithium-treated podocytes and KD-overexpressing

2750 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics

Figure 7 Paxillin interacts with GSK3b as its putative substrate in podocytes. A: Differentiated podocytes were fixed and subjected to dual-color immu- nocytochemical staining for GSK3b (green) and paxillin (red). A high-powered view of normal podocytes by confocal fluorescence microscopy revealed a close spatial association and co-localization (arrowheads) between a discrete pool of GSK3b and paxillin in the xy and z planes. B: Lysates of cultured podocytes and homogenates of glomeruli isolated from normal murine kidneys by the magnetic beadsebased approach were subjected to immunoprecipitation (IP) assay by using an anti-GSK3b antibody or the preimmune IgG. Subsequently, immunoprecipitates were processed for immunoblot analysis for paxillin. Arrow indicates the band for paxillin. C: In silico analysis demonstrated that amino acid residues S126, S226, S328, and S336 of paxillin reside in the consensus motifs for phos- phorylation by GSK3b, denoting paxillin as a cognate substrate for GSK3b. D: Characteristics of consensus GSK3b phosphorylation motifs, including the predicted phosphorylation sites, prediction confidence scores, and sequences in paxillin, as estimated by in silico analysis. Scale bar Z 5 mm(A). IB, immunoblot. podocytes displayed comparable migration activity on doxoru- high-confidence matches to GSK3b phosphorylation motifs bicin injury (Figure 6E). (Figure 7, C and D). Collectively, these data suggest that paxillin is a putative cognate substrate for GSK3b. Paxillin Co-Localizes and Physically Interacts with GSK3b as Its Putative Substrate in Podocytes A Single Low Dose of Lithium Ameliorates Podocyte To further decipher the mechanistic essence of the GSK3b- Foot Process Effacement, Attenuates Proteinuria, and mediated regulation of paxillin phosphorylation and acti- Improves Glomerulosclerosis in Experimental vation, the subcellular physical association between GSK3b Doxorubicin Nephropathy and paxillin was examined by dual-color fluorescence immunocytochemical staining. A high-powered view of To further explore whether the GSK3b-regulated FA dy- normal podocytes by confocal fluorescence microscopy namics are involved in podocytopathy in vivo and also to revealed a close spatial association and co-localization be- assess the possible effect of therapeutic targeting of GSK3b,we tween a discrete pool of GSK3b and paxillin in the xy and z used the mouse model of doxorubicin nephropathy, which is planes (Figure 7A). To validate this morphologic observa- accounted for, in part, by podocyte hypermotility and re- tion, immunoprecipitation was performed and demonstrated capitulates key features of podocytopathy and focal and that GSK3b evidently coprecipitated with paxillin in lysates segmental glomerulosclerosis in humans, including podocyte of cultured podocytes and in homogenates of glomeruli foot process effacement, massive proteinuria, and progressive isolated from murine kidneys, suggesting that GSK3b glomerulosclerosis.39,40 Mice were injured with an intravenous physically interacts with paxillin in podocytes in vivo and injection of 10 mg/kg doxorubicin 6 hours after an i.p. injection in vitro (Figure 7B). To further define the mechanism by of a low dose of 40 mg/kg of lithium chloride or an equal molar which GSK3b regulates paxillin phosphorylation, the amino amount (1 mEq/kg) of sodium chloride saline. Doxorubicin acid sequences of paxillin (UniProtKB/Swiss-Prot accession injury elicited heavy proteinuria that peaked on days 5 and 7 number Q8VI36.1) were subjected to computational active and then partially receded on day 14 as determined by urine site analysis (http://scansite.mit.edu/motifscan_seq.phtml, electrophoresis and urine albumin/creatinine ratios (Figure 8,A last accessed December 28, 2013) for putative consensus and B) and was associated with progressive glomerulosclerosis phosphorylation motifs for GSK3b. In silico analysis on periodic acideSchiff staining and with extensive foot pro- deduced that residues S126, S226, S328, and S336 of cess effacement on electron microscopy (Figure 8, C and D). paxillin reside in the consensus motifs for phosphorylation Lithium therapy considerably attenuated proteinuria, amelio- by GSK3b, with prediction scores higher than 0.5 denoting rated glomerulosclerosis, and substantially improved foot

The American Journal of Pathology - ajp.amjpathol.org 2751 Xu et al

Figure 8 Lithium attenuates podocyte effacement and ameliorates proteinuria and progressive glomerulosclerosis in experimental Adriamycin (ADR; doxorubicin) nephropathy. A: Mice were injured with ADR or an equal volume of vehicle 6 hours after a single i.p. injection of 40 mg/kg of lithium chloride (LiCl) or an equal molar amount (1 mEq/kg) of sodium chloride as saline. Urine was collected at the indicated time points and was subjected to SDS-PAGE and staining with Coomassie Brilliant Blue. Bovine serum albumin (BSA), 5, 10, 20, and 40 mg, served as standard control. Urine samples (1.5 mL) collected on the indicated postinjury days from each group were loaded. B: Quantification of urine albumin levels adjusted with urine creatinine concentrations. C: Repre- sentative micrographs demonstrated periodic acideSchiff staining mouse kidneys. ADR-induced injury was featured by glomerular matrix accumulation and protein casts. D: Electron microscopy of kidney specimens procured from animals on day 7. Podocyte injury featured by extensive foot process effacement is evident in ADR-treated kidney, and lithium therapy significantly attenuates this lesion. E: Morphometric analysis of glomerulosclerosis scores on kidney sections prepared on day 14. F: Absolute count of the number of foot processes per unit length of glomerular basement membrane (GBM) on electron mi- crographs of kidney specimens. Data are given as means SD. n Z 6(B, E, and F). *P < 0.05 versus control group (B, E, and F); yP < 0.05 versus LiCl þ ADR (B, E, and F). Scale bars: 20 mm(C); 2 mm(D).

2752 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics

Figure 9 Lithium counteracts the Adriamycin (ADR; doxorubicin)-induced GSK3b overactivity and paxillin hyperphosphorylation in glomeruli and re- instates actin cytoskeleton integrity in glomerular podocytes. A: Glomeruli were isolated from kidneys from differently treated animals by the magnetic beadsebased approach and were homogenized for immunoblot analysis for phosphorylated GSK3b, phosphorylated paxillin, total GSK3b, total paxillin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Densitometric Western blot analysis estimates the relative levels of phosphorylated GSK3b/total GSK3b ratios and phosphorylated pax- illin/total paxillin ratios in isolated glomeruli from different groups. C: Frozen kidney sections procured on day 14 were subjected to phalloidin labeling of F- actin (red) as well as immunofluorescence staining for synaptopodin (green), a podocyte marker. Confocal microscopy images. ADR injury not only reduces synaptopodin expression but also di- minishes the integrated pixel density of the merged areas (yellow), where F-actin co-localizes with syn- aptopodin, suggesting a disorganized actin cyto- skeletal network in the remnant intact podocytes. Computerized morphometric analysis of the ratios of integrated pixel densities between yellow signal to green signal in immunofluorescence micrographs obtained in C and D. Data are given as means SD. n Z 6(B and D). *P < 0.05 versus control group (B) or versus all other groups (D); yP < 0.05 versus LiCl þ ADR (B). Scale bar Z 20 mm(C). process effacement in doxorubicin-injured mice, consistent from animals on day 14 were subjected to phalloidin labeling with a podocyte protective and antiproteinuric effect. Control for F-actin and to immunofluorescence staining for synapto- mice were treated with sodium or lithium 6 hours before podin, a podocyte marker (Figure 9C). Confocal fluorescence vehicle injection, and no noticeable changes in proteinuria or microscopy demonstrated that intense F-actin was found to renal histologic features were noted. These morphologic find- locate extensively to all over the glomerular tufts in normal ings were further corroborated by the semiquantitative kidneys. Podocyte expression of F-actin was highlighted by morphometric measurements of glomerulosclerosis scores and co-localization of F-actin signals (red) with synaptopodin the absolute count of the number of foot processes per unit staining (green). Lithium treatment alone barely affected length of glomerular basement membrane (Figure 8,EandF). either F-actin expression or synaptopodin expression in glomeruli and podocytes. In contrast, doxorubicin induced Lithium Mitigates GSK3b Overactivity, Prevents prominent podocyte injury, as evidenced by reduced syn- Paxillin Activation, and Reinstates Actin Cytoskeleton aptopodin expression, and also diminished F-actin expression Integrity in Doxorubicin-Injured Glomeruli in the periphery of glomerular tufts, consistent with podocyte localization. In agreement, the co-localization of F-actin with To examine the molecular changes associated with the synaptopodin was considerably lessened in doxorubicin- lithium-induced remission of proteinuria, glomeruli were injured kidneys, suggestive of a disorganized actin cytoskel- isolated from kidneys by the magnetic beadsebased approach etal network in the remnant intact podocytes. Lithium therapy and were homogenized for immunoblot analysis. Lithium largely abrogated this injurious effect of doxorubicin, pre- treatment substantially enhanced the inhibitory phosphory- vented the reduction in synaptopodin expression, and rein- lation of GSK3b, suggestive of repressed GSK3b activity, stated F-actin and synaptopodin coexpression in podocytes concomitant with diminished phosphorylation of paxillin (Figure 9D). (Figure 9, A and B). In contrast, doxorubicin injury signifi- cantly enhanced the activity of GSK3b, as marked by the Discussion reduced inhibitory phosphorylation of GSK3b associated with accentuated phosphorylation of paxillin on all observed A growing body of evidence indicates that podocytes are time points. This effect was largely abolished by lithium motile cells and that podocyte foot process motility is vital treatment. To examine the effect of lithium on the ensuing for maintaining the structural and functional homeostasis of actin cytoskeleton organization, kidney specimens procured the glomerular filtration barrier.1,5,6 FAs, by which cells are

The American Journal of Pathology - ajp.amjpathol.org 2753 Xu et al anchored to the extracellular matrix, are a major determinant of actin cytoskeleton dynamics and cellular motility.15 Thus, high FA turnover is associated with high motility of cells.20 To our knowledge, the present study is the first attempt to explore the GSK3b-controlled FA turnover and the ensuing effects on actin cytoskeleton organization and cell motility in podocytes (Figure 10). We demonstrated that lithium, a selective GSK3b inhibitor, protected podocytes from doxo- rubicin injury in vitro and in vivo. Although a variety of other mechanisms might also contribute, it seems that lithium conferred this podocyte protective action, at least in part, by counteracting the doxorubicin-elicited GSK3b overactivity; intercepting the GSK3b-directed hyperphosphorylation and overactivation of paxillin, a core structural component of FAs; and subsequently impeding FA turnover and overriding podocyte hypermotility (Figure 10). GSK3b situates at the nexus of multiple crucial cell signaling pathways and is centrally involved in the pathogenesis of dis- ease in multifaceted organ systems, including the kidney.41 As a redox-sensitive signaling transducer, the activity of GSK3b could be substantially enhanced on oxidative stress induced by a multitude of podocyte-injurious mediators, such as doxoru- bicin and chronic kidney injuries.42 We recently uncovered that expression of GSK3b is aberrantly up-regulated in diseased human kidneys in tubules and glomeruli.43 Similarly, Waters and Koziell44 also noted up-regulation of GSK3b in human podocytes in association with specific NPHS1 mutations. Consistently, gene-targeted knock-in mice with mutated uninhibitable GSK3 developed albuminuria and podocyte Figure 10 Schematic diagram detailing the mechanism of the GSK3b- injury, suggesting a detrimental role of GSK3 in podocyte governed FA dynamics in the pathogenesis of podocytopathy. GSK3b plays a injury.45 In contrast, studies exploiting selective small mole- key role in the regulation of FA turnover and podocyte motility by directing cule inhibitors of GSK3b reached conflicting conclusions. For paxillin phosphorylation and activation and subsequently controlling actin cytoskeleton dynamics. As a redox-sensitive signaling transducer, the ac- example, inhibition of GSK3b by the selective small molecule 0 tivity of GSK3b could be reinforced on oxidative stress induced by a variety of inhibitor 6-bromoindirubin-3 -oxime (BIO) at a low dose podocyte-injurious mediators, including Adriamycin (doxorubicin). GSK3b dramatically normalized proteinuria and attenuated histologic overactivity will elicit paxillin hyperphosphorylation and overactivation and injury of glomeruli in rat models of diabetic nephropathy, thus cause the ensuing actin cytoskeleton disorganization and podocyte although hyperglycemia was not corrected, implying direct hypermotility, ultimately resulting in podocyte foot process effacement, massive proteinuria, and progressive glomerulosclerosis. GSK3b is a drug- antiproteinuric and renoprotective action.46 However, Matsui 47 gable target that could be blocked by lithium, a selective inhibitor of GSK3b et al found that high-dose BIO exacerbated proteinuria and and US Food and Drug Administrationeapproved mood stabilizer that has loss of glomerular nephrin in puromycin-injured rats. Another been safely used for >50 years as a first-line therapy for affective psychiatric study by Dai et al48 reported that a transient and low level of disorders. Lithium treatment could override the Adriamycin-elicited GSK3b proteinuria followed by a rapid spontaneous remission was overactivity, counteract paxillin hyperphosphorylation and overactivation, and thereby obliterate podocyte hypermotility and reinstate actin cyto- provoked by an ultrahigh dose of lithium chloride (16 mmol/ skeleton integrity. Consequently, lithium therapy could ameliorate the kg), which is almost two times the median lethal dose of lithium Adriamycin-induced podocyte foot process effacement, induce proteinuria chloride in mice. In contrast, we demonstrated that low-dose remission, and improve glomerulosclerosis. lithium conferred prominent protection against podocyte injury. Of note, as typical chemical inhibitors of kinases, GSK3b blockers, including lithium, BIO, and 4-benzyl-2- Lithium, a selective inhibitor of GSK3b, has been methyl-1,2,4-thiadiazolidine-3,5-dione, if used at high doses, commonly and safely used for the past 50 years as a US Food could have nonselective off-target effects and could induce and Drug Administrationeapproved first-line drug to treat cytotoxicity and even lethality.49 Thus, the most likely expla- bipolar affective disorders.50,51 Recent evidence revealed nation for these conflicting findings might be the difference in that blockade of GSK3b by lithium reduces cellular motility the doses of GSK3b inhibitor. Collectively, accumulating ev- in a variety of cells, including vascular smooth muscle idence indicates that GSK3b promotes podocyte injury and cells,26 glioma cells,27 gastric cancer cells,28 and airway proteinuria, and inhibition of GSK3b by low-dose inhibitors epithelial cells,52 suggesting that lithium might be a choice of might be beneficial for podocytopathy. therapy for diseases associated with cellular hypermotility,

2754 ajp.amjpathol.org - The American Journal of Pathology GSK3b Regulates Podocyte FA Dynamics such as malignant tumor metastasis. Podocyte hypermotility attenuated the doxorubicin-elicited paxillin phosphorylation is a central pathogenic mechanism accounting for nephrotic and rapid FA turnover, reinstated actin cytoskeleton integrity, glomerulopathy induced by a variety of mediators, including and overrode podocyte hypermotility. In experimental doxo- soluble urokinase-type plasminogen activator receptor,12 rubicin nephropathy, a single low dose of lithium effectively proteases, and nephrotoxins such as doxorubicin.9,10 suppressed the overactivity of GSK3b and paxillin, recovered Correction of podocyte hypermotility via therapeutic target- actin cytoskeleton in glomerular podocytes, prevented podo- ing of FA dynamics, a prerequisite of cell migration and cyte foot process effacement, and attenuated proteinuria motility, has been shown to successfully override podocyte (Figure 10). Collectively, this study suggests that the GSK3b- injuries induced by podocytopathic mediators and to governed FA dynamics might serve as a novel therapeutic improve the podocyte structure and function.23 In this study, target for podocytopathy. lithium, through stabilizing FA dynamics, also counteracted doxorubicin-elicited podocyte hypermotility and consis- tently resulted in a podocyte-protective and antiproteinuric Supplemental Data effect in doxorubicin nephropathy. Apparently, this study might have an immediate implication for clinical translation Supplemental material for this article can be found at into prophylactic treatment for recurrent focal and segmental http://dx.doi.org/10.1016/j.ajpath.2014.06.027. glomerulosclerosis in kidney transplant patients, which has been attributed to a rapid podocytic injury associated with References podocyte hypermotility caused by circulating permeability factors, such as soluble urokinase-type plasminogen acti- 1. Welsh GI, Saleem MA: The podocyte cytoskeleton: key to a functioning 12,53 vator receptor. glomerulus in health and disease. Nat Rev Nephrol 2012, 8:14e21 Of note, a basal level of podocyte motility is essential for 2. Mundel P, Reiser J: Proteinuria: an enzymatic disease of the podo- e sustaining the glomerular filtration barrier homeostasis. cyte? Kidney Int 2010, 77:571 580 3. Mundel P, Shankland SJ: Podocyte biology and response to injury. J Podocyte motility that is too low secondary to genetic de- Am Soc Nephrol 2002, 13:3005e3015 fects of cytoskeleton structural or regulatory molecules has 4. Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular been associated with cytopathic changes in podocytes that podocyte. Physiol Rev 2003, 83:253e307 ultimately also result in focal and segmental glomerulo- 5. Peti-Peterdi J, Sipos A: A high-powered view of the filtration barrier. e sclerosis.1 Therefore, provided the observation that the J Am Soc Nephrol 2010, 21:1835 1841 6. Kistler AD, Altintas MM, Reiser J: Podocyte GTPases regulate kid- lithium represses FA turnover and podocyte motility in ney filter dynamics. Kidney Int 2012, 81:1053e1055 normal podocytes, one of the conceivable concerns would 7. Wang L, Ellis MJ, Gomez JA, Eisner W, Fennell W, Howell DN, be the potential podocytopathic effect of lithium therapy. Ruiz P, Fields TA, Spurney RF: Mechanisms of the proteinuria Indeed, long-term lithium therapy primarily for psychiatric induced by Rho GTPases. Kidney Int 2012, 81:1075e1085 disorders has been complicated by some renal adverse ef- 8. Faul C, Asanuma K, Yanagida-Asanuma E, Kim K, Mundel P: Actin up: regulation of podocyte structure and function by components of fects, such as nephrotic syndrome, glomerular disease, and e 54 the actin cytoskeleton. Trends Cell Biol 2007, 17:428 437 interstitial nephritis, as reported by Markowitz et al in a 9. Koshikawa M, Mukoyama M, Mori K, Suganami T, Sawai K, case series report. However, according to a large-scale Yoshioka T, Nagae T, Yokoi H, Kawachi H, Shimizu F, Sugawara A, epidemiology study,55 the incidence of chronic kidney dis- Nakao K: Role of p38 mitogen-activated protein kinase activation in ease in lithium-treated patients is actually comparable with podocyte injury and proteinuria in experimental nephrotic syndrome. J Am Soc Nephrol 2005, 16:2690e2701 that in the general population, suggesting that the lithium- 10. Liu H, Gao X, Xu H, Feng C, Kuang X, Li Z, Zha X: Alpha-Actinin- associated renal adverse effects are uncommon. Further- 4 is involved in the process by which dexamethasone protects actin more, patients with lithium-associated kidney diseases cytoskeleton stabilization from adriamycin-induced podocyte injury. usually have received lithium therapy at the psychiatric high Nephrology (Carlton) 2012, 17:669e675 dose for a long time (usually >10 years). In the present 11. Lennon R, Singh A, Welsh GI, Coward RJ, Satchell S, Ni L, Mathieson PW, Bakker WW, Saleem MA: Hemopexin induces study, the single dose of lithium used (40 mg/kg) is much nephrin-dependent reorganization of the actin cytoskeleton in podo- lower than the standard dose of lithium that has been safely cytes. J Am Soc Nephrol 2008, 19:2140e2149 and routinely used for neurobiology research (120 mg/kg) in 12. Wei C, El Hindi S, Li J, Fornoni A, Goes N, Sageshima J, Maiguel D, rodents, and no detectable changes in glomerular histologic Karumanchi SA, Yap HK, Saleem M, Zhang Q, Nikolic B, features or function were observed in control mice, sug- Chaudhuri A, Daftarian P, Salido E, Torres A, Salifu M, Sarwal MM, Schaefer F, Morath C, Schwenger V, Zeier M, Gupta V, Roth D, gesting that low-dose lithium might be protective for Rastaldi MP, Burke G, Ruiz P, Reiser J: Circulating urokinase re- podocyte injury. Therefore, it seems that short-term use of ceptor as a cause of focal segmental glomerulosclerosis. Nat Med low-dose lithium is safe in humans and might be a prom- 2011, 17:952e960 ising approach for preventing podocytopathies. 13. Koukouritaki SB, Tamizuddin A, Lianos EA: Enhanced expression of In summary, GSK3b plays an important role in the regula- the cytoskeletal-associated protein, paxillin, in experimental nephrotic syndrome. J Investig Med 1998, 46:284e289 tion of FA turnover and podocyte motility by directing paxillin 14. Carragher NO, Frame MC: Focal adhesion and actin dynamics: a phosphorylation and activation and subsequently controlling place where kinases and proteases meet to promote invasion. Trends actin cytoskeleton dynamics. Lithium, an inhibitor of GSK3b, Cell Biol 2004, 14:241e249

The American Journal of Pathology - ajp.amjpathol.org 2755 Xu et al

15. Lauffenburger DA, Horwitz AF: Cell migration: a physically inte- 35. Matthew E, Berginski SMG: The Focal Adhesion Analysis Server: a web grated molecular process. Cell 1996, 84:359e369 tool for analyzing focal adhesion dynamics. F1000Res 2013, 2:68 16. Gardel ML, Schneider IC, Aratyn-Schaus Y, Waterman CM: Me- 36. Committee for the Update of the Guide for the Care and Use of chanical integration of actin and adhesion dynamics in cell migration. Laboratory Animals; National Research Council: Guide for the Care Annu Rev Cell Dev Biol 2010, 26:315e333 and Use of Laboratory Animals: Eighth Edition. Washington, DC, 17. Wehrle-Haller B: Structure and function of focal adhesions. Curr National Academies Press, 2011 Opin Cell Biol 2012, 24:116e124 37. Takemoto M, Asker N, Gerhardt H, Lundkvist A, Johansson BR, 18. Wehrle-Haller B: Assembly and disassembly of cell matrix adhesions. Saito Y, Betsholtz C: A new method for large scale isolation of Curr Opin Cell Biol 2012, 24:569e581 kidney glomeruli from mice. Am J Pathol 2002, 161:799e805 19. Worth DC, Parsons M: Adhesion dynamics: mechanisms and mea- 38. Endlich N, Simon O, Gopferich A, Wegner H, Moeller MJ, surements. Int J Biochem Cell Biol 2008, 40:2397e2409 Rumpel E, Kotb AM, Endlich K: Two-photon microscopy reveals 20. Abou Zeid N, Valles AM, Boyer B: Serine phosphorylation regulates stationary podocytes in living zebrafish larvae. J Am Soc Nephrol paxillin turnover during cell migration. Cell Commun Signal 2006, 4:8 2014, 25:681e686 21. Dai C, Stolz DB, Bastacky SI, St-Arnaud R, Wu C, Dedhar S, Liu Y: 39. Lee VW, Harris DC: Adriamycin nephropathy: a model of focal Essential role of integrin-linked kinase in podocyte biology: bridging segmental glomerulosclerosis. Nephrology (Carlton) 2011, 16: the integrin and slit diaphragm signaling. J Am Soc Nephrol 2006, 30e38 17:2164e2175 40. Wang Y, Wang YP, Tay YC, Harris DC: Progressive adriamycin 22. Topham PS, Haydar SA, Kuphal R, Lightfoot JD, Salant DJ: Com- nephropathy in mice: sequence of histologic and immunohisto- plement-mediated injury reversibly disrupts glomerular epithelial cell chemical events. Kidney Int 2000, 58:1797e1804 actin microfilaments and focal adhesions. Kidney Int 1999, 55: 41. Wang Z, Havasi A, Gall J, Bonegio R, Li Z, Mao H, Schwartz JH, 1763e1775 Borkan SC: GSK3beta promotes apoptosis after renal ischemic 23. Ma H, Togawa A, Soda K, Zhang J, Lee S, Ma M, Yu Z, Ardito T, injury. J Am Soc Nephrol 2010, 21:284e294 Czyzyk J, Diggs L, Joly D, Hatakeyama S, Kawahara E, Holzman L, 42. Wang SH, Shih YL, Kuo TC, Ko WC, Shih CM: Cadmium toxicity Guan JL, Ishibe S: Inhibition of podocyte FAK protects against pro- toward autophagy through ROS-activated GSK-3beta in mesangial teinuria and foot process effacement. J Am Soc Nephrol 2010, 21: cells. Toxicol Sci 2009, 108:124e131 1145e1156 43. Gong R, Ge Y, Chen S, Liang E, Esparza A, Sabo E, Yango A, 24. Sun T, Rodriguez M, Kim L: Glycogen synthase kinase 3 in the world Gohh R, Rifai A, Dworkin LD: Glycogen synthase kinase 3beta: a of cell migration. Dev Growth Differ 2009, 51:735e742 novel marker and modulator of inflammatory injury in chronic renal 25. Wu X, Shen QT, Oristian DS, Lu CP, Zheng Q, Wang HW, Fuchs E: allograft disease. Am J Transplant 2008, 8:1852e1863 Skin stem cells orchestrate directional migration by regulating 44. Waters A, Koziell A: Activation of canonical Wnt signaling meets microtubule-ACF7 connections through GSK3beta. Cell 2011, 144: with podocytopathy. J Am Soc Nephrol 2009, 20:1864e1866 341e352 45. Boini KM, Amann K, Kempe D, Alessi DR, Lang F: Proteinuria in 26. Wang Z, Zhang X, Chen S, Wang D, Wu J, Liang T, Liu C: Lithium mice expressing PKB/SGK-resistant GSK3. Am J Physiol Renal chloride inhibits vascular smooth muscle cell proliferation and Physiol 2009, 296:F153eF159 migration and alleviates injury-induced neointimal hyperplasia via 46. Lin CL, Wang JY, Huang YT, Kuo YH, Surendran K, Wang FS: induction of PGC-1alpha. PLoS One 2013, 8:e55471 Wnt/beta-catenin signaling modulates survival of high glucose- 27. Nowicki MO, Dmitrieva N, Stein AM, Cutter JL, Godlewski J, stressed mesangial cells. J Am Soc Nephrol 2006, 17:2812e2820 Saeki Y, Nita M, Berens ME, Sander LM, Newton HB, 47. Matsui I, Ito T, Kurihara H, Imai E, Ogihara T, Hori M: Snail, a Chiocca EA, Lawler S: Lithium inhibits invasion of glioma cells; transcriptional regulator, represses nephrin expression in glomerular possible involvement of glycogen synthase kinase-3. Neuro Oncol epithelial cells of nephrotic rats. Lab Invest 2007, 87:273e283 2008, 10:690e699 48. Dai C, Stolz DB, Kiss LP, Monga SP, Holzman LB, Liu Y: Wnt/beta- 28. Ryu YK, Lee YS, Lee GH, Song KS, Kim YS, Moon EY: Regu- catenin signaling promotes podocyte dysfunction and albuminuria. lation of glycogen synthase kinase-3 by thymosin beta-4 is asso- J Am Soc Nephrol 2009, 20:1997e2008 ciated with gastric cancer cell migration. Int J Cancer 2012, 131: 49. Cohen P, Goedert M: GSK3 inhibitors: development and therapeutic 2067e2077 potential. Nat Rev Drug Discov 2004, 3:479e487 29. Peng J, Ramesh G, Sun L, Dong Z: Impaired wound healing in 50. Marmol F: Lithium: bipolar disorder and neurodegenerative dis- hypoxic renal tubular cells: roles of hypoxia-inducible factor-1 and eases: possible cellular mechanisms of the therapeutic effects of glycogen synthase kinase 3beta/beta-catenin signaling. J Pharmacol lithium. Prog Neuropsychopharmacol Biol Psychiatry 2008, 32: Exp Ther 2012, 340:176e184 1761e1771 30. Wang Z, Ge Y, Bao H, Dworkin L, Peng A, Gong R: Redox-sensitive 51. Yang ES, Wang H, Jiang G, Nowsheen S, Fu A, Hallahan DE, Xia F: glycogen synthase kinase 3beta-directed control of mitochondrial Lithium-mediated protection of hippocampal cells involves permeability transition: rheostatic regulation of acute kidney injury. enhancement of DNA-PK-dependent repair in mice. J Clin Invest Free Radic Biol Med 2013, 65C:849e858 2009, 119:1124e1135 31. Shankland SJ, Pippin JW, Reiser J, Mundel P: Podocytes in culture: 52. Wang WC, Kuo CY, Tzang BS, Chen HM, Kao SH: IL-6 augmented past, present, and future. Kidney Int 2007, 72:26e36 motility of airway epithelial cell BEAS-2B via Akt/GSK-3beta 32. Cho JH, Johnson GV: Primed phosphorylation of tau at Thr231 by signaling pathway. J Cell Biochem 2012, 113:3567e3575 glycogen synthase kinase 3beta (GSK3beta) plays a critical role in 53. Morath C, Wei C, Macher-Goeppinger S, Schwenger V, Zeier M, regulating tau’s ability to bind and stabilize microtubules. J Neuro- Reiser J: Management of severe recurrent focal segmental glomer- chem 2004, 88:349e358 ulosclerosis through circulating soluble urokinase receptor modifica- 33. Quizi JL, Baron K, Al-Zahrani KN, O’Reilly P, Sriram RK, tion. Am J Ther 2013, 20:226e229 Conway J, Laurin AA, Sabourin LA: SLK-mediated phosphorylation 54. Markowitz GS, Radhakrishnan J, Kambham N, Valeri AM, of paxillin is required for focal adhesion turnover and cell migration. Hines WH, D’Agati VD: Lithium nephrotoxicity: a progressive Oncogene 2013, 32:4656e4663 combined glomerular and tubulointerstitial nephropathy. J Am Soc 34. Gong R, Rifai A, Ge Y, Chen S, Dworkin LD: Hepatocyte growth Nephrol 2000, 11:1439e1448 factor suppresses proinflammatory NFkappaB activation through 55. Bendz H, Schon S, Attman PO, Aurell M: Renal failure occurs in GSK3beta inactivation in renal tubular epithelial cells. J Biol Chem chronic lithium treatment but is uncommon. Kidney Int 2010, 77: 2008, 283:7401e7410 219e224

2756 ajp.amjpathol.org - The American Journal of Pathology