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Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity

Implications for the Protective Effect of Low-Dose Lithium in Podocytopathy
  • Weiwei Xu
    Affiliations
    National Clinical Research Center of Kidney Disease, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China

    Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island
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  • Yan Ge
    Affiliations
    Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island
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  • Zhihong Liu
    Affiliations
    National Clinical Research Center of Kidney Disease, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China
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  • Rujun Gong
    Correspondence
    Address correspondence to Rujun Gong, M.D., Ph.D., Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, 593 Eddy St., Providence, RI 02903.
    Affiliations
    Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island
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      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 signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion–associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β 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 GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy.
      Glomerular visceral epithelial cells or podocytes are a core structural constituent of the glomerular filtration barrier, with elaborate interdigitating foot processes that envelop the capillaries of the glomeruli in the kidney, control glomerular permselectivity, and prevent protein in the bloodstream from leaking into the urine.
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      Focal adhesions (FAs), by which cells are anchored to the extracellular matrix, are a crucial determinant of actin cytoskeleton integrity and cell motility.
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      Molecules from FA structures connect the extracellular matrix to bundles of actin filaments, enabling the growing actin network to push the plasma membrane and the contractile stress fibers to pull the cell body, corresponding to protrusive and retractive activities.
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      Dynamic turnover of FAs is indispensable for constant motility and reorganization of cell edges that manifest as boundary curvature waves.
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      Structure and function of focal adhesions.
      A cycle of cellular boundary motion commences with the formation of nascent adhesions, which initiate actin assembly and, thus, allow the growing actin network to push the cell protrusion forward. The nascent adhesion or focal complex, a precursor of the FA, is smaller in size, with weaker adhesive force and rapid turnover.
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      Subsequently, nascent adhesions will either disassemble rapidly or mature to be FAs. The FAs usually contain multiple structural and regulatory molecules, among which paxillin acts as a pivotal adaptor protein to provide docking sites for cytoskeletons and to recruit FA regulators that control actin dynamics and FA stability.
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      Serine phosphorylation regulates paxillin turnover during cell migration.
      The rate of FA turnover determines cell motility and governs the podocyte foot process dynamics. Consistently, targeted manipulation of FA turnover in podocytes by enhancing or intercepting the activity of FA regulatory molecules incurred foot process effacement and podocyte dysfunction.
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      Inhibition of podocyte FAK protects against proteinuria and foot process effacement.
      Glycogen synthase kinase 3β (GSK3β), a well-conserved and ubiquitously expressed serine/threonine protein kinase, plays a key role in the regulation of cytoskeleton organization and cellular motility.
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      Glycogen synthase kinase 3 in the world of cell migration.
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      In the kidney, GSK3β has lately been implicated in acute kidney injury and repair.
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      However, its role in podocyte injury and foot process cytoskeletal disarrangement remains unknown. This study examined the role of GSK3β in a model of hypermotility-associated podocytopathy induced by doxorubicin injury in vivo and in vitro. The potential intervention effect of GSK3β blockade by a single low dose of lithium, a selective GSK3β inhibitor and US Food and Drug Administration–approved mood stabilizer, on podocyte motility and dysfunction was accordingly delineated.

      Materials and Methods

      Cell Culture and Transient Transfection

      Conditionally immortalized mouse podocytes in culture were a gift from Dr. Stuart Shankland (University of Washington, Seattle, WA).
      • Shankland S.J.
      • Pippin J.W.
      • Reiser J.
      • Mundel P.
      Podocytes in culture: past, present, and future.
      The cells between passages 21 to 25 were used. Podocytes were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies), 0.075% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO), 0.075% sodium pyruvate (Sigma-Aldrich), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Life Technologies) in a humidified incubator with 5% CO2. The cells were cultured at 33°C with 50 U/mL of recombinant mouse interferon-γ (Millipore, Billerica, MA) on collagen-coated Petri dishes. The cells were transferred to 37°C incubators without interferon-γ to induce differentiation, which took 14 days, and then were treated with doxorubicin (0.25 μg/mL; Sigma-Aldrich) and/or lithium chloride (10 mmol/L; Sigma-Aldrich). Podocytes were cultured under permissive conditions (33°C) and were prepared for immunoblot analysis after sodium chloride (10 mmol/L) treatment for 24 hours. Subcultures of the immortalized mouse podocytes were maintained under nonpermissive conditions (37°C) to induce differentiation for 14 days and then were treated with lithium chloride (10 or 20 mmol/L) for 24 or 48 hours. As control, cells were treated with sodium chloride (10 mmol/L) for 24 hours. Cells were subsequently collected and prepared for Western blot analysis and immunohistochemical staining. The expression vectors encoding the constitutively active GSK3β mutant (S9A-GSK3β-HA/pcDNA3), kinase-dead GSK3β mutant (KD-GSK3β-HA/pcDNA3), and wild-type GSK3β (WT-GSK3β-HA/pcDNA3) were a gift from Dr. Gail V.W. Johnson (University of Alabama at Birmingham, Birmingham, AL).
      • Cho J.H.
      • Johnson G.V.
      Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3beta (GSK3beta) plays a critical role in regulating tau's ability to bind and stabilize microtubules.
      The vector encoding green fluorescent protein–paxillin was a gift from Dr. Luc Sabourin (Ottawa Hospital Research Institute, Ottawa, ON, Canada).
      • Quizi J.L.
      • Baron K.
      • Al-Zahrani K.N.
      • O'Reilly P.
      • Sriram R.K.
      • Conway J.
      • Laurin A.A.
      • Sabourin L.A.
      SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration.
      Podocytes were transfected by using Lipofectamine 2000 reagent (Life Technologies) as previously described.
      • Gong R.
      • Rifai A.
      • Ge Y.
      • Chen S.
      • Dworkin L.D.
      Hepatocyte growth factor suppresses proinflammatory NFkappaB activation through GSK3beta inactivation in renal tubular epithelial cells.

      Cell Migration Assay

      Confluent monolayers of differentiated podocytes were scraped with a 10-μL pipette after different treatments. Images of the same area were acquired at indicated time points using an inverted microscope and were analyzed using the ImageJ version 1.48 (NIH, Bethesda, MD) image processing program. The percentage of cell migration area was calculated as
      (Area0hour-Areaindicatedtime)/Area0hour
      (1)


      Time-Lapse Fluorescence Microscopy

      Podocytes transected with green fluorescent protein–paxillin were subjected to different treatments and placed in a heating chamber (37°C) on the stage of a time-lapse fluorescence microscope (Axiovert; Zeiss, Cologne, Germany). Images were taken at 2-minute intervals. The FA turnover rate was calculated using the Focal Adhesion Analysis Server web tool (http://faas.bme.unc.edu, last accessed October 7, 2013), as described previously.
      • Matthew E.
      • Berginski S.M.G.
      The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics.

      Western Immunoblot Analysis

      Cultured cells were lyzed and animal tissues homogenized in radioimmunoprecipitation assay buffer supplemented with protease inhibitors and samples were processed for immunoblot analysis. The antibodies against paxillin, GSK3β, p-GSK3β (S9), synaptopodin, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and those against paxillin, phosphorylated paxillin (S126), and nephrin were acquired from Cell Signaling Technology Inc. (Danvers, MA) and Progen Biotechnik GmbH (Heidelberg, Germany), respectively.

      Animal Experiment Design

      Animal studies were approved by the Rhode Island Hospital Animal Care and Use Committee, and they conform to the US Department of Agriculture regulations and the NIH's Guide for the Care and Use of Laboratory Animals.
      Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council
      Guide for the Care and Use of Laboratory Animals: Eighth Edition.
      Male BALB/c mice weighing 20 to 25 g and aged 8 weeks were randomly assigned to the following treatments. A single dose of lithium chloride (40 mg/kg) or an equal molar amount (1 mEq/kg) of sodium chloride as saline was given via i.p. injection on day 0. Doxorubicin (10 mg/kg) or an equal volume of vehicle was given as a tail vein injection 6 hours later. Groups control, LiCl, NaCl + ADR, and LiCl + ADR refer to sodium chloride + vehicle, lithium chloride + vehicle, sodium chloride + doxorubicin, and lithium chloride + doxorubicin treatments, respectively. Spot urine was collected on postinjury days 0, 1, 3, 5, 7, 10, and 14. Mice were sacrificed on days 3, 7, and 14. Six mice were randomly assigned to each group for each observed time point.

      Urine Analyses

      To discern the protein compositions in urine, equal amounts of urine samples were subjected to SDS-PAGE followed by Coomassie Blue (Sigma-Aldrich) staining. Urine albumin concentration was measured using a mouse albumin enzyme-linked immunosorbent assay quantitation kit (Bethyl Laboratories Inc., Montgomery, TX). Urine creatinine concentration was measured by a creatinine assay kit (BioAssay Systems, Hayward, CA).

      Morphologic Studies

      Formalin-fixed kidneys were embedded in paraffin and prepared sections (3-μm thick). For general histologic analysis, sections were processed for periodic acid–Schiff staining. The morphologic features of all the sections were assessed by a single observer (W.X.) in a blinded manner. A semiquantitative glomerulosclerosis score was used to evaluate the degree of glomerulosclerosis. The severity of sclerosis for each glomerulus was graded from 0 to 4 as follows: 0 represents no lesions; 1, sclerosis of <25% of the glomerulus; and 2, 3, and 4, sclerosis of 25% to 50%, >50% to 75%, and >75% of the glomerulus, respectively. A whole-kidney average glomerulosclerosis score was obtained by averaging scores from all glomeruli on one section.

      Immunofluorescence Staining

      Podocytes or cryosections of kidneys were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized, and stained with primary antibodies against paxillin, GSK3β, and synaptopodin, followed by Alexa fluorophore–conjugated secondary antibody staining (Life Technologies). Filamentous actin (F-actin) was stained by rhodamine phalloidin (Cytoskeleton Inc., Denver, CO). Finally, cells were counterstained with DAPI, mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and visualized using a fluorescence microscope. As a negative control, the primary antibody was replaced by preimmune serum from the same species. The confocal images were acquired using an LSM 710 Meta confocal microscope (Zeiss). For dual-color staining, images were acquired sequentially to avoid dye interference. ImageJ software was used for postprocessing of the images, eg, scaling, merging, and co-localization analysis.

      Glomerular Isolation

      Mice were anesthetized and perfused by infusing the abdominal artery with 5 mL of phosphate-buffered saline containing 8 × 107 Dynabeads M-450 beads (Dynal Biotech ASA, Oslo, Norway). After perfusion, the kidneys were removed, minced into 1-mm3 pieces, and digested in collagenase A, and the glomeruli-containing Dynabeads were collected using a magnetic particle concentrator as described previously.
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      A new method for large scale isolation of kidney glomeruli from mice.

      Transmission Electron Microscopy

      For transmission electron microscopy, kidney cortical tissues were cut into small pieces (1 mm3), fixed with 2.5% glutaraldehyde, and embedded in Epon 812 (Polysciences Inc., Warrington, PA). Conventional electron micrographs were obtained using an EM-10 microscope (Zeiss) operated at 60 kV. The podocyte foot process density was estimated by dividing the total length of glomerular basement membrane by the total number of foot processes present in each micrograph.

      Statistical Analysis

      For immunoblot analysis, bands were scanned and the integrated pixel density was determined using a densitometer and the ImageJ analysis program. All in vitro studies and immunoblot analyses were performed with triplicate samples and were repeated three to six times. All the data are expressed as means ± SD or as otherwise indicated. Statistical analysis of the data from multiple groups was performed by analysis of variance followed by Student-Newman–Keuls tests. Data from two groups were compared by Student's t-test or Wilcoxon rank sum test. Linear regression analysis was applied to examine possible relationships between two parameters. P < 0.05 was considered significant.

      Results

      Lithium Abrogates Podocyte Hypermotility Induced by Doxorubicin Stimulation

      The conditionally immortalized differentiated murine podocytes in culture exhibited typical arborized morphologic features and were characterized as expressing multiple podocyte markers (Supplemental Figure S1). Evidence suggests that podocytes are motile cells with considerable constitutive motility.
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      • Endlich K.
      Two-photon microscopy reveals stationary podocytes in living zebrafish larvae.
      Indeed, as revealed by a traditional cell migration assay for assessing cellular motility, podocytes under basal conditions possessed a substantial migratory capacity 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 doxorubicin 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).
      Figure thumbnail gr1
      Figure 1Lithium treatment abrogates Adriamycin (ADR; doxorubicin)-elicited podocyte hypermotility as assessed by cell migration assay. A: Differentiated podocytes were stimulated with 0.25 μg/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), and subsequently scratch was processed using a 10-μL pipette. Control cells were treated with NaCl and vehicle. The observation was made immediately after scratch (0:00 hour) and at 24:00 hours. B: Quantification of the cell migration area by computerized morphometric analysis. Data are given as means ± SD. n = 20 areas from three independent experiments. P < 0.05 versus all other groups; P < 0.05 versus sodium treatment alone. Original magnification, ×200.

      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.
      • Lauffenburger D.A.
      • Horwitz A.F.
      Cell migration: a physically integrated molecular process.
      To understand whether the effects of doxorubicin and lithium on podocyte motility were associated with alterations in FA and the ensuing changes in actin cytoskeleton, 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 demonstrated 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 primary antibody was replaced with preimmune IgG (Figure 2A). Lithium treatment alone slightly reduced the number of FAs but barely affected the size of FAs, suggesting a stabilized FA. Correspondingly, lithium alone–treated podocytes exhibited a stretched cellular shape and a ventral stress fiber with long paralleled cortical stress fibers as major F-actin that were indistinguishable from the morphologic features of control podocytes. In contrast, in doxorubicin-injured podocytes, the number of FAs was substantially increased, and FAs dramatically shrunk to the size that was likely tantamount to that of focal complexes or nascent adhesions, denoting a more dynamic FA. In agreement, doxorubicin-injured podocytes exhibited an asterlike cell shape and actin cytoskeleton disruption that manifested as increased expression of cortical filaments, diminished ventral stress fibers, more transverse arcs, and sporadic short dorsal stress fibers that were connected to the FA only at one end. This cytopathic effect is reminiscent of the actin changes that are observed in foot process effacement in vivo in doxorubicin podocytopathy.
      • Welsh G.I.
      • Saleem M.A.
      The podocyte cytoskeleton: key to a functioning glomerulus in health and disease.
      • Mundel P.
      • Reiser J.
      Proteinuria: an enzymatic disease of the podocyte?.
      • Mundel P.
      • Shankland S.J.
      Podocyte biology and response to injury.
      • Pavenstadt H.
      • Kriz W.
      • Kretzler M.
      Cell biology of the glomerular podocyte.
      Lithium treatment strikingly prevented the doxorubicin-induced increase in FA numbers and shrinkage in FA sizes, retained stress fibers, and largely preserved actin cytoskeleton integrity. These morphologic findings were subsequently corroborated by morphometric measurements of FA sizes and absolute FA counts (Figure 2, B and C).
      Figure thumbnail gr2
      Figure 2Lithium preserves FAs and actin cytoskeleton integrity in Adriamycin (ADR; doxorubicin)-injured podocytes. Differentiated podocytes were stimulated with 0.25 μg/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 alone–treated 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 μm2 to <1 μm2, 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 = 30 cells from three independent experiments. P < 0.05 versus control; P < 0.05 versus LiCl + ADR. Scale bar = 10 μm (A). Original magnification: ×800 (boxed regions in A).

      Lithium Normalizes the Doxorubicin-Accelerated Dynamics of FA Turnover in Podocytes

      Fixed podocytes provide only a brief snapshot of FA expression. By observing alive podocytes, however, additional insights into FA dynamics could be gained to validate or complement the findings from fixed cells. To further define the functional impact of lithium- and doxorubicin-regulated FA numbers and sizes on FA dynamics, live imaging of podocytes was performed. Green fluorescent protein–conjugated paxillin was ectopically expressed in podocytes by transient transfection so that turnover of FAs could be visualized and documented by time-lapse fluorescence microscopy. After transfection and time-lapse microscopy, control cells retained podocyte morphology and evidently expressed typical podocyte marker molecules, including synaptopodin (Figure 3A), suggesting that podocytes were maintained in a healthy state. Consistent with a constitutive motility, basal FA dynamics were evidently noted in podocytes under normal conditions and were barely affected by vehicle and sodium treatment (Figure 3B). Doxorubicin injury substantially accelerated FA turnover, as reflected by a reduced number of microscopic image frames showing the temporal evolution of an individual FA (Figure 3B). In contrast, lithium treatment resulted in more stable FA dynamics and prominently counteracted the effect of doxorubicin in the observed podocytes. Computerized morphometric analysis of FA turnover rates revealed that both assembly and disassembly rates of FA were significantly elevated in doxorubicin-injured cells. Concomitant lithium treatment largely prevented the effect of doxorubicin and normalized the FA assembly and disassembly rates to the levels of normal podocytes (Figure 3, C and D).
      Figure thumbnail gr3
      Figure 3Lithium 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)–paxillin and were subjected to time-lapse microscopy for 1 hour, followed by fluorescence immunocytochemical staining for synaptopodin. Representative fluorescent 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. Subsequently, 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 individual 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 assembly rate, which was significantly obliterated by lithium pretreatment. D: Quantification of FA disassembly rates. ADR markedly increased the FA disassembly rate, and lithium pretreatment prevented 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 = 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 = 10 μm (A and B).

      Lithium Obliterates Doxorubicin-Elicited GSK3β Overactivity and Paxillin Hyperphosphorylation

      Next we tested whether lithium counteracted the effect of doxorubicin on FA turnover through a direct action on FA molecules, such as paxillin. Differentiated podocytes were pretreated with lithium or sodium for 6 hours, followed by doxorubicin injury or vehicle treatment. Inhibitory phosphorylation of GSK3β was evidently increased by lithium, in agreement with the role of lithium as a specific inhibitor of GSK3β (Figure 4A). Doxorubicin injury prominently diminished GSK3β phosphorylation at all observed time points, denoting GSK3β overactivity. This effect was largely abrogated by lithium treatment. Paxillin phosphorylation, on the contrary, exhibited opposing tendencies in response to doxorubicin injury or lithium treatment: doxorubicin enhanced whereas lithium abolished paxillin phosphorylation. Densitometric analysis of immunblots confirmed these findings and revealed an inverse correlation between the changes in GSK3β phosphorylation and the changes in paxillin phosphorylation (Figure 4, B and C).
      Figure thumbnail gr4
      Figure 4Inhibitory phosphorylation of GSK3β 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 GSK3β but enhanced paxillin phosphorylation, whereas lithium chloride (LiCl), as a selective inhibitor of GSK3β, counteracted this effect, potentiated GSK3β 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 GSK3β/total GSK3β ratios at different time points. C: Linear regression analysis showed a negative correlation between inhibitory phosphorylation of GSK3β 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 = 6 separate experiments (B); n = 6 representative experiments (C). P < 0.05 versus control; P < 0.05 versus LiCl + ADR.

      GSK3β Is Necessary and Sufficient for Paxillin Overactivation, FA Instability, and Podocyte Hypermotility

      To examine a possible causal relationship between GSK3β and paxillin phosphorylation and activation as well as the ensuing changes in FA dynamics and podocyte motility, the activity of GSK3β was selectively manipulated by forced expression of vectors encoding the hemagglutinin-conjugated wild-type GSK3β, a dominant negative kinase dead mutant (KD) or a constitutively active mutant (S9A) of GSK3β. Immunofluorescence staining for hemagglutinin revealed a satisfactory transfection efficiency (>70%). Forced expression of KD reduced paxillin phosphorylation (Figure 5, A and B) and diminished the doxorubicin-elicited podocyte injury, marked by an increased number and reduced size of FAs as well as actin cytoskeleton disorganization that manifested as increased expression of cortical filaments and diminished stress fibers, reminiscent of the protective effect of lithium (Figure 5, C–E). In contrast, ectopic expression of S9A prominently elicited hyperphosphorylation and overactivation of paxillin (Figure 5, A and B) under basal conditions, associated with an increased number and reduced size of FAs as well as disruption of actin cytoskeleton integrity, mimicking the effect of doxorubicin (Figure 5, C–E). Moreover, on doxorubicin injury, the protective effect of lithium on FAs and actin cytoskeleton was largely abolished in cells expressing S9A (Figure 5, C–E), suggesting that inhibitory phosphorylation of GSK3β is, at least in part, responsible for the protection conferred by lithium. Consistently, on doxorubicin injury, lithium-treated podocytes and KD-overexpressing podocytes displayed comparable numbers and sizes of FAs (Figure 5F). Consistent with the role of FA and actin cytoskeleton in cellular motility, forced expression of S9A reinforced, whereas ectopic expression of KD mitigated, the doxorubicin-accelerated closure of the gap between the leading edges of the migrating podocyte sheets as assessed by the cell migration assay, thus inferring enhanced and impeded podocyte motility, respectively (Figure 6, A–D). Moreover, the suppressive effect of lithium on doxorubicin-elicited cell migration was substantially abrogated in S9A-overexpressing podocytes injured with doxorubicin (Figure 6, B and D), again suggesting that GSK3β inhibition is an indispensable and key mechanism accounting for the effect of lithium on podocyte motility. Consistently, lithium-treated podocytes and KD-overexpressing podocytes displayed comparable migration activity on doxorubicin injury (Figure 6E).
      Figure thumbnail gr5
      Figure 5GSK3β regulates paxillin phosphorylation in podocytes and determines the ensuing FA dynamics. A: Differentiated podocytes were transfected with vectors encoding the hemagglutinin (HA)-conjugated wild-type (WT) GSK3β or a dominant negative KD or a constitutively active (S9A) mutant of GSK3β. 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: Densitometric 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 μg/mL of Adriamycin (ADR; doxorubicin). C: Podocytes were fixed 8 hours after injury and were subjected to phalloidin labeling of F-actin (red) and immunofluorescence 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. Arrowheads 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 treatment (A) or in ADR-injured KD-expressing podocytes (C) by computerized morphometric analysis; not statistically significant between the two groups. Data are given as means ± SD. n = 6 representative experiments (B); n = 25 cells, three independent experiments (DF). P < 0.05 versus all other groups (B) or versus vehicle treated WT-expressing cells (D and E); P < 0.05 versus ADR-injured WT-expressing cells (D and E), P < 0.05 versus ADR-injured and LiCl-treated WT-expressing cells (D and E). Scale bar = 5 μm (C).
      Figure thumbnail gr6
      Figure 6Modulation of GSK3β 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 GSK3β 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 μg/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 (A) 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 = 20 areas from three independent experiments (C and E); n = 6 areas, three independent experiments (D). P < 0.05 versus vehicle-treated WT-expressing cells (C and D); P < 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).

      Paxillin Co-Localizes and Physically Interacts with GSK3β as Its Putative Substrate in Podocytes

      To further decipher the mechanistic essence of the GSK3β-mediated regulation of paxillin phosphorylation and activation, the subcellular physical association between GSK3β and paxillin was examined by dual-color fluorescence immunocytochemical staining. A high-powered view of normal podocytes by confocal fluorescence microscopy revealed a close spatial association and co-localization between a discrete pool of GSK3β and paxillin in the xy and z planes (Figure 7A). To validate this morphologic observation, immunoprecipitation was performed and demonstrated that GSK3β evidently coprecipitated with paxillin in lysates of cultured podocytes and in homogenates of glomeruli isolated from murine kidneys, suggesting that GSK3β physically interacts with paxillin in podocytes in vivo and in vitro (Figure 7B). To further define the mechanism by which GSK3β regulates paxillin phosphorylation, the amino acid sequences of paxillin (UniProtKB/Swiss-Prot accession number Q8VI36.1) were subjected to computational active site analysis (http://scansite.mit.edu/motifscan_seq.phtml, last accessed December 28, 2013) for putative consensus phosphorylation motifs for GSK3β. In silico analysis deduced that residues S126, S226, S328, and S336 of paxillin reside in the consensus motifs for phosphorylation by GSK3β, with prediction scores higher than 0.5 denoting high-confidence matches to GSK3β phosphorylation motifs (Figure 7, C and D). Collectively, these data suggest that paxillin is a putative cognate substrate for GSK3β.
      Figure thumbnail gr7
      Figure 7Paxillin interacts with GSK3β as its putative substrate in podocytes. A: Differentiated podocytes were fixed and subjected to dual-color immunocytochemical staining for GSK3β (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 GSK3β 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 beads–based approach were subjected to immunoprecipitation (IP) assay by using an anti-GSK3β 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 phosphorylation by GSK3β, denoting paxillin as a cognate substrate for GSK3β. D: Characteristics of consensus GSK3β phosphorylation motifs, including the predicted phosphorylation sites, prediction confidence scores, and sequences in paxillin, as estimated by in silico analysis. Scale bar = 5 μm (A). IB, immunoblot.

      A Single Low Dose of Lithium Ameliorates Podocyte Foot Process Effacement, Attenuates Proteinuria, and Improves Glomerulosclerosis in Experimental Doxorubicin Nephropathy

      To further explore whether the GSK3β-regulated FA dynamics are involved in podocytopathy in vivo and also to assess the possible effect of therapeutic targeting of GSK3β, we used the mouse model of doxorubicin nephropathy, which is accounted for, in part, by podocyte hypermotility and recapitulates key features of podocytopathy and focal and segmental glomerulosclerosis in humans, including podocyte foot process effacement, massive proteinuria, and progressive glomerulosclerosis.
      • Lee V.W.
      • Harris D.C.
      Adriamycin nephropathy: a model of focal segmental glomerulosclerosis.
      • Wang Y.
      • Wang Y.P.
      • Tay Y.C.
      • Harris D.C.
      Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.
      Mice were injured with an intravenous injection of 10 mg/kg doxorubicin 6 hours after an i.p. injection of a low dose of 40 mg/kg of lithium chloride or an equal molar amount (1 mEq/kg) of sodium chloride saline. Doxorubicin injury elicited heavy proteinuria that peaked on days 5 and 7 and then partially receded on day 14 as determined by urine electrophoresis and urine albumin/creatinine ratios (Figure 8, A and B) and was associated with progressive glomerulosclerosis on periodic acid–Schiff staining and with extensive foot process effacement on electron microscopy (Figure 8, C and D). Lithium therapy considerably attenuated proteinuria, ameliorated glomerulosclerosis, and substantially improved foot process effacement in doxorubicin-injured mice, consistent with a podocyte protective and antiproteinuric effect. Control mice were treated with sodium or lithium 6 hours before vehicle injection, and no noticeable changes in proteinuria or renal histologic features were noted. These morphologic findings were further corroborated by the semiquantitative morphometric measurements of glomerulosclerosis scores and the absolute count of the number of foot processes per unit length of glomerular basement membrane (Figure 8, E and F).
      Figure thumbnail gr8
      Figure 8Lithium 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 μg, served as standard control. Urine samples (1.5 μL) collected on the indicated postinjury days from each group were loaded. B: Quantification of urine albumin levels adjusted with urine creatinine concentrations. C: Representative micrographs demonstrated periodic acid–Schiff 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 micrographs of kidney specimens. Data are given as means ± SD. n = 6 (B, E, and F). P < 0.05 versus control group (B, E, and F); P < 0.05 versus LiCl + ADR (B, E, and F). Scale bars: 20 μm (C); 2 μm (D).

      Lithium Mitigates GSK3β Overactivity, Prevents Paxillin Activation, and Reinstates Actin Cytoskeleton Integrity in Doxorubicin-Injured Glomeruli

      To examine the molecular changes associated with the lithium-induced remission of proteinuria, glomeruli were isolated from kidneys by the magnetic beads–based approach and were homogenized for immunoblot analysis. Lithium treatment substantially enhanced the inhibitory phosphorylation of GSK3β, suggestive of repressed GSK3β activity, concomitant with diminished phosphorylation of paxillin (Figure 9, A and B). In contrast, doxorubicin injury significantly enhanced the activity of GSK3β, as marked by the reduced inhibitory phosphorylation of GSK3β associated with accentuated phosphorylation of paxillin on all observed time points. This effect was largely abolished by lithium treatment. To examine the effect of lithium on the ensuing actin cytoskeleton organization, kidney specimens procured from animals on day 14 were subjected to phalloidin labeling for F-actin and to immunofluorescence staining for synaptopodin, a podocyte marker (Figure 9C). Confocal fluorescence microscopy demonstrated that intense F-actin was found to locate extensively to all over the glomerular tufts in normal kidneys. Podocyte expression of F-actin was highlighted by co-localization of F-actin signals (red) with synaptopodin staining (green). Lithium treatment alone barely affected either F-actin expression or synaptopodin expression in glomeruli and podocytes. In contrast, doxorubicin induced prominent podocyte injury, as evidenced by reduced synaptopodin expression, and also diminished F-actin expression in the periphery of glomerular tufts, consistent with podocyte localization. In agreement, the co-localization of F-actin with synaptopodin was considerably lessened in doxorubicin-injured kidneys, suggestive of a disorganized actin cytoskeletal network in the remnant intact podocytes. Lithium therapy largely abrogated this injurious effect of doxorubicin, prevented the reduction in synaptopodin expression, and reinstated F-actin and synaptopodin coexpression in podocytes (Figure 9D).
      Figure thumbnail gr9
      Figure 9Lithium counteracts the Adriamycin (ADR; doxorubicin)-induced GSK3β overactivity and paxillin hyperphosphorylation in glomeruli and reinstates actin cytoskeleton integrity in glomerular podocytes. A: Glomeruli were isolated from kidneys from differently treated animals by the magnetic beads–based approach and were homogenized for immunoblot analysis for phosphorylated GSK3β, phosphorylated paxillin, total GSK3β, total paxillin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Densitometric Western blot analysis estimates the relative levels of phosphorylated GSK3β/total GSK3β ratios and phosphorylated paxillin/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 diminishes the integrated pixel density of the merged areas (yellow), where F-actin co-localizes with synaptopodin, suggesting a disorganized actin cytoskeletal 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 = 6 (B and D). P < 0.05 versus control group (B) or versus all other groups (D); P < 0.05 versus LiCl + ADR (B). Scale bar = 20 μm (C).

      Discussion

      A growing body of evidence indicates that podocytes are motile cells and that podocyte foot process motility is vital for maintaining the structural and functional homeostasis of the glomerular filtration barrier.
      • Welsh G.I.
      • Saleem M.A.
      The podocyte cytoskeleton: key to a functioning glomerulus in health and disease.
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      A high-powered view of the filtration barrier.
      • Kistler A.D.
      • Altintas M.M.
      • Reiser J.
      Podocyte GTPases regulate kidney filter dynamics.
      FAs, by which cells are anchored to the extracellular matrix, are a major determinant of actin cytoskeleton dynamics and cellular motility.
      • Lauffenburger D.A.
      • Horwitz A.F.
      Cell migration: a physically integrated molecular process.
      Thus, high FA turnover is associated with high motility of cells.
      • Abou Zeid N.
      • Valles A.M.
      • Boyer B.
      Serine phosphorylation regulates paxillin turnover during cell migration.
      To our knowledge, the present study is the first attempt to explore the GSK3β-controlled FA turnover and the ensuing effects on actin cytoskeleton organization and cell motility in podocytes (Figure 10). We demonstrated that lithium, a selective GSK3β inhibitor, protected podocytes from doxorubicin 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 GSK3β overactivity; intercepting the GSK3β-directed hyperphosphorylation and overactivation of paxillin, a core structural component of FAs; and subsequently impeding FA turnover and overriding podocyte hypermotility (Figure 10).
      Figure thumbnail gr10
      Figure 10Schematic diagram detailing the mechanism of the GSK3β-governed FA dynamics in the pathogenesis of podocytopathy. GSK3β plays a key role in the regulation of FA turnover and podocyte motility by directing paxillin phosphorylation and activation and subsequently controlling actin cytoskeleton dynamics. As a redox-sensitive signaling transducer, the activity of GSK3β could be reinforced on oxidative stress induced by a variety of podocyte-injurious mediators, including Adriamycin (doxorubicin). GSK3β overactivity will elicit paxillin hyperphosphorylation and overactivation and thus cause the ensuing actin cytoskeleton disorganization and podocyte hypermotility, ultimately resulting in podocyte foot process effacement, massive proteinuria, and progressive glomerulosclerosis. GSK3β is a druggable target that could be blocked by lithium, a selective inhibitor of GSK3β and US Food and Drug Administration–approved mood stabilizer that has been safely used for >50 years as a first-line therapy for affective psychiatric disorders. Lithium treatment could override the Adriamycin-elicited GSK3β overactivity, counteract paxillin hyperphosphorylation and overactivation, and thereby obliterate podocyte hypermotility and reinstate actin cytoskeleton integrity. Consequently, lithium therapy could ameliorate the Adriamycin-induced podocyte foot process effacement, induce proteinuria remission, and improve glomerulosclerosis.
      GSK3β situates at the nexus of multiple crucial cell signaling pathways and is centrally involved in the pathogenesis of disease in multifaceted organ systems, including the kidney.
      • Wang Z.
      • Havasi A.
      • Gall J.
      • Bonegio R.
      • Li Z.
      • Mao H.
      • Schwartz J.H.
      • Borkan S.C.
      GSK3beta promotes apoptosis after renal ischemic injury.
      As a redox-sensitive signaling transducer, the activity of GSK3β could be substantially enhanced on oxidative stress induced by a multitude of podocyte-injurious mediators, such as doxorubicin and chronic kidney injuries.
      • Wang S.H.
      • Shih Y.L.
      • Kuo T.C.
      • Ko W.C.
      • Shih C.M.
      Cadmium toxicity toward autophagy through ROS-activated GSK-3beta in mesangial cells.
      We recently uncovered that expression of GSK3β is aberrantly up-regulated in diseased human kidneys in tubules and glomeruli.
      • Gong R.
      • Ge Y.
      • Chen S.
      • Liang E.
      • Esparza A.
      • Sabo E.
      • Yango A.
      • Gohh R.
      • Rifai A.
      • Dworkin L.D.
      Glycogen synthase kinase 3beta: a novel marker and modulator of inflammatory injury in chronic renal allograft disease.
      Similarly, Waters and Koziell
      • Waters A.
      • Koziell A.
      Activation of canonical Wnt signaling meets with podocytopathy.
      also noted up-regulation of GSK3β in human podocytes in association with specific NPHS1 mutations. Consistently, gene-targeted knock-in mice with mutated uninhibitable GSK3 developed albuminuria and podocyte injury, suggesting a detrimental role of GSK3 in podocyte injury.
      • Boini K.M.
      • Amann K.
      • Kempe D.
      • Alessi D.R.
      • Lang F.
      Proteinuria in mice expressing PKB/SGK-resistant GSK3.
      In contrast, studies exploiting selective small molecule inhibitors of GSK3β reached conflicting conclusions. For example, inhibition of GSK3β by the selective small molecule inhibitor 6-bromoindirubin-3′-oxime (BIO) at a low dose dramatically normalized proteinuria and attenuated histologic injury of glomeruli in rat models of diabetic nephropathy, although hyperglycemia was not corrected, implying direct antiproteinuric and renoprotective action.
      • Lin C.L.
      • Wang J.Y.
      • Huang Y.T.
      • Kuo Y.H.
      • Surendran K.
      • Wang F.S.
      Wnt/beta-catenin signaling modulates survival of high glucose-stressed mesangial cells.
      However, Matsui et al
      • Matsui I.
      • Ito T.
      • Kurihara H.
      • Imai E.
      • Ogihara T.
      • Hori M.
      Snail, a transcriptional regulator, represses nephrin expression in glomerular epithelial cells of nephrotic rats.
      found that high-dose BIO exacerbated proteinuria and loss of glomerular nephrin in puromycin-injured rats. Another study by Dai et al
      • Dai C.
      • Stolz D.B.
      • Kiss L.P.
      • Monga S.P.
      • Holzman L.B.
      • Liu Y.
      Wnt/beta-catenin signaling promotes podocyte dysfunction and albuminuria.
      reported that a transient and low level of proteinuria followed by a rapid spontaneous remission was provoked by an ultrahigh dose of lithium chloride (16 mmol/kg), which is almost two times the median lethal dose of lithium chloride in mice. In contrast, we demonstrated that low-dose lithium conferred prominent protection against podocyte injury. Of note, as typical chemical inhibitors of kinases, GSK3β blockers, including lithium, BIO, and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, if used at high doses, could have nonselective off-target effects and could induce cytotoxicity and even lethality.
      • Cohen P.
      • Goedert M.
      GSK3 inhibitors: development and therapeutic potential.
      Thus, the most likely explanation for these conflicting findings might be the difference in the doses of GSK3β inhibitor. Collectively, accumulating evidence indicates that GSK3β promotes podocyte injury and proteinuria, and inhibition of GSK3β by low-dose inhibitors might be beneficial for podocytopathy.
      Lithium, a selective inhibitor of GSK3β, has been commonly and safely used for the past 50 years as a US Food and Drug Administration–approved first-line drug to treat bipolar affective disorders.
      • Marmol F.
      Lithium: bipolar disorder and neurodegenerative diseases: possible cellular mechanisms of the therapeutic effects of lithium.
      • Yang E.S.
      • Wang H.
      • Jiang G.
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      • Fu A.
      • Hallahan D.E.
      • Xia F.
      Lithium-mediated protection of hippocampal cells involves enhancement of DNA-PK-dependent repair in mice.
      Recent evidence revealed that blockade of GSK3β by lithium reduces cellular motility in a variety of cells, including vascular smooth muscle cells,
      • Wang Z.
      • Zhang X.
      • Chen S.
      • Wang D.
      • Wu J.
      • Liang T.
      • Liu C.
      Lithium chloride inhibits vascular smooth muscle cell proliferation and migration and alleviates injury-induced neointimal hyperplasia via induction of PGC-1alpha.
      glioma cells,
      • Nowicki M.O.
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      • Stein A.M.
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      • Nita M.
      • Berens M.E.
      • Sander L.M.
      • Newton H.B.
      • Chiocca E.A.
      • Lawler S.
      Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3.
      gastric cancer cells,
      • Ryu Y.K.
      • Lee Y.S.
      • Lee G.H.
      • Song K.S.
      • Kim Y.S.
      • Moon E.Y.
      Regulation of glycogen synthase kinase-3 by thymosin beta-4 is associated with gastric cancer cell migration.
      and airway epithelial cells,
      • Wang W.C.
      • Kuo C.Y.
      • Tzang B.S.
      • Chen H.M.
      • Kao S.H.
      IL-6 augmented motility of airway epithelial cell BEAS-2B via Akt/GSK-3beta signaling pathway.
      suggesting that lithium might be a choice of therapy for diseases associated with cellular hypermotility, such as malignant tumor metastasis. Podocyte hypermotility is a central pathogenic mechanism accounting for nephrotic glomerulopathy induced by a variety of mediators, including soluble urokinase-type plasminogen activator receptor,
      • Wei C.
      • El Hindi S.
      • Li J.
      • Fornoni A.
      • Goes N.
      • Sageshima J.
      • Maiguel D.
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      • Roth D.
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      • Burke G.
      • Ruiz P.
      • Reiser J.
      Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis.
      proteases, and nephrotoxins such as doxorubicin.
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      • Sawai K.
      • Yoshioka T.
      • Nagae T.
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      • Sugawara A.
      • Nakao K.
      Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome.
      • Liu H.
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      • Feng C.
      • Kuang X.
      • Li Z.
      • Zha X.
      Alpha-Actinin-4 is involved in the process by which dexamethasone protects actin cytoskeleton stabilization from adriamycin-induced podocyte injury.
      Correction of podocyte hypermotility via therapeutic targeting of FA dynamics, a prerequisite of cell migration and motility, has been shown to successfully override podocyte injuries induced by podocytopathic mediators and to improve the podocyte structure and function.
      • Ma H.
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      • Ishibe S.
      Inhibition of podocyte FAK protects against proteinuria and foot process effacement.
      In this study, lithium, through stabilizing FA dynamics, also counteracted doxorubicin-elicited podocyte hypermotility and consistently resulted in a podocyte-protective and antiproteinuric effect in doxorubicin nephropathy. Apparently, this study might have an immediate implication for clinical translation into prophylactic treatment for recurrent focal and segmental glomerulosclerosis in kidney transplant patients, which has been attributed to a rapid podocytic injury associated with podocyte hypermotility caused by circulating permeability factors, such as soluble urokinase-type plasminogen activator receptor.
      • Wei C.
      • El Hindi S.
      • Li J.
      • Fornoni A.
      • Goes N.
      • Sageshima J.
      • Maiguel D.
      • Karumanchi S.A.
      • Yap H.K.
      • Saleem M.
      • Zhang Q.
      • Nikolic B.
      • Chaudhuri A.
      • Daftarian P.
      • Salido E.
      • Torres A.
      • Salifu M.
      • Sarwal M.M.
      • Schaefer F.
      • Morath C.
      • Schwenger V.
      • Zeier M.
      • Gupta V.
      • Roth D.
      • Rastaldi M.P.
      • Burke G.
      • Ruiz P.
      • Reiser J.
      Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis.
      • Morath C.
      • Wei C.
      • Macher-Goeppinger S.
      • Schwenger V.
      • Zeier M.
      • Reiser J.
      Management of severe recurrent focal segmental glomerulosclerosis through circulating soluble urokinase receptor modification.
      Of note, a basal level of podocyte motility is essential for sustaining the glomerular filtration barrier homeostasis. Podocyte motility that is too low secondary to genetic defects of cytoskeleton structural or regulatory molecules has been associated with cytopathic changes in podocytes that ultimately also result in focal and segmental glomerulosclerosis.
      • Welsh G.I.
      • Saleem M.A.
      The podocyte cytoskeleton: key to a functioning glomerulus in health and disease.
      Therefore, provided the observation that the lithium represses FA turnover and podocyte motility in normal podocytes, one of the conceivable concerns would be the potential podocytopathic effect of lithium therapy. Indeed, long-term lithium therapy primarily for psychiatric disorders has been complicated by some renal adverse effects, such as nephrotic syndrome, glomerular disease, and interstitial nephritis, as reported by Markowitz et al
      • Markowitz G.S.
      • Radhakrishnan J.
      • Kambham N.
      • Valeri A.M.
      • Hines W.H.
      • D'Agati V.D.
      Lithium nephrotoxicity: a progressive combined glomerular and tubulointerstitial nephropathy.
      in a case series report. However, according to a large-scale epidemiology study,
      • Bendz H.
      • Schon S.
      • Attman P.O.
      • Aurell M.
      Renal failure occurs in chronic lithium treatment but is uncommon.
      the incidence of chronic kidney disease in lithium-treated patients is actually comparable with that in the general population, suggesting that the lithium-associated renal adverse effects are uncommon. Furthermore, patients with lithium-associated kidney diseases usually have received lithium therapy at the psychiatric high dose for a long time (usually >10 years). In the present study, the single dose of lithium used (40 mg/kg) is much lower than the standard dose of lithium that has been safely and routinely used for neurobiology research (120 mg/kg) in rodents, and no detectable changes in glomerular histologic features or function were observed in control mice, suggesting that low-dose lithium might be protective for podocyte injury. Therefore, it seems that short-term use of low-dose lithium is safe in humans and might be a promising approach for preventing podocytopathies.
      In summary, GSK3β plays an important role in the regulation of FA turnover and podocyte motility by directing paxillin phosphorylation and activation and subsequently controlling actin cytoskeleton dynamics. Lithium, an inhibitor of GSK3β, attenuated the doxorubicin-elicited paxillin phosphorylation and rapid FA turnover, reinstated actin cytoskeleton integrity, and overrode podocyte hypermotility. In experimental doxorubicin nephropathy, a single low dose of lithium effectively suppressed the overactivity of GSK3β and paxillin, recovered actin cytoskeleton in glomerular podocytes, prevented podocyte foot process effacement, and attenuated proteinuria (Figure 10). Collectively, this study suggests that the GSK3β-governed FA dynamics might serve as a novel therapeutic target for podocytopathy.

      Supplemental Data

      • Supplemental Figure S1

        Lithium potentiates nephrin expression in differentiated conditionally immortalized murine podocytes. A: Differentiated podocytes in culture were treated with 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) for 24 hours, followed by fluorescence immunocytochemical staining for the podocyte-specific marker nephrin. Representative micrographs demonstrated that lithium treatment up-regulates the fluorescence immunocytochemical staining for nephrin in cultured podocytes. Arrowheads indicate nephrin staining. B: Differentiated (37°C) or undifferentiated (33°C) podocytes were treated with 10 or 20 mmol/L LiCl or 10 mmol/L NaCl for 24 or 48 hours, followed by collection of cell lysates and immunoblot analysis for nephrin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative immunoblots indicated that lithium treatment increases the expression of nephrin in cultured podocytes in a dose- and time-dependent manner. C: Densitometric analysis of immunoblots quantified the relative abundance of nephrin normalized to GAPDH as folds of the group of undifferentiated podocytes. Data are given as means ± SD. n = 3 separate experiments. P < 0.05 versus NaCl-treated differentiated podocytes. Scale bar = 5 μm (A).

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