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Converging evidence points to glycogen synthase kinase (GSK) 3 as a key player in the pathogenesis of podocytopathy and proteinuria. However, it remains unclear if GSK3 is involved in podocyte autonomous injury in glomerular disease. In normal kidneys, the β isoform of GSK3 was found to be the major GSK3 expressed in glomeruli and intensely stained in podocytes. GSK3β expression in podocytes was markedly elevated in experimental or human proteinuric glomerulopathy. Podocyte specific somatic ablation of GSK3β in adult mice attenuated proteinuria and ameliorated podocyte injury and glomerular damage in experimental adriamycin (ADR) nephropathy. Mechanistically, actin cytoskeleton integrity in podocytes was largely preserved in GSK3β knockout mice following ADR insult, concomitant with a correction of podocyte hypermotility and lessened phosphorylation and activation of paxillin, a focal adhesion associated adaptor protein. In addition, GSK3β knockout diminished ADR induced NFκB RelA/p65 phosphorylation selectively at serine 467, suppressed de novo expression by podocytes of NFκB dependent podocytopathic mediators, including B7-1, cathepsin L and MCP-1, but barely affected the induction of NFκB target prosurvival factors, such as Bcl-xL. Moreover, the ADR-elicited podocytopenia and podocyte death was significantly attenuated in GSK3β knockout mice, associated with a protection against podocyte mitochondrial damage and reduced phosphorylation and activation of cyclophilin F, a structural component of mitochondria permeability transition pores. Overall, our findings suggest that the β isoform of GSK3 mediates autonomous podocyte injury in glomerulopathy by integrating multiple podocytopathic signalling pathways.
Regardless of the original aetiology, the pathogenic basis of persistent proteinuria is generally attributable to podocyte dysfunction or injury . Converging evidence recently points to glycogen synthase kinase (GSK) 3 as a novel therapeutic target for podocytopathy and proteinuria [2-4]. In accordance with this, blockade of GSK3 has been shown to attenuate proteinuria, podocyte injury and glomerulosclerosis in a number of experimental glomerular diseases, including diabetic nephropathy , lupus nephritis , and podocytopathy elicited by adriamycin (ADR) or LPS [2-4]. GSK3 is a highly conserved, ubiquitously expressed, and constitutively active multitasking serine/threonine protein kinase, situated at the nexus of a multitude of essential pathways in the complex cell signalling network and acting on a multitude of cognate substrates, including NFκB, β-catenin, paxillin, cyclophilin F (Cyp-F), and cyclin D1 [3,4,7-11]. GSK3 exists as two isoforms, GSK3α (51 kDa) and GSK3β (47 kDa), which are encoded by separate genes that produce highly homologous proteins with significant difference only in their N- and C-terminal regions . Although displaying 84% structural homology, GSK3α and GSK3β are not functionally interchangeable, and each possesses unique and distinct biological actions [13-17]. The two isoforms exert similar or distinct roles in diverse cellular processes, including glycogen metabolism, cell cycle progression, cytoskeletal organization, inflammation and immunity, and mitochondria permeability transition, and have been recently identified as a putative target for the treatment of disorders including diabetes, cancer, Alzheimer's disease and glomerulopathy [17-21].
Despite burgeoning evidence suggesting a role for GSK3 in podocyte injury, previous data are not conclusive due to the following limitations. First, most studies relied exclusively on the use of small molecule inhibitors of GSK3β, which acts in a concentration-dependent way and may possess additional unknown properties, such as off-target activities. Secondly, chemical inhibitors developed so far all possess a poor selectivity between the isoforms of GSK3, even though it remains unknown if the two isoforms of GSK3 are equally expressed in glomerular podocytes or play the same role in the pathogenesis of podocyte injury. Thirdly, podocyte injury and proteinuria in most glomerular diseases result from both podocyte autonomous and non-autonomous pathogenic processes. Thus, with systemic use of chemical inhibitors of GSK3β in vivo it is impossible to discriminate a direct podocyte effect from an effect secondary to systemic actions, such as immunomodulation . Genetic knockout of GSK3β would be a conclusive approach with great targeting selectivity, but unfortunately has caused embryonic lethality . To circumvent these issues, inducible deletion of GSK3β in mature glomerular podocytes offers an ideal and preferred approach to avoid potential congenital defects of the kidney. In this study, mice with the doxycycline-inducible podocyte specific somatic ablation of GSK3β were exposed to ADR injury and the role of GSK3β in podocyte injury and proteinuria was delineated in knockout mice in vivo and in primary podocytes in vitro.
Animal studies were approved by the institution's Animal Care and Use Committee and they conformed to the United States Department of Agriculture regulations and the National Institutes of Health guidelines for humane care and use of laboratory animals.
Mice with the floxed GSK3β gene (GSK3βfl/+), on a genetic background of C57/B6J, were used previously [15,24] and backcrossed for 10 generations onto an FVB/N congenic background. To generate mice with doxycycline-inducible podocyte-specific GSK3β KO genotype (NPHS2rtTA/TRECre/GSK3βfl/fl), GSK3βfl/+ mice were respectively crossed with TREcre mice and NPHS2rtTA mice on a genetic background of FVB/N (Jackson Laboratory, Bar Harbor, ME). All animals were born normally at the expected Mendelian frequency. For the induction of podocyte-specific GSK3β deletion, 8- to 10-week-old mice with KO genotype received oral doxycycline (TCI, Tokyo, Japan; 2mg/ml with 5% sucrose, protected from light) for 2 wk and were subsequently designated as KO mice. Littermates with the same KO genotype but not treated with doxycycline served as control (Ctrl). Littermates with the NPHS2rtTA/GSK3βfl/fl genotype were treated with doxycycline for 2 wk and served as Cre-negative control (Cre-Ctrl). A routine PCR protocol was performed (Figure S1) for genotyping tail DNA samples using transgene-specific primers (Supplementary Table S1).
KO mice, Ctrl and Cre-Ctrl littermates aged 10 to 12 wk were randomly assigned to receive ADR (25 mg/kg) or vehicle treatment via tail vein injection on day 0. Spot urine was collected and mice were sacrificed on post injury days 0, 2, 6, and 14. Six mice were randomly assigned to each group for each observed time point. For additional experiments, male Balb/c mice (Jackson Laboratory) aged 8 wk received a low dose of ADR (10mg/kg) or vehicle via tail vein injection and were sacrificed 4 wk later.
This study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board of Shanghai Tenth People's Hospital of Tongji University School of Medicine. Written informed consent was obtained from each subject. Archived formalin-fixed paraffin-embedded kidney biopsy tissues were randomly chosen for analysis from three patients diagnosed with each of the following renal diseases, including idiopathic focal segmental glomerulosclerosis (FSGS), class V lupus nephritis [LN(V)] and tubulointerstitial nephritis (TIN). Morphologically normal tissues from the unaffected areas of renal tumour nephrectomy specimens were used as controls.
Urine samples were subjected to SDS-PAGE followed by Coomassie Brilliant Blue (Sigma) staining. Urine albumin concentration was measured using a mouse albumin ELISA quantitation kit (Bethyl Laboratories Inc., Montgomery, TX, USA). Urine and serum creatinine concentration was measured by a creatinine assay kit (BioAssay Systems, Hayward, CA, USA).
Formalin-fixed mouse kidneys were embedded in paraffin and 3μm-thick sections prepared. Sections were processed for PAS staining or immunohistochemical staining. A semiquantitative glomerular damage index [25-28] was used to evaluate the degree of glomerular damage. The detailed protocol is provided in the Supplementary Methods.
Glomeruli were isolated from mouse kidneys by a magnetic beads based approach as described previously . Primary podocytes were prepared from the isolated glomeruli as reported before [29-31]. A detailed protocol is provided in the Supplementary Methods.
Cultured cells were lysed and animal tissues homogenized for immunoblot analysis or immunoprecipitation. Detailed protocols are provided in the Supplementary Methods.
Fixed cells were subjected to TUNEL staining by using a commercial kit (Roche Molecular Biochemicals, Mannheim, Germany) as described previously .
For immunoblot analysis, bands were scanned and the integrated pixel density was determined using a densitometer and the ImageJ analysis program. All data are expressed as means ± SD. Data from two groups were compared by the t test. For multiple comparisons of values, ANOVA was performed followed by the Student-Newman-Keuls test or the Scheffé test; p < 0.05 was considered significant.
The two isoforms of GSK3, GSK3α and GSK3β, have been recently shown to have distinct distributions in distal nephrons . In mouse kidney, both the α and the β isoforms were evidently expressed in mouse renal parenchyma as shown by immunoperoxidase staining (Figure 1A). In glomeruli, the β rather than the α isoform was mainly detected and was located within the mesangium and the periphery of glomerular tufts, consistent with a podocyte distribution. Immunoblot analysis demonstrated that GSK3β was expressed in much greater abundance than GSK3α in isolated glomeruli, primary podocytes and an immortalized podocyte cell line (Figure 1B). In contrast, in whole kidney homogenates, GSK3α was detected at a level almost equal to GSK3β (Figure 1B). Likewise, in normal human kidney specimens, immunostaining also revealed predominant GSK3β expression in glomeruli, with intense staining clearly localized within podocytes at the periphery of glomerular tufts (Figure 1C). Computerized morphometric analysis (Figure 1D, E) precisely quantified the integrated pixel densities of the staining and indicated that the level of GSK3β expression was 1.5-fold greater than GSK3α in glomeruli of human kidney specimens (Figure 1E).
Increased expression of GSK3β in renal tubules has been implicated in the pathogenesis of progressive chronic kidney disease . Whether glomerular expression of GSK3β alters in glomerular disease was explored next. We first assessed experimental FSGS in ADR-injured Balb/c mice [35,36]. Four wk after ADR injury, representing the chronic stage of ADR nephropathy, glomerular staining of GSK3β was markedly increased with intense immunostaining located in podocytes at the periphery of glomerular tufts (Figure 2A). To determine if the elevated glomerular expression of GSK3β also occurs in human proteinuric glomerular disease, kidney biopsy tissues were examined. As shown in Figure 2B, the staining of GSK3β in glomeruli was substantially enhanced in two proteinuric glomerulopathies, FSGS and LN(V), with strong staining localised to podocytes at the periphery of glomerular tufts, compatible with the findings in experimental ADR nephropathy. In contrast, only a weak level of induced glomerular expression of GSK3β was noted in TIN. The morphological findings were further corroborated by the computerized morphometric analysis, which demonstrated 1.6 fold induction in mouse ADR nephropathy as compared with control mice (Figure 2C), and 1.1, 2.6 and 3.1 fold induction respectively in TIN, FSGS and LN(V) as compared with normal human kidney tissues (Figure 2D) .
To further substantiate a possible role of GSK3β in the pathogenesis of proteinuria and podocyte injury, the doxycycline-inducible Cre/loxP mediated gene targeting strategy was adopted to ablate GSK3β selectively in mature podocytes (Figure S2A). Following doxycycline treatment, KO mice exhibited no detectable abnormalities in development, kidney gross morphology, function (Figure S3) or histology (Figure 3) as compared with Ctrl littermates. Dual colour immunofluorescent staining revealed striking loss of GSK3β immunoreactivity specifically in synaptopodin-positive podocytes in KO mice (Figure S2A), inferring a successful knockout. To confirm this finding, primary podocytes were isolated and cultured from glomeruli prepared from WT, KO and Ctrl mice and subjected to immunoblot analysis (Figure S2B) and fluorescent immunostaining (Figure S2C). GSK3β was drastically ablated, whereas GSK3α expression was barely affected (Figure S2B) in primary KO podocytes that were characterized by the expression of typical podocyte markers, including podocin, Wilms' tumor 1 (WT1) and synaptopodin (Figure S2C). Of note, GSK3β knockout seemed barely to affect β-catenin signalling, a major downstream pathway of GSK3, because the levels of total β-catenin and nuclear translocation of β-catenin were comparable between KO podocyte and Ctrl or WT podocytes, as determined by immunoblot analysis (Figure S2B).
To examine the effect of GSK3β knockout on podocyte injury in vivo, ADR nephropathy was developed in KO, Ctrl and Cre-Ctrl mice. In Cre-Ctrl and Ctrl mice, ADR injury was equally effective at eliciting a heavy proteinuria that peaked on day 6 and partially receded on day 14, as estimated by urine electrophoresis and quantified by urine albumin to creatinine ratios (Figure 3A, B). This was associated with prominent renal histological injury on PAS staining (Figure 3C), featured by large protein casts in tubules, glomerular synechiae, mesangial extracellular matrix accumulation and progressive glomerulosclerosis (Figure 3C). In addition, ultrastructural injuries in podocytes, such as extensive podocyte foot process effacement, were evident from transmission electron microscopy images (Figure 3D). These morphological findings were further corroborated by the semiquantitative morphometric measurements of glomerular damage index (Figure 3E) and by absolute count of the number of foot process per unit length of glomerular basement membrane (Figure 3F). In KO mice, the ADR induced proteinuria was drastically attenuated (Figure 3A, B), concomitant with lessened histological injury in glomeruli and tubulointerstitium (Figure 3C, E) and improved podocyte injury and foot process effacement (Figure 3D, F). In parallel, focal lesions of fibrosis and inflammatory infiltration in the kidney 14 d after ADR injury were ameliorated in KO mice, as shown by immunofluorescent staining (Figure 3G) and immunoblot analysis of kidney homogenates (Figure 3H) for fibronectin and the leukocyte common antigen CD45.
Disruption of actin cytoskeleton integrity is the molecular basis for podocyte foot process effacement [37-40]. To determine if the attenuated podocyte foot process effacement found in KO mice after ADR injury is attributable to any improvement in actin cytoskeleton in podocytes, kidney specimens procured from animals on day 6 were subjected to both phalloidin labelling for filamentous actin (F-actin) and immunofluorescence staining for synaptopodin (Figure 4A). Confocal fluorescence microscopy demonstrated that intense F-actin staining was present extensively all over the glomerular tufts of normal kidneys from KO and Ctrl mice. Podocyte specific F-actin was highlighted by co-localization of F-actin signals (red) with synaptopodin expression (green) in glomeruli. ADR injury in Ctrl mice resulted in a remarkable reduction in F-actin and synaptopodin co-localization, indicative of disruption of actin cytoskeleton integrity in podocytes. This injurious effect of ADR was significantly abrogated in KO mice. To corroborate the morphological findings, computerized morphometric analysis was performed and revealed that the ratio of integrated pixel densities between the signal of podocyte specific F-action (yellow signal) and the signal of podocytes (green signal) was decreased in Ctrl mice but considerably preserved in KO mice following ADR injury (Figure 4B). Evidence suggests that GSK3β dictates the phosphorylation and activation of paxillin [3,41], which is a core structural and regulatory component of focal adhesions, the crucial determinant of cellular actin cytoskeleton dynamics and motility. In consistency, knockout of GSK3β dramatically abolished the ADR elicited paxillin hyperphosphorylation in glomeruli, shown by immunoblot analysis of the isolated glomeruli (Figure 4C).
To understand if the protective effect on podocyte cytoskeleton observed in KO mice may be ascribed primarily to a direct effect on podocytes, primary podocytes were prepared from Ctrl or KO mice and examined. Podocytes are motile cells with considerable constitutive motility . Indeed, as shown by a traditional cell migration assay, podocytes derived from Ctrl mice possessed a substantial migratory capacity under basal conditions that lessened the distances between the leading edges of the migrating podocyte sheets (Figure 4D). This basal migratory capacity was slightly reduced in podocytes isolated from KO mice, as quantified by morphometric analysis of cell migration areas (Figure 4E). ADR injury strikingly accelerated the closure of the gap between the invading fronts of podocytes from Ctrl mice, indicative of podocyte hypermotility. This effect of ADR was markedly abrogated in podocytes from KO mice, suggesting that loss of GSK3β impedes podocyte hypermotility. The difference seen in the motility assay was unlikely to be attributable to distinct proliferative ability, because, as terminally differentiated cells, primary podocytes exhibited a low proliferative ability that was comparable between KO and WT podocytes as shown by immunoblotting for proliferating cell nuclear antigen (Figure S4). Focal adhesion turnover is a prerequisite for cell spreading and migration [43-45]. As a key adaptor and structural component of the focal adhesions, paxillin was hyperphosphorylated in Ctrl podocytes upon ADR injury, as measured by immunoblot analysis. This effect was markedly mitigated in KO podocytes (Figure 4F). To visualize the changes in actin cytoskeleton, fluorescent immunocytochemistry for paxillin and phalloidin labelling for F-actin was carried out in primary cells. ADR injury in Ctrl podocytes caused a typical disruption of actin cytoskeleton, manifested as increased distribution of cortical filaments, diminished ventral stress fibres, more transverse arcs, and sporadic short dorsal stress fibres that were connected to focal adhesions only at one end, marked by colocalization of F-actin with paxillin. In contrast, KO podocytes largely preserved F-actin stress fibres following ADR and retained the pattern of focal adhesion distribution (Figure 4G).
Evidence suggests that podocyte de novo expression of multiple podocytopathic mediators, such as B7-1  and cathepsin L , is implicated in podocyte cytoskeleton disorganization and foot process effacement. Of note, expression of these podocytopathic molecules is under the control of NFκB [48,49]. In agreement, ADR injury in Ctrl mice elicited marked expression of a multitude of NFκB target molecules, including monocyte chemoattractant protein-1 (MCP-1), B7-1, cathepsin L and the prosurvival protein Bcl-xL, concomitant with an induced phosphorylation of NFκB RelA/p65 at all serine residues (serine 276, 467 and 536) as assessed by immunoblot analysis of the isolated glomeruli (Figure 5A). The expression of these molecules in glomeruli was predominantly localised to podocytes that were positive for synaptopodin or WT1, as revealed by fluorescent immunohistochemistry, implying a podocyte specific induction of NFκB dependent molecules involved in both podocytopathy and prosurvival (Figure 5B). Our latest data indicate that the serine 467 residue of RelA/p65 lies within the consensus motif for GSK3β phosphorylation and serves as a cognate substrate of GSK3β . In podocytes, GSK3β fine tunes NFκB activation and directs the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL . Congruously in this study, the ADR elicited expression of podocytopathic factors, like MCP-1, B7-1 and cathepsin L, was considerably abrogated in KO mice, but the Bcl-xL induction in podocytes was largely preserved, associated with a diminished phosphorylation of NFκB RelA/p65 selectively at serine 467. To verify if loss of GSK3β affects proinflammatory NFκB activation and de novo expression of podocytopathic mediators in a podocyte autonomous fashion, primary podocytes were examined next. Shown in Figure 5C, ADR injury induced RelA/p65 phosphorylation at all serine residues in Ctrl podocytes and elicited the expression of diverse NFκB target molecules, including both prosurvival Bcl-xL and podocytopathic mediators, like MCP-1, B7-1 and cathepsin L, as measured by immunoblot analysis. In contrast in KO podocytes, the ADR induced RelA/p65 phosphorylation was selectively mitigated at serine 467, associated with minimal induction of podocytopathic mediators, like MCP-1, B7-1 and cathepsin L, but a preserved up-regulation of prosurvival Bcl-xL. Fluorescent immunocytochemistry demonstrated prominent de novo expression of B7-1 and cathepsin L in Ctrl podocytes but minimal expression in KO podocytes following ADR injury (Figure 5D).
As a prototypical pro-oxidant, ADR is known to cause podocyte loss by inducing oxidative stress and podocyte mitochondrial dysfunction, ensued by podocyte loss and podocytopenia . In concordance with this fluorescence immunohistochemistry (Figure 6A) and immunoblot analysis of isolated glomeruli for WT1 (Figure 6B) indicated that ADR injury resulted in striking podocytopenia in Ctrl mice on day 6, associated with evident mitochondria damage in podocytes, characterized by decreased number of mitochondria, mitochondrial swelling and fragmentation, destruction of the inner mitochondrial membrane and disruption of mitochondrial cristae on electron microscopy (Figure 6C). In contrast, the ADR elicited podocytopenia and podocyte mitochondria damage was ameliorated in KO mice. To explore if GSK3β knockout affects autonomous podocyte injury, primary podocytes were examined. Upon ADR injury, KO versus Ctrl podocytes developed much less apoptosis as evaluated TUNEL staining (Figure 6D) and validated by immunoblot analysis of cell lysates for cleaved caspase 3 (Figure 6E). Under basal condition, MitoTracker Red labelling of Ctrl or KO podocytes (Figure 6F) revealed a healthy complex network of mitochondria of elongated, branched and non-uniform shape spreading throughout the whole podocyte cell body and stretching out to podocyte extensions. Upon ADR injury, the mitochondria network in Ctrl podocytes was drastically disrupted into smaller mitochondria in a shape of small spheres or short rods that were mainly concentrated in the perinuclear area. This effect was dramatically attenuated in KO podocytes, suggesting a direct protective effect of GSK3β knockout on mitochondria injury. Dual colour staining for cleaved caspase-3 and mitochondria demonstrated that mitochondrial damage preceded overt apoptosis, and both were sequentially mitigated in KO podocytes upon ADR insult, inferring that the lessened mitochondrial damage in KO podocytes is a primary effect of GSK3β ablation rather than an effect secondary to the anti-apoptotic action. Our latest data indicated that Cyp-F, a core structural constituent of the mitochondria permeability transition (MPT) pore, is a substrate for GSK3β . In agreement, loss of GSK3β in KO podocytes abolished the ADR elicited phosphorylation of Cyp-F, as shown by immunoblot analysis of immunoprecipitated Cyp-F for phosphorylated serine (Figure 6G).
As a key structural constituent of the glomerular filtration barrier, podocyte dictates glomerular permselectivity and its injury has been implicated to play a central role in the pathogenesis of proteinuria and progression of chronic renal disease [1,51-53]. This study examined the effect of podocyte specific ablation of GSK3β on podocytopathy and the underlying mechanisms. Our results suggest that GSK3β signalling is responsible, at least in part, for podocyte autonomous injury.
The finding in this study that podocyte specific ablation of GSK3β attenuated proteinuria and podocyte injury is consistent with the data using chemical inhibitors, like lithium or TDZD-8 [2,4]. However, it seems that pharmacologic blockade of GSK3β attained a pronounced proteinuria-reducing efficacy. This discrepancy might be ascribed to the difference in the mechanism of action between systemic and podocyte specific GSK3β inhibition. In addition to an effect on podocyte autonomous injury, systemic blockade of GSK3β by chemical inhibitors is also able to regulate global immunity  and thus possibly corrects the ADR impaired systemic immune homeostasis, which contributes to podocytopathy and proteinuria by generating podocyte injurious immunogenic factors . Alternatively, systemic inhibition of GSK3β might protect other kidney parenchymal cells from injury, like glomerular endothelial cells and mesangial cells. Apart from podocytes, GSK3β also plays a detrimental role in mediating injuries in other kidney cells and nephron segments [33,54]. At least, amelioration of glomerular endothelial injury has been shown to improve glomerular filtration barrier and attenuate proteinuria in experimental ADR nephropathy .
The glomerular filtration barrier is a highly dynamic structure. In support of this, perfusion of rat kidneys with the polycation protamine sulphate elicits foot process effacement and proteinuria within a few minutes, which can be fully reversed within 15 min by the administration of the polyanion heparin . These rapid changes of podocyte foot processes architecture require foot processes to retract or elongate, hence to “move” quickly along the glomerular basement membrane. Most importantly, podocyte movement was directly observed via multiphoton imaging in injured glomeruli in rats with experimental FSGS, suggesting that podocyte motility occurs in vivo . Built upon this observation, podocyte cultures have been used to study their motility patterns in vitro . Stimuli that induce foot process effacement in vivo (e.g., LPS or ADR) have been found to induce hypermotility of cultured podocytes in cell migration assays. In our study, the ADR-elicited hypermotility was attenuated in KO podocytes, indicative of a pro-motility effect of GSK3β. GSK3β seems to exert this effect by regulating multiple structural components involved in the control of cellular motility, including cytoskeletons and focal adhesions. Indeed, GSK3β is able to directly catalyse the phosphorylation and activation of paxillin, a focal adhesion associated adaptor protein, and thereby accelerate focal adhesion turnover, resulting in podocyte hypermotility and disruption of actin cytoskeleton integrity. Besides, GSK3β was found to catalyse the phosphorylation of serine 467 of NFκB RelA, which specifies the transcription of a subset of NFκB dependent molecules involved in podocyte cytoskeleton disorganization, like MCP-1, B7-1, and cathepsin L (Figure 6H). Moreover, GSK3β is also likely involved in MPT. Knockout of GSK3β reduced the activation of clyclophilin F, a key structural component of the MPT pore, resulting in a lessened mitochondrial damage and apoptosis in podocytes (Figure 6H).
In addition to the regulation of podocyte cytoskeletons, NFκB-dependent acquisition of proinflammatory phenotypes, and MPT, GSK3β is also a crucial element of the canonical Wnt/β-catenin pathway, which has been recently implicated in podocytopathy [59,60]. However, a drastic inhibition of GSK3 seems to be essential to activate the β-catenin pathway. In support of this, Doble et al. revealed a functional redundancy of GSK3α and GSK3β in Wnt/β-catenin signalling by using compound knockouts of GSK3α and β . They found that gene deletion of at least three of the four alleles of both isoforms of GSK3α and β is required to show an appreciable change in β-catenin activation. In consistency, our study demonstrated that knockout of GSK3β barely activate β-catenin in podocytes.
In summary, podocyte specific ablation of GSK3β attenuated podocyte injury and ameliorated proteinuria in ADR nephropathy. Our data suggest that GSK3β mediates podocyte autonomous injury in proteinuric glomerulopathy possibly by integrating multiple podocytopathic signalling pathways.
Figure S1. Genotyping transgenic mice by PCR.
Figure S2. Podocyte specific somatic ablation of glycogen synthase kinase 3β (GSK3β) in adult mice is achieved by the doxycycline inducible Cre/loxP mediated gene targeting system.
Figure S3. Podocyte specific knockout of GSK3β in adult mice results in no noticeable difference in development, gross kidney morphology, and kidney function under physiological condition.
Figure S4. Primary podocyte derived from Ctrl and KO mice exhibit a low but equal proliferative ability.
Table S1. Nucleotide sequences of the primers used for PCR genotyping
This work was supported in part by the U.S. National Institutes of Health grant R01DK092485, the Natural Science Foundation of China 81270136/H0111, the Foundation for Health and the International Society of Nephrology (ISN) Sister Renal Center Trio Program. The authors are indebted to the technical assistance provided by Dr. Pei Wang and Sijie Zhou. Changbin Li is an ISN fellow and recipient of the ISN fellowship.
Disclosure: All the authors declared no competing interests.
Statement of Author Contributions: C.L. and R.G. conceived and designed the study, contributed to data acquisition and interpretation, and drafted the manuscript. C.L. and Y.G. performed experiments and analysed data. L.D. and A.P. contributed to study design and data interpretation. All authors were involved in drafting the manuscript and had final approval of the submitted and published versions.