Developmental genetic loss of podocyte/nephrocyte GSK3 is catastrophic

To study the developmental importance of GSK3, podocyte-specific GSK3α, GSK3β and combined GSK3 α/β knockout (podGSK3DKO) transgenic mice were generated. This was achieved by crossing floxed GSK3α16 and/or GSK3β mice17 with a podocin Cre mouse31 (Supplementary Fig. 1a). Mice were genotyped and genomic excision of GSK3α and β DNA verified (Supplementary Fig. 1b). Furthermore, GSK3 isoform protein loss was confirmed using IHC (Supplementary Fig. 1c).

All genotypes were born with normal Mendelian frequency (Supplementary Table 1) indicating that there was no pre-natal lethality. Single isoform podocyte-specific deletion of GSK3α or β, as well as deletion of three out of four of the GSK3 alleles (i.e. podCreGSK3αfl/fl βfl/wt or podCreGSK3αfl/wtβfl/fl) caused no discernible phenotypes, with mice surviving normally to two years of age. However, when both isoforms were simultaneously deleted (i.e. all four floxed GSK3 alleles inactivated [podCreGSK3αfl/flβfl/fl or podGSK3DKO]) the mice all died between postnatal day 10 and 16 (p = 0.003 [Fig. 1a]). Prior to death these mice were indistinguishable from their littermates with regard to external appearance, weight and behaviour (Supplementary Fig. 1e). However, at time of death they had pronounced renal involvement with enlarged pale kidneys (Supplementary Fig. 1f), renal failure (Fig. 1b), acidosis (Fig. 1c), and high levels of albuminuria (Fig. 1d; Supplementary Fig. 1g). Renal disease evolved rapidly after birth as evidenced by a 25-fold increase in albuminuria from P2 to P10 (Fig. 1d). Histological examination using light and transmission electron microscopy (TEM) also revealed that glomerular and renal abnormalities were initially subtle in podGSK3DKO mice, but these rapidly progressed over the first 10 days of life to show glomerulosclerosis, multiple tubular protein casts and major disruption of the glomerular filtration barrier on TEM (Fig. 1e). By P10 highly abnormal, cystic and vacuolated, glomeruli were evident in the podGSK3DKO mice (Fig. 1f).

Fig. 1 Developmental loss of podocyte/nephrocyte GSK3 is catastrophic. a Kaplan–Meier survival plot. Log-rank (Mantel-Cox test) p = 0.00307 (Cre negative controls n = 3; podGSK3αKO n = 3; podGSK3βKO n = 3; podGSK3DKO n = 19 mice). b podGSK3DKO mice are in renal failure with elevated urea levels at day 11 (one-way ANOVA, Tukey’s post hoc test **p < 0.01; ***p < 0.001 Cre negative controls n = 25; podGSK3βKO n = 3; podGSK3DKO n = 6 mice). c podGSK3DKO mice are acidotic at day 11 with significantly lower bicarbonate levels (one-way ANOVA, Tukey’s post hoc test ***p < 0.001 Cre negative controls n = 16; podGSK3βKO n = 4; podGSK3 DKO n = 3 mice). d Rapid progression of kidney disease in the first 11 days of life in podGSK3DKO mice. Top: a Coomassie gel showing progression of albuminuria in an illustrative knockout and control mouse (2 μl of urine loaded in each lane). See also Supplementary Fig. 1 g. Bottom: graph showing the albumin:creatinine ratio (ACR) in a population of podGSK3DKO and control mice (t test **p < 0.01; ***p < 0.001 Cre negative controls P2 n = 8; podGSK3DKO P2 n = 3; Cre negative controls P5 n = 5; podGSK3DKO P5 n = 3; Cre negative control P10 n = 10; podGSK3DKO P10 n = 10 mice). e PAS (scale bar 25 μm) and transmission electron microscopy (scale bar = 1 μm) of podGSK3DKO and control mice at P1, P5 and P10. f H & E at P10 of podGSK3DKO and control mouse shows tubular protein casts and vacuolated glomeruli Scale bar = 25 μm. g Specific shaggy knockdown and control nephrocytes immunostained with dumbfounded (cyan). Scale bar = 100 µm. HT heart tube. h Nephrocyte number n = 6–10 flies per genotype; ANOVA, ***p < 0.001 for the effect of genotype. Data are presented as the mean + /-SEM Full size image

To assess whether GSK3 plays similar roles in Drosophila nephrocytes we used the dKlf15-Gal4 driver to silence the GSK3 gene here, which is called shaggy. This resulted in a total loss of pericardial nephrocytes, indicative of a critical requirement for shaggy during nephrocyte development (Fig. 1g, h). Interestingly, complete absence of nephrocytes was not overtly detrimental to Drosophila.

Mature genetic loss of podocyte/nephrocyte GSK3 is detrimental

After establishing the developmental importance of GSK3 in the podocyte/nephrocyte we evaluated the role of GSK3 in mature cells. Podocin RtTA -tet-o-Cre GSK3αfl/flβfl/fl (mpodGSK3DKO) mice were generated and given doxycycline from 4 weeks of age to specifically delete GSK3 in their podocytes after glomerular development was complete (Supplementary Figs. 2a–d). These mice were compared to Cre negative and non-doxycycline treated control littermates. All but one of the mpodGSK3DKO mice developed albuminuric kidney disease (Fig. 2a, b), but none of the control mice did. This occurred rapidly in the majority of mpodGSK3DKO mice with many developing albuminuria that was detectable within one week of finishing doxycycline (Fig. 2a, b). There was variability in the severity of their renal disease (Fig. 2a; Supplementary Fig. 3a), with some mpodGSK3DKO mice only mildly albuminuric at 22 weeks of age, but in all there was evidence of mesangial hypercellularity and glomerular fibrosis on trichrome staining (Fig. 2c, Supplementary Fig. 3a). In contrast ~30% of animals had raised serum creatinine relative to controls (Supplementary Fig. 3b), associated with global glomerulosclerosis and widespread interstitial fibrosis throughout the kidney (Fig. 2c; Supplementary Fig. 3a). One mouse was found to have severe hypertension (204 mmHg (SEM ± 2.67) / 158 mmHg (SEM ± 2.37) c/w systolic 115 (SEM ± 3.34) and diastolic 85 (SEM ± 2.46) in controls). mpodGSK3DKO mice had fewer podocytes and reduced nephrin expression compared to littermate controls (Supplementary Figs. 3c, d). Fascinatingly, approximately 20% of mpodGSK3DKO mice developed a florid crescentic glomerulopathy (Fig. 2d). To elucidate the cellular origin of the infiltrating crescentic cells we crossed the mpodGSK3DKO mouse with the mT/mG fluorescent reporter mouse to lineage tag the podocytes. This revealed extracapillary cells were of podocyte origin (Fig. 2e).

Fig. 2 Mature loss of podocyte/nephrocyte GSK3 is detrimental. a Significant albuminuria in mpodGSK3DKO mice. Population characteristics shown. Mann-Whitney Analysis of groups ***p < 0.001 (n = 9–10). b Representative Coomassie staining of urine from mice. 2 μl urine loaded. c Spectrum of renal involvement in model. PAS and trichrome staining. Left panels = control Middle panel = least albuminuric mouse. Right = most albuminuric mouse with global glomerulosclerosis. This mouse was in renal failure with severe hypertension. Scale bar = 25 μm. See also Supplementary Fig. 3. d Representative picture of glomerular crescents in mpodGSK3DKO mice. Scale bar = 50 μm. e Lineage tagging of mpodGSK3DKO podocytes by crossing with mT/mG reporter shows extracapillary cells are originating from podocytes. GFP and merge show green fluorescent protein (GFP) tagged podocytes in Bowman’s space (arrowed). Scale bar = 25 μm. f Glomerular filtration barrier TEM from two 22-week mpodGSK3DKO mice. Shows podocyte foot process retraction, mesangial hypercellularity and glomerular basement membrane thickening. Scale bar top panel = 10 μm; bottom panel = 500 nm. g Confocal micrographs of adult Drosophila heart stained with phalloidin (to visualise the heart tube [HT]) and antibodies to the nephrocyte marker protein Duf. Adults were reared at 18 °C until eclosure, then transferred to 29 °C. This temperature shift permits the expression of RNAi to shaggy specifically in nephrocytes at the higher but not the lower temperature. At 29 °C, nephrocytes in the sgg RNAi flies developed an enlarged morphology and abnormal foci of Duf immunoreactive staining across the cell surface (arrow). Controls at the same temperature exhibit a wild-type morphology (three nephrocytes are shown). Scale bar = 50 μm. h Graphs show number of nephrocytes in adult flies and percentage that were immunopositive for anti-Duf antisera. ANOVA *p < 0.05; n = 6–8 flies for each genotype at each temperature. Data are presented as the mean ± SEM Full size image

High-powered TEM demonstrated foot process effacement in mpodGSK3DKO podocytes together with basement membrane thickening in some mice. Low power TEM images confirmed our observations of mesangial hypercellularity and enlarged glomeruli. However, glomerular endothelial appearance and fenestrations were unchanged (Fig. 2f).

We also temporally deleted GSK3 (shaggy) from the mature nephrocyte in adult Drosophila using the dkfl15-TARGET system. This caused cellular dysfunction with reduced nephrocyte number and a significant loss and mislocalisation of the NEPH1 homolog dumfounded expression on the cells (Fig. 2g, h).

Pharmacological suppression of podocyte/nephrocyte GSK3 is detrimental

Complete genetic deletion of GSK3α and β in the podocyte was highly detrimental. However, of more clinical relevance is the pharmacological inhibition of GSK3 as there are multiple pharmacological compounds that can do this, and there is great interest in their potential use in a variety of important diseases including diabetes, cancer and dementia32,33. The most common agent of use in clinical practice is lithium, which is used for bipolar conditions. It is unknown how lithium precisely inhibits GSK3 activity but it is proposed that it has a dual inhibitory action by interfering with a secondary magnesium binding site as well as increasing inhibitory phosphorylation of both serine 21 for GSK3α, and serine 9 for GSK3β10. We initially confirmed that lithium caused phosphorylation of these inhibitory sites in conditionally immortalised human and murine podocytes (Supplementary Figs. 4a–d). To assess the functional significance of prolonged podocyte GSK3 inhibition we then studied Wistar rats that were chronically treated with lithium for 6-months. This caused inhibitory phosphorylation of the GSK3 isoforms, together with stabilization of β-catenin specifically in their podocytes within their glomeruli (Fig. 3a). β-catenin activation occurs when GSK3 is sufficiently inhibited and thus unable to regulate the phosphorylation-dependent degradation of β-catenin via its ubiquitinylation and destruction by the 26S proteasome21. Lithium-treated rats developed significant proteinuria (Fig. 3b) and glomerulosclerosis (Fig. 3c, d) in comparison to their age and sex matched controls.

Fig. 3 Pharmacological suppression of podocyte/nephrocyte GSK3. a Wistar rats fed lithium for 6-months caused inhibitory phosphorylation of GSK3α/β (left panel) and nuclear activation of β-catenin within their podocytes (right panel) (arrowed). Scale bar = 25 μm. b Lithium-treated rats developed significant proteinuria. t test ***p < 0.001, control n = 6; lithium treated n = 7. c, d Lithium-treated rats had evidence of glomerulosclerosis. Representative glomerular pictures shown, scale bar = 25 μm (c) together with blinded scoring of glomerulosclerosis index (d) in the 6-month treated Lithium and control group (n = 6–7). Unpaired two-tailed t-test*** p < 0.001. e At 24 h there is a dose response increase in nephrocyte size with increased Duf expression. ANOVA, ***p < 0.001, n = 6 individual flies per dose. f Altered cellular location of Dumbfounded in the nephrocyte after 24 h in a dose-dependent manner. Z-projected confocal stacks of controls show short linear arrays of Duf staining, presumed to be openings of individual slit diaphragms (arrowed). Cells treated with LiCl show disrupted Duf staining. g LiCl treatment affects endocytic Dextran uptake of dextran after 48 h. View through midpoint of cell. 1 mM demonstrates increased dextran signal uptake. At 20 mM severe disruption of dextran uptake. Cyan = dextran. Magenta = Wheat Germ Agglutinin (WGA). Scale bar = 10 μm. h The depth coloured images show nephrocytes stained with WGA after incubation for 48 h with or without 20 mM lithium. The uppermost region of the cell is red, the lower most is blue. In the control WGA (green) is located at the cell surface in shallow lacunae, whereas in the lithium treated cell the WGA associates with wider, deeper lacunae. Lower panels show a transection through the cell with a lacuna highlighted by an arrow. i Prolonged exposure of nephrocytes to lithium causes nephrocyte loss, reduced Duf expression and increased WGA nephrocyte accumulation (t test ***p < 0.001; n = 6–7 flies after 1-week exposure to 5 mM of lithium) DRAQ 5 was used to visualise nuclei. Scale bar top panel = 100 μm; bottom panel = 10 μm. j Immunohistochemistry showing increased inhibitory phosphorylation of GSK3αβ in the glomerulus from a patient on long-term lithium therapy when compared with a control biopsy. Scale bar = 50 μm. Data are presented as the mean ± SEM Full size image

We then examined the direct impact of lithium on nephrocyte function in Drosophila using an ex vivo culture system involving semi-intact fly preparations. Within 48 h of lithium treatment dose-dependent nephrocyte hypertrophy was observed (Fig. 3e), coupled with mislocalisation of dumbfounded from uniformly short linear arrays, consistent with previous reports34, and to an accumulation of bright, punctate Duf foci across the nephrocyte surface (Fig. 3f). Furthermore, lithium caused severe functional abnormalities to the nephrocytes’ endocytic function. At lower doses (1 mM), there was an increase in the Dextran signal within nephrocytes (Fig. 3g). However, when the cells were exposed to 20 mM lithium for 48 h it resulted in major issues with their endocytic capabilities with the formation of large lacunae and uptake of large Wheat Germ Agglutinin (WGA) (Fig. 3h). In similar experiments, flies provided with food containing 5 mM lithium for 1 week showed nephrocyte loss, reduced Duf staining and increased large protein WGA accumulation (Fig. 3i). These phenotypes are consistent with progressive loss of diaphragm integrity and comparable to foot process effacement seen in injured mammalian podocytes (Supplementary Fig. 4e). Finally, we examined a kidney biopsy from a patient who had been on long-term lithium therapy and found that they also had inhibitory GSK3 phosphorylation in their podocytes compared to control human kidney samples (Fig. 3j).

Podocyte GSK3 loss activates Wnt-β-catenin but this is not responsible for pathology

Genetic deletion of GSK3αβ in the podocytes of podGSK3DKO mice caused substantive activation and nuclear translocation of β-catenin in this cell type. Interestingly β-catenin was also activated throughout the kidney in the tubular compartment (Fig. 4a, b; Supplementary Fig. 5a), which we subsequently identified was probably a consequence of massive albuminuria (Supplementary Fig. 5b). Similarly, pharmacological inhibition of GSK3 also activated β-catenin in the podocyte (Fig. 3a).

Fig. 4 Podocyte GSK3 loss activates Wnt-β-catenin but this is not responsible for pathology. a β-catenin is expressed in podocytes and throughout the kidney in podGSK3DKO and mpodGSK3DKO mice but not in single isoform podocyte-specific knockout mice. Representative IHC pictures, scale bar = 25 μm. b Western blot of kidney lysates shows increased activated β-catenin in podGSK3DKO mice but not in control and single isoform podocyte-specific knockout mice, n = 3 experiments. See also Supplementary Fig. 5a. (c) No difference in survival of the podGSK3DKO/β-catenin KO compared to the podGSK3DKO mice. (Kaplan–Meier survival curve. Log-rank (Mantel-Cox test non-significant) podGSK3DKO n = 14; podGSK3DKO/β-catenin KO n = 12 mice. d No difference in the histological appearance of the podGSK3DKO/β-cateninKO compared to the podGSK3DKO mice at day 10 of life. Representative images of PAS staining, scale bar = 25 μm. e No difference in the level of albuminuria in podGSK3DKO/β-cateninKO compared to the podGSK3DKO mice. (Cre negative control n = 10; podGSK3DKO n = 10; podGSK3DKO/β-catenin KO n = 7 mice. Kruskel Wallis test not significant). f No difference in the level of albuminuria in mpodGSK3DKO/β-cateninKO compared to the mpodGSK3DKO mice, n = 5–7. g Inhibiting the Wnt pathway has no effect on cipodGSK3DKO cell survival (n = 3 experiments). h Inhibiting the Notch pathway has no effect on cipodGSK3DKO cell survival (n = 3 experiments). i Inhibiting the Hedgehog pathway in cipodGSK3DKO cells increases cell death (unpaired two-tailed t test *p < 0.05, n = 3 experiments). Data are presented as the mean ± SEM Full size image

As β-catenin activation and transcription of its target genes have been shown to be detrimental in other cell types when GSK3 is lost35,36, we hypothesized that β-catenin activation was the critical factor causing kidney damage in this model. We therefore generated transgenic mice models in which GSK3α, GSK3β and β-catenin were all contemporaneously deleted specifically from the podocyte both developmentally and in maturity. We confirmed that β-catenin was knocked down in these models using polymerase chain reaction (PCR), immunohistochemistry (IHC) and immunofluorescence (IF) (Supplementary Figs. 5c–e). However, a loss of podocyte β-catenin did not improve survival or kidney involvement in the developmental model (Fig. 4c–e) or renal damage in the mature model (Fig. 4f).

To support these findings, and given the contribution of Wnt/β-catenin along with Notch and Hedgehog pathways to the detrimental phenotype observed in other cells lacking GSK335 we also used pharmacological inhibitors of these pathways in a genetic GSK3αβ podocyte knockout cell line we developed (described in detail in next section). We inhibited Wnt signalling with iCRT3, NOTCH with DAPT and Hedgehog with GANT 61 but none of these improved cell survival (Fig. 4g–i; Fig. 8c). Interestingly, and unexpectedly, inhibiting Hedgehog signalling significantly increased cell death in this model (Fig. 4i; Fig. 8c).

Podocyte GSK3 loss causes mitotic catastrophe

To elucidate the mechanisms causing cellular damage when GSK3 was lost we generated a conditionally immortalised temperature-sensitive SV40 antigen podocyte cell line from a GSK3αfl/flβfl/fl mouse. This allowed us to initially culture healthy podocytes and then temporally delete GSK3 using lentiviral delivered Cre recombinase to make a GSK3α/β knockout cell line (cipodGSK3DKO) (Supplementary Figs. 6a–c). Three days after Cre transduction there was ~85% reduction of GSK3α and a ~70% reduction of GSK3β (Fig. 5a; Supplementary Fig. 6d) but the podocytes appeared healthy without any detectable cell loss. Therefore, we elected to interrogate this time point to identify early GSK3 driven mechanistic processes. More prolonged loss of GSK3, for 7 days, caused podocytes to be lost and microscopically appear unhealthy (Fig. 5b; Supplementary Fig. 6e).

Fig. 5 Podocyte GSK3 loss causes mitotic catastrophe. a Western blot showing robust knock down of GSK3α and GSK3β in lentiviral Cre transduced floxed podocytes (n = 4). Controls of Cre transduced wild-type podocytes (n = 3) and non-cre transduced GSK3 floxed podocytes (n = 3) also shown. See also Supplementary Fig. 5d. b Only 50% of ciGSK3DKO cells survive 7 days after Cre lenti transduction. ANOVA, Tukey’s post hoc test ***p < 0.001, n = 3 experiments. c Volcano plot of proteomic data revealed that at day-3 following lentiviral transduction, GSK3α and GSK3β levels were significantly reduced (unpaired two-tailed t test p < 0.01). Numerous proteins associated with cell cycle entry were up-regulated. d Phase contrast microscopy shows ciGSK3DKO cells have significantly more multinucleate cells than controls at day 5, unpaired two-tailed t test *p < 0.05. Three fields of view per group, n = 3 experiments. e Representative western blots of cipodGSK3DKO at day 5 reveals increased expression of Cyclin B1 and phosphorylation of Cdk1 and Histone 3 when compared with control cells, n = 3 experiments. See also Supplementary Fig. 6f. f Representative western blots of cipodGSK3DKO and control cells at day 7 reveals apoptosis in knockout cells. Increased levels of cleaved Caspase 3 and cleaved PARP shown, n = 4–5 experiments. See also Supplementary Fig. 6g. g Representative immunohistochemistry showing increased PCNA staining in glomerulus of mpodGSK3DKO mice. Scale bar = 25 μm. h Histology showing a mitotic figure an mpodGSK3DKO mouse podocyte. Scale bar top panel = 25 μm; bottom panel = 10 μm. Data are presented as the mean ± SEM Full size image

Non-biased Tandom Mass tagged (TMT) LC-MS/MS proteomic analysis of the cells 3 days after Cre transduction identified 486 proteins that were up-regulated more than 30% in cipodGSK3DKO cells compared to their controls (non-Cre treated GSK3αfl/flβfl/fl podocytes or lentiviral Cre expressing wild-type podocytes) at a statistical significance p < 0.01. STRING ( s earch t ool for the r etrieval of in teracting g enes/proteins) was used to visualise the protein–protein interactions and biological processes enriched in this data set. It revealed enrichment of multiple proteins involved in the cell cycle, including many involved in mitosis and mitotic spindle formation (Fig. 5c; Supplementary Fig. 7; Supplementary Table 3). To explore these findings further we performed fixed (Fig. 5d), and live (Supplementary Movie 1 and 2), phase contrast light microscopy of the cipodGSK3DKO cells. These confirmed that the cells were re-entering the cell cycle and attempting to divide as shown by significantly increased numbers of bi-nucleate and multi-nucleate cells in comparison to controls. However, many podocytes were unable to complete cytokinesis and appeared to be undergoing apoptosis and dying (Supplementary Movie 1 and 2). Detailed analysis of ciGSK3DKO cell numbers using an IN-Cell analyser confirmed these observations (Fig. 5b). The presence of multiple nuclei suggested that mitosis was occurring in ciGSK3DKO cells and western blot analysis of ciGSK3DKO cells 5 days after GSK3 knockdown showed a significant increase in phosphorylated Histone H3 (Fig. 5e and Supplementary Fig. 6f), a commonly used marker of mitosis associated with chromatin condensation37. Cyclin B1 is not usually detectable in healthy, mature podocytes38 but our proteomic analysis revealed that it was one of the most up-regulated proteins in ciGSK3DKO cells (Fig. 5c and Supplementary Table 3). This was confirmed by western blotting along with increased phosphorylation of Cdk1 (Fig. 5e; Supplementary Fig. 6f). Levels of the Cyclin B1/Cdk1 complex increase during G2 of the cell cycle with dephosphorylation occurring at the G2/M transition39. The absence of this dephosphorylation event suggests impaired mitotic progression in ciGSK3DKO cells. cipodGSK3DKO cells then appeared to undergo apoptosis as Western blot analysis of cells at 7 days revealed increased cleavage of caspase 3 and PARP (Fig. 5f; Supplementary Fig. 6g). Collectively, these data demonstrate that cipodGSK3DKO cells were undergoing mitotic catastrophe. We also looked at our podGSK3DKO and mpodGSK3DKO models and found evidence of DNA accumulation in their podocytes with nuclear PCNA staining suggesting they were attempting to re-enter the cell cycle (Fig. 5g). Furthermore, in the mature mpodGSK3DKO mice we detected occasional mitotic figures in their differentiated podocytes (Fig. 5h).

Fig. 6 Hippo signalling is disrupted in GSK3 deficient podocytes in vitro. a Summary of proteomics results for Ajuba in Cre treated wild-type (CreWT n = 3), Cre treated floxed GSK3α/β (GSK3KO n = 4) and non-Cre treated floxed GSK3α/β (GSK3FL n = 3) podocytes. ANOVA p = 0.004, Tukey post hoc analysis **p < 0.01 ***p < 0.001. b Representative western blot of analysis of ciGSK3DKO cells shows increased expression of Ajuba relative to controls, n = 3–4. See also Supplementary Fig. 7a. c, d Representative western blots of wild-type mouse podocytes incubated with 20 mM LiCl (c) and 3 μM CHIR99201 (d) showing increased expression of ajuba and Cyclin B1, n = 3. See also Supplementary Figs. 7b and c. e–g Immunofluorescence analysis showing increased nuclear YAP/TAZ staining in ciGSK3DKO cells 24 h after induction of gene knockout (e) n = 3, wild-type podocytes after 24 h 20 mM LiCl (f) n = 4 and 24 h 3 μM CHIR99201 (g) n = 8. Unpaired t test *p < 0.05. Data are presented as the mean ± SEM Full size image

Hippo signalling is disrupted in GSK3 deficient podocytes

Proteomic analysis also identified that an important protein in the Hippo-signalling pathway, Ajuba, was significantly increased by 200–300% in the ciGSK3DKO cells (Figs. 5c and 6a). This was validated by western blotting in the ciGSK3DKO cells (Fig. 6b; Supplementary Fig. 8a). Furthermore, an increased level of Ajuba was also detected in podocytes which had GSK3 pharmacologically supressed using either lithium and a more specific GSK3 inhibitor called CHIR99201 (Fig. 6c and d; Supplementary Figs. 8b and 8c). The Hippo pathway leads to phosphorylation of YAP/TAZ proteins and prevents these proteins from translocating from the cytoplasm into the nucleus, where they bind with TEAD transcription co-activators and ultimately promote transcription of a number of genes that are associated with proliferation and apoptosis40. Our proteomic analysis revealed significant up-regulation of a range of YAP/TAZ TEAD targets41 (Supplementary Table 4). Classically, when Ajuba increases it inhibits Hippo signalling by preventing the phosphorylation of YAP/TAZ thereby resulting in the nuclear translocation of these proteins42. In support of this mechanism we detected nuclear YAP/TAZ translocation in ciGSK3DKO cells (Fig. 6e), as well as the lithium and CHIR99201 treated podocytes (Fig. 6f and g). This was associated with induction of Cyclin B1 in all these models (Figs. 5e and 6c, d; Supplementary Figs. 6f and 8c). Analysis of our in vivo podGSK3DKO and mpodGSK3DKO mouse models also revealed significant up-regulation of Ajuba and nuclear YAP/TAZ translocation in their podocytes (Fig. 7a–d; Supplementary Fig. 9a). This was associated with an increased number of cells positive for Cyclin A2, a cell cycle protein and a recognised target of YAP/TAZ-TEAD40 (Fig. 7e). We also detected an increase of another YAP/TAZ TEAD target, c-myc43 in both podGSK3DKO and mpodGSK3DKO mice (Fig. 7f and Supplementary Figs. 9c, d). Finally, we examined the glomeruli of our lithium treated rats and human biopsy. These both showed increased podocyte Ajuba staining (Fig.7g).

Fig. 7 Hippo signalling is disrupted in GSK3 deficient podocytes in vivo. a, b Western blot (a) and quantification (b) of glomeruli isolated from mpodGSK3DKO and littermate control mice showing increased expression of Ajuba, n = 2 mice per group. c, d Immunohistochemistry showing increased Ajuba (top panels) and YAP/TAZ nuclear translocation (arrowed, bottom panels) in the podocytes of podGSK3DKO (c) and mpodGSK3DKO (d) mice. Representative immunohistochemistry and quantification shown (>5 glomeruli per mouse analysed, 3 mice per group, ANOVA ***p < 0.001. Scale bar = 25 μm). e Immunohistochemistry showing an increase in the number of cells positive for the YAP/TAZ TEAD target Cyclin A2. Three mice from each group analysed, t test *p < 0.5. f Immunofluorescence staining showing increased expression of c-myc in podGSK3DKO and mpodGSK3DKO mice. Three mice analysed per group, t test ***p < 0.001. Scale bar = 50 μm. g Representative immunohistochemistry using an anti-Ajuba antibody in a glomerulus from a rat given high dose lithium for 6 months and in a biopsy from a patient on long-term lithium therapy. Ajuba expression is increased with lithium treatment relative to control tissue. Scale bar = 25 μm. Data are presented as the mean ± SEM Full size image

Nuclear translocation of YAP/TAZ also occurred in the podGSK3DKO/β-cateninKO and mpodGSK3DKO/β-cateninKO mice (Supplementary Fig. 9b) indicating that this process is independent of Wnt- β-catenin signalling.

We then investigated if inhibiting YAP/TAZ activity in the nuclei of GSK3α/β knockdown podocytes was beneficial. Treating ciGSK3DKO podocytes with the YAP/TAZ-TEAD inhibitor verteporfin, improved cell survival by approximately 50% demonstrating that at least some of the adverse effects of podocyte GSK3 loss are mediated through the Hippo pathway (Fig. 8a, c; Supplementary Fig. 10a). Western blot analysis showed that verteporfin was also able to attenuate Cyclin B1 accumulation in ciGSK3DKO cells (Fig. 8b; Supplementary Fig. 10b). This was also the case in lithium and CHIR99201 treated podocytes (Fig. 8d, e; Supplementary Figs 10c,10d).

Fig. 8 Verteporfin attenuates the effects of GSK3 loss in cultured podocytes. a Inhibiting YAP/TAZ activity in the nuclei of cipodGSK3DKO cells with verteporfin improves cell survival. n = 4 independent experiments, unpaired two-tailed t test, *p < 0.05. See also Supplementary Fig. 10a. b Representative western blot showing that increased expression of Cyclin B1 in ciGSK3DKO cells is reduced by verteporfin, n = 3 experiments. See also Supplementary Fig. 10b. c Comparison of the effect of signalling pathway inhibitors on cipodGSK3DKO cell survival compared with vehicle. Inhibition of Wnt signalling with iCRT3 and Notch signalling with DAPT have no effect on cell survival while inhibition of Hedgehog signalling using GANT 61 significantly increases cell death (unpaired two-tailed t test *p < 0.05, n = 3 experiments except verteporfin n = 4 experiments). d, e Representative western blot of wild-type mouse podocytes incubated for 24 h with 20 mM LiCl or 3 μM CHIR99201 showing that Cyclin B1 accumulation is reversed by treatment with 1.25 μM verteporfin, n = 3 experiments. See also Supplementary Figs. 10c and d. Data are presented as the mean ± SEM Full size image

Finally, we assessed the role of Hippo signalling in the nephrocytes of Drosophila. Verteporfin protected inducible genetic shaggy knockout nephrocytes from the loss and mis-localisation of dumfounded (Fig. 9a–c) and also lithium induced nephrocyte damage (Fig. 9d, e). Collectively this data supports a key role of dysregulated Hippo signalling when podocyte or nephrocyte GSK3 activity is lost (Fig. 9f).