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A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury

Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI.


Supplementary
: Characterization of phospho-Thr-169 Cdkl5 antibody. (a) BUMPT cells were transiently transfected with FLAG-tagged WT or T169A Cdkl5 plasmids and 24 hours later, lysates were collected, followed by Cdkl5 immunoprecipitation using anti-FLAG beads. Immunoprecipitates were either left untreated (-) or phosphatase treatment (+) was carried out for 3 hours. Subsequently western blot analysis was carried out with phospho-Cdkl5 and FLAG antibodies. Blot is representative of two independent experiments. (b) Cellular lysates from BUMPT cells stably transfected with indicated shRNAs and treated with cisplatin for 6 hours were used for western blot analysis of total and phospho-Cdkl5 protein levels. Blots are representative of two independent experiments. Source data are provided as a Source Data file. Figure 3: Characterization of Ggt1-Cre mice. The ROSA mT/mG mice were crossed with the renal tubular epithelial cell-specific Ggt1-Cre (gamma-glutamyltransferase 1) mice to generate transgenic mice that express membranelocalized GFP in the tubular epithelial cells. Before Cre-mediated recombination, all cell types express membrane localized Td tomato. Indicated tissues from Cre positive mT/mG mice were examined for Cre-mediated expression of EGFP. Representative image showing EGFP expression in renal tubules (arrow), while other tissues did not express EGFP. Images were taken with confocal microscope at 60X magnification. Scale bar: 100 µm. Figure 4: Stress responsive and cell cycle independent activation of CDKL5. (a-c) Rhabdomyolysis was induced in male wild-type (C57BL/6) mice by glycerol injection (7.5 ml/kg 50% glycerol) in the hind-leg muscles followed by measurement of renal function (BUN and Creatinine) and renal Cdkl5 activity (Kinase assay). Data are presented as individual data points (n = 4 biologically independent samples), from one out of two independent experiments, all producing similar results.

Supplementary
(d-f) Male wild-type (FvB) mice were injected with folic acid (250 mg/kg, i.p.) followed by measurement of renal function (BUN and Creatinine) and renal CDKL5 activity (Kinase assay). Data are presented as individual data points (n = 4 biologically independent samples), from one out of two independent experiments, both producing similar results. (g) Renal tissues from a previously described porcine model of ischemic acute kidney injury were subjected to CDKL5 kinase assay. Data are presented as individual data points (n = 4 biologically independent samples), from one out of two repeat experiments, both producing similar results. (h) Cellular stress was induced in primary tubular cells by cisplatin, hydrogen peroxide, hemin or 1% hypoxia treatment followed by Cdkl5 kinase assays. Data are presented as individual data points (n = 5 biologically independent samples), from one out of three independent experiments, all producing similar results. (i) primary tubular cells were synchronized by double thymidine block and then released into nocodazole-containing medium. Non-synchronized and synchronized cells were then treated with hydrogen peroxide for 1 hour, followed by assessment of CDKL5 kinase activity. Data are presented as individual data points (n = 3 biologically independent samples), from one out of two independent experiments, all producing similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's (a,b, h, and i) or students t-test (c,d,e, f, and g) was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 5: Effect of gene knockout on renal function. Renal function (BUN and Creatinine) was examined in littermates with indicated genotypes from 6-24 weeks of age under baseline conditions. These results show that germline Cdkl5 knockout (a-b), renal tubule specific Cdkl5 knockout (d-e) or conditional Sox9 gene knockout (g-h) does not affect kidney function under normal conditions. Data are presented as individual data points (n = 4 biologically independent samples), from a single long-term experiment. Source data are provided as a Source Data file. Figure 6: Ischemic and nephrotoxic AKI is mitigated in Cdkl5 knockout mice. (a) Representative western blot confirming Cdkl5 gene ablation in renal tissues. Bilateral renal ischemia was induced in wild-type and Cdkl5 -/y mice for 28 minutes followed by examination of renal function and damage post-reperfusion. (b) Blood urea nitrogen (c) Serum creatinine (d) renal Kim1 mRNA expression (e) renal histological analysis (H&E) showed that Cdkl5 deficiency confers protection from ischemia-associated AKI. Data presented (b-e) is cumulative of three independent experiment (n=7). Wild-type and Cdkl5 -/y mice were treated with cisplatin (25 mg/kg) followed by examination of renal function. (f) Blood urea nitrogen (g) Serum creatinine (h) renal Kim1 mRNA expression (i) renal histological analysis (H&E) showed that Cdkl5 contributes to cisplatin-mediated AKI. Data presented (f-i) is cumulative of two out of four independent experiment (n=8), that showed similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. , or FLAG tagged WT as well as S199A and S199D Sox9 plasmids. One day later, cellular fractionation was carried out using Thermo Scientific Subcellular Protein Fractionation Kit. Lysates from various fractions were then subjected to western blot analysis of indicated proteins. Blot is representative of two independent experiment. (b-c) BUMPT cells were transiently transfected with FLAG tagged wild-type or S199A (non-phosphorylatable ) and S199D (phospho-mimetic) Sox9 mutant constructs. One day later, cells were incubated with or without cycloheximide (100 μg/ml) for the indicated times, followed by western blot analysis of FLAG-tagged Sox9. Densitometric analysis was then performed to examine Sox9 protein stability. Graph represents data (n=3 biologically independent samples) from one out of three independent experiment. *indicates p<0.05 as compared to the WT group. Source data are provided as a Source Data file. Figure 9: Validation of phospho-Ser-199 Sox9 antibody. (a) In vitro kinase assays were carried out with cold ATP using recombinant CDKL5-kinase and wild-type or SOX9-S199A mutant proteins as substrates followed by western blot analysis of indicated proteins. In one group phosphatase treatment was carried out for 3 hours. Blot is representative of two independent experiment. (b) Ischemic renal tissues from control and Sox9 PT-/mice were subjected to immunoblot analysis of indicated proteins. Blots are representative of two independent experiments. Source data are provided as a Source Data file.

Supplementary Figure 10: Increased Cdkl5 phosphorylation in the TEY motif during AKI. (a-b) Littermate control and
Cdkl5 conditional knockout mice (Cdkl5 PT-/y ) were challenged with bilateral renal ischemia followed by western blot analysis of total and phospho-Thr-169 Cdkl5 protein in the renal cortical tissues as shown in the representative blots in Figure 4e. The graphs represent densitometric analysis (normalized to β-actin levels) of five distinct samples from three independent experiments. (c-d) Control and Cdkl5 PT-/y male mice were challenged with cisplatin injection, followed by western blot analysis of total and phospho-Thr-169 Cdkl5 protein in the renal cortex as shown in the representative blots in Figure 4d. The graphs represent densitometric analysis (normalized to β-actin levels) of five distinct samples from three independent experiments. No positive signals were detected by both the total and phospho-Thr-169 antibody in the Cdkl5 PT-/y tissues, confirming the antibody specificity. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's multiple-comparisons test (b-d, vs. WT 0hrs. group) was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 11: Cdkl5 gene ablation does not influence AKI-induced Sox9 mRNA upregulation. qPCR analysis of Sox9 mRNA was carried in the renal cortical tissues of control and mice with renal injury at the indicated timepoints. AKI-induced Sox9 mRNA up-regulation was not altered by Cdkl5 deficiency. Graph represents data (n=4 biologically independent samples) from one out of two independent experiments. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant, while ns indicates not significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 12: Effect of Sox9 mutations at S199 site on RTEC survival. (a-b) Primary murine RTECs were isolated from Sox9 conditional knockout mice. After culturing for one week, lentiviral mediated transduction was carried out with empty vector (mock) or FLAG-tagged WT or mutant Sox9 (S199A and S199D). Two days after transduction, cells were treated with 50 μM Cisplatin for 24 hours followed by western blot analysis of FLAG tagged Sox9. The blots are representative of three independent experiments. The graph depicts densitometric analysis of Sox9 protein levels (FLAG). (c-e) Primary murine RTECs were isolated from WT and Sox9 conditional knockout mice. After culturing for one week, lentiviral mediated transduction was carried out with empty vector (mock) or FLAG-tagged WT or mutant Sox9 (S199A and S199D). Two days after transduction, cells were treated with 50 μM Cisplatin for 24 hours followed by cell viability assessment and caspase assays using cellular lysates. Graph represents data (n=5 biologically independent samples) from one out of three independent experiments. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant, while ns indicates not significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Supplementary Figure 13: Identification of Sox9 target genes in renal tubular cells. (a) Sox9
Chromatin immunoprecipitations (ChIP) were carried out in indicated ischemic renal tissues followed by qPCR analysis to determine target gene binding. Data are shown as mean of 3 replicates and representative of two independent experiments. (b-e) Sox9 binding to putative target gene (Gadd45a, Wwp2, Myof and Sema3e) promoter regions was assesed in three distinct models of AKI, namely, cisplatin nephrotoxicity, rhabdomyolysis and folic acid-associated AKI. Data are presented as individual data points (n = 3-4 biologically independent samples), from one out of three independent experiments, all producing similar results. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant, while ns indicates not significant. 1-way ANOVA followed by Dunnett's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 14: Gene expression analysis of putative Sox9 target genes in Sox9 conditional knockout mice. Bilateral renal ischemia was induced in wild-type and Sox9 PT-/mice for 30 minutes or wild-type and Sox9 PT-/mice were treated with cisplatin (30 mg/kg) followed by qPCR based analysis of gene expression in renal cortical tissues. These results indicate that Sox9 potentially suppresses Gadd45a expression (a-b) during AKI. On the other hand Sox9 seems to be essential for upregulation of Wwp2 (c-d), Myof (e-f) and Sema3e (g-h) during AKI. Data are presented as individual data points (n = 4 biologically independent samples), from one out of two independent experiments, all producing similar results. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant, while ns indicates not significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 15: Effect of Cdkl5 deficiency on the expression of putative Sox9 target genes. Ischemic or nephrotoxic AKI was induced in wild-type and Cdkl5 PT-/mice followed by qPCR based analysis of gene expression in renal cortical tissues. These results indicate that Cdkl5 deficiency leads to suppression of Gadd45a expression (a-b) during AKI. On the other hand Cdkl5 deficiency leads to increased upregulation of Wwp2 (c-d), Myof (e-f) and Sema3e (g-h) during AKI. Data are presented as individual data points (n = 4 biologically independent samples), from one out of two independent experiments, all producing similar results. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant, while ns indicates not significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 16: Reporter assays for detection of SOX9 binding at putative target gene promoters. HEK293 cells, which express low levels of endogenous SOX9 protein were stably transfected with empty plasmid or SOX9 construct. The mock transfected cells (-) and SOX9 expressing cells (+) were then used for luciferase based reporter assays. Promoter sequences (-2000bp from TSS) for the four target genes were cloned in a luciferase reporter construct. Mock (-) and SOX9 (+) expressing HEK293 cells were transiently co-transfected with reporter renila luciferase contructs (empty, Myof, Sema3e, Wwp2, and Gadd45a) and reference cypridina luciferase (normalizing control), followed by measurement of luciferase activity at 24 hours. The normalized luciferase activity of mock (-) group was then compared with the SOX9 (+) expression group for each target gene. Data in the graphs are presented as individual data points (n = 5 biologically independent samples), from one out of three independent experiments, all producing similar results. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant. Student's t test was used and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 17: Functional role of putative Sox9 target genes in renal epithelial cell death. BUMPT cells were transfected with indicated siRNAs (one control non-specific siRNA and two distinct siRNAs targeting the same gene, indicated as siRNA1 and 2) followed by cisplatin treatment for 24 hours and subsequent assessment of cell viability and caspase activity. Myoferlin (a-d), Wwp2 (e-h), and Sema3e (i-l) knockdown sensitizes BUMPT cells to cisplatin associated cell death. On the other hand Gadd45a knockdown (m-p) provided protection from cisplatin mediated cell death. Western blots (a, e, I, and m) of siRNA transfected and untreated cells are representative of two independent experiments. Data in the graphs are presented as individual data points (n = 4 biologically independent samples), from one out of three independent experiments, all producing similar results. In the bar graph, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was considered as statistically significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Supplementary Figure 18: AST-487 protects RTECs in a Cdkl5-dependent manner. (a-d) BUMPT cells were treated with 25
μM Cisplatin for 24 hours. At the same time, cells were co-treated with either vehicle or AST-487. After 24 hours, Cdkl5 kinase assay, cell viability and caspase activity assays were carried out, which showed that AST-487 protects BUMPT cells from cisplatin-associated cell death. (e) BUMPT cells were transfected with FLAG-tagged vector or CDKL5 WT or CDKL5 gatekeeper mutant (indicated as mut). After 24 hours, cell lysates were prepared followed by FLAG-immunoprecipitation and CDKL5 kinase assay in the presence or absence of 100 nM AST-487. The results indicate that the gatekeeper mutation confers resistance to AST-487 mediated CDKL5 inhibition. (f-h) BUMPT cells transfected with indicated constructs were treated with vehicle or 25 μM cisplatin and 100 nM AST-487 for 24 hours. After the incubation period, cell viability and caspase activity assays were carried out, which showed that AST-487 protects BUMPT cells from cisplatin-associated cell death in a CDKL5 dependent manner. Data are presented as individual data points (n = 3-5 biologically independent samples), from one out of 2-3 independent experiments, all producing similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's (a-c) or Tukey's multiplecomparisons test (d-g) was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 19: AST-487 mediated renal protection is Cdkl5-dependent. Wild type and Cdkl5 PT-/y mice were injected with cisplatin (30 mg/kg, i.p.) followed by treatment with either vehicle (-) or AST-487 (+). Subsequently, the renal tissues were collected after 24 hours followed by Cdkl5 immunoprecipitation, Cdkl5 kinase assays and immunoblot analysis of indicated proteins in the Cdkl5 immunoprecipitates and input lysates (whole tissue). (a) The representative immunoblots (input) show that AST-487 treatment resulted in reduced Cdkl5 phosphorylation in the TEY motif, while having no significant effect on total Cdkl5 protein levels. (b) Cdkl5 kinase assays showed that AST-487 can inhibit Cdkl5 activity in the renal tissues of the WT mice. Data presented (four biological replicates) are representative of two independent experiment that showed similar results. (c-e) Wild type and Cdkl5 PT-/y mice were injected with cisplatin (30 mg/kg, i.p.) followed by treatment with either vehicle or AST-487 (25 mg/kg, oral gavage) 6 hours later, and subsequent assessment of renal function (BUN and serum creatinine) and histology (H&E, renal damage score) at indicated time-points. Data presented are cumulative of two independent experiments (n=7), that showed similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's (b) or Tukey's multiple-comparisons test (ce) was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 20: AST-487 mediated Cdkl5 inhibition results in reduced Sox9 phosphorylation and increased Sox9 protein levels. Western blot analysis of renal tissues (representative blots shown in Figure 6i) indicated that AST-487 suppress Sox9 phosphorylation and increases Sox9 protein levels in vivo. Blots are representative of three independent experiments. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 21: Pharmacological or genetic Cdkl5 inhibition provides long-term protection from AKI. (a) Wildtype mice were treated with a single cisplatin (30 mg/kg) injection followed by administration with either vehicle or AST-487 (25 mg/kg, p.o.) and examination of overall survival up to 30 days. (b) Wild-type and Cdkl5 PT-/y mice were treated with cisplatin (30 mg/kg) followed by examination of overall survival over a period of 30 days. Data is presented as Kaplan-Meier survival curves and Mantel-Cox test was performed to determine statistical significance. Source data are provided as a Source Data file.

Supplementary Figure 22: Cdkl5 regulates cisplatin-associated AKI in a Sox9 dependent and independent manner.
Littermate control and Sox9 PT-/were treated with cisplatin (30 mg/kg) followed by administration of either vehicle or AST-487 (25 mg/kg, oral gavage, 6 hours post-cisplatin injection). At 72 hours renal function and damage were assessed through (a) Blood urea nitrogen (b) Serum creatinine (c) renal histological analysis (H&E). Age-matched Wild type, Cdkl5 PT-/y , Sox9 PT-/-, and Cdkl5 PT-/y Sox9 PT-/-(double knock out mice indicated as dKO PT ) were treated with cisplatin (30 mg/kg) followed by assessment of renal structure and function at 72 hours through (d) Blood urea nitrogen (e) Serum creatinine (f) renal histological analysis (H&E). Data presented (a-f) are cumulative of three independent experiment (n=6). In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 23: Cdkl5 regulates cisplatin-associated RTEC cell death in a Sox9 dependent and independent manner. Primary murine RTECs were cultured from littermate control and Sox9 PT-/mice followed by co-treatment with 50 µM Cisplatin and either vehicle (-) or 100nM AST-487 (+) for 24 hours followed by assessment of cellular viability using Trypan Blue (a) and MTT (b) assays as well as Caspase activity (c). (d) Primary murine RTECs were cultured from Wild type control, Cdkl5 f/f , Sox9 f/f , and Cdkl5 f/f Sox9 f/f mice. Subsequently RTECs were transduced with Cre expressing lentivirus and western blot analysis of untreated cells indicated successful knockout of floxed genes. (e-g) Primary murine RTECs from WT, Cdkl5 f/f , Sox9 f/f , and Cdkl5 f/f Sox9 f/f mice were transduced with either control (mock) or Cre expressing lentivirus. Subsequently, the RTECs were treated with 50 µM Cisplatin for 24 hours followed by assessment of cell viability and caspase activity assay. Data presented are representative of three independent experiment (n=5-6 biological replicates). In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 24: Genetic or pharmacological Cdkl5 inhibition mitigates Ischemia-associated AKI in female mice. Control and Cdkl5 PT-/female littermates underwent bilateral renal ischemic surgery (35 minutes) followed by examination of renal damage. (a) Blood urea nitrogen (b) Serum creatinine (c) renal histological analysis (H&E) showed that Cdkl5 contributes to Ischemia-associated AKI. Data presented (a-c) are cumulative of three independent experiments (n=6) that showed similar results. (d) Renal tissue lysates were used for Cdkl5 immunoprecipitation followed by kinase assay at indicated time-points. The results showed that Cdkl5 activity increases during ischemic-AKI. The data presented is representative of two independent experiment (n=4 biological replicates). Eight weeks old wild type C57B6 female mice underwent bilateral renal ischemic surgery for 35 minutes, followed by either vehicle or 25 mg/kg (p.o.) AST-487 administration (6 hours post-IR) and subsequent examination of renal damage two days later. (e) Blood urea nitrogen (f) Serum creatinine (g) renal histological analysis (H&E) showed that Cdkl5 contributes to Ischemia-associated AKI. Data presented (e-g) are cumulative of three independent experiments (n=6) that showed similar results. . In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's or Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 25: Genetic or pharmacological Cdkl5 inhibition mitigates Cisplatin-mediated AKI in female mice. Control and Cdkl5 PT-/female littermates were treated with 15 mg/kg Cisplatin, followed by examination of renal damage. (a) Blood urea nitrogen (b) Serum creatinine (c) renal histological analysis (H&E) showed that Cdkl5 contributes to cisplatinassociated AKI. Data presented (a-c) are representative of two independent experiments (n=5) that showed similar results. (d) Renal tissue lysates were used for Cdkl5 immunoprecipitation followed by kinase assay at indicated time-points. The results showed that Cdkl5 activity increases during cisplatin nephrotoxicity. The data presented is representative of two independent experiment (n=4 biological replicates). Eight weeks old wild type C57B6 female mice were treated with 15 mg/kg Cisplatin, followed by either vehicle or 25 mg/kg (p.o.) AST-487 administration (6 hours post-cisplatin injection) and subsequent examination of renal damage three days later. (e) Blood urea nitrogen (f) Serum creatinine (g) renal histological analysis (H&E) showed that Cdkl5 contributes to Cisplatin-associated AKI. Data presented (e-g) are cumulative of representative of two independent experiments (n=4) that showed similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Dunnett's or Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Figure 26: Cdkl5 regulates Sox9 phosphorylation in female mice. Control, cisplatin and ischemic renal tissues from control and Cdkl5 PT-/female mice were subjected to immunoblot analysis of indicated proteins. Blots (a) are representative of three independent experiments. (b) Densitometric analysis of Sox9 and p-Ser-199 Sox9 protein levels (normalized to β-actin levels). Graph represents cumulative results (n=3 independent biological samples) from three independent experiments. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar=1 s.e. and p<0.05 was indicated as statistically significant. 1-way ANOVA followed by Tukey's multiple-comparisons test was carried out and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.