Role of the CDKL1-SOX11 signaling axis in acute kidney injury

Abstract

The biology of the cyclin-dependent kinase-like (CDKL) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with mitogen-activated protein kinases and glycogen synthase kinase-3, although their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse that exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia-reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, this study has unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI.NEW & NOTEWORTHY Identifying and targeting pathogenic protein kinases holds potential for drug discovery in treating acute kidney injury. Our study, using novel germline knockout mice, revealed that Cdkl1 kinase deficiency does not affect mouse viability but provides protection against acute kidney injury. This underscores the importance of Cdkl1 kinase in kidney injury and supports the development of targeted small-molecule inhibitors as potential therapeutics.

Keywords: acute kidney injury; cyclin-dependent kinase-like 1; ischemia; renal tubular epithelial cells; rhabdomyolysis.

Graphical abstract

Figure 1.

Cdkl1 is activated in the kidneys during acute kidney injury. A: schematic representation for the CDKL family of kinases. The red, blue, and green triangles represent the ATP-binding site, TXY motif, and putative nuclear localization signal, respectively. B–H: C57B/6 wild-type male mice (8–12 wk old) were either injected with 50% glycerol into the hindlimb muscle (Rhabdo) or challenged with bilateral kidney ischemia for 30 min followed by reperfusion (IRI) to induce kidney injury. Kidneys were collected 24 h after injury followed by analysis of BUN (B) and serum creatinine (C) to assess the extent of damage. Sham, Rhabdo, and IRI kidney tissue lysates were used for immunoprecipitation of Cdkl1 (D), Cdkl2 (E), Cdkl3 (F), Cdkl4 (G), and Cdkl5 (H), followed by in vitro kinase assays. In the bar graphs (n = 8 biological replicates from 3 repeated experiments), the experimental values are presented as means ± SD. The height of the error bar = 1 SD. P < 0.05 was indicated as statistically significant. Student’s t test was carried out, and statistical significance is indicated by ****P < 0.0001. BUN, blood urea nitrogen; CDKL, cyclin-dependent kinase-like; IRI, ischemia-reperfusion injury; NS, not significant; Rhabdo, rhabdomyolysis.

Figure 2.

Cdkl1 germline deletion provides protection from rhabdomyolysis- and IRI-associated acute kidney injury. Cdkl1 knockout mice were created by deleting exon (E)3 in the Cdkl1 gene to create a null transcript. A: illustration of the Cdkl1 knockout mouse gene structure with exon 3 deletion in red. B: representative Western blots of kidney tissues showing successful gene deletion. C–F: Cdkl1 knockout mice or littermate WT controls (8–12 wk old) were subjected to rhabdomyolysis-associated acute kidney injury by injecting 50% glycerol into the hind muscle or subjected to bilateral ischemia for 30 min followed by reperfusion. Blood and kidneys were collected at 24 h, and kidney function and damage were examined by BUN (C), serum creatinine (D), and histological analysis (H&E staining) (E and F). Asterisks indicate damaged tubules. Scale bar = 100 μm. In the bar graphs (n = 6 biological replicates from 3 repeated experiments), the experimental values are presented as means ± SD. The height of the error bar = 1 SD. P < 0.05 was indicated as statistically significant. Student’s t test was carried out, and statistical significance is indicated by *P < 0.05. BUN, blood urea nitrogen; CDKL, cyclin-dependent kinase-like; H&E, hematoxylin and eosin; IRI, ischemia-reperfusion injury; NS, not significant; Rhabdo, rhabdomyolysis; WT, wild-type.

Figure 3.

Cdkl1 phosphorylates Sox11 at site Ser²⁵³. A: bilateral kidney ischemic injury, rhabdomyolysis-associated injury, or sham kidney tissues lysates from Cdkl1^−/− mice or control littermates were used for Western blot analysis of the indicated proteins. Kidney cortical lysates were also immunoprecipitated by Sox11 antibody to assess Sox11 and phospho-serine levels in WT and Cdkl1^−/− mice (bottom). B: purified Cdkl1, WT Sox11, and mutant Sox11 were coincubated for an in vitro kinase assay followed by Western blot and autoradiography of Sox11 phosphorylation. C: schematic of the mouse Sox11 protein sequence. HMG-Box indicates the high mobility group box domain, AR indicates the autoregulatory domains, and TAD indicates the transactivation domain. Protein sequence analysis showed the sequence surrounding Ser²⁵³ was highly conserved among organisms. D and E: analysis of Sox11 protein stability was performed by carrying out the cycloheximide (CHX) chase assay in BUMPT cells transfected with Flag-tagged WT and S253D phosphomimetic Sox11 mutant. The results indicated that the phosphomimetic mutant had reduced stability compared with WT Sox11. In the bar graph (n = 3 biological replicates from 3 repeated experiments), the experimental values are presented as means ± SD. The height of the error bar = 1 SD. P < 0.05 was indicated as statistically significant. Student’s t test was carried out, and statistical significance is indicated by *P < 0.05. CDKL, cyclin-dependent kinase-like; IP, immunoprecipitation; IRI, ischemia-reperfusion injury; Rhabdo, rhabdomyolysis; WT, wild-type.

Figure 4.

Tubular Sox11 knockout mice exhibit elevated acute kidney injury. A: tubular-specific Sox11 conditional knockout (Sox11^cKO) mice were created by crossing Sox11-flox mice with Ggt1-Cre mice. B: representative Western blots of Rhabdo kidney tissues showing successful gene deletion. C–F: Sox11^cKO mice or littermate controls (8–12 wk old) were subjected to Rhabdo-associated acute kidney injury by injection of 50% glycerol into the hind muscle or subjected to bilateral ischemia for 30 min followed by reperfusion. Blood and kidneys were collected at 24 h, and kidney function and damage were examined by BUN (C), serum creatinine (D), and histological analysis (H&E staining) (E and F). Asterisks indicate damaged tubules. Scale bar = 100 μm. In the bar graphs (n = 6 biological replicates from 3 repeated experiments), the experimental values are presented as means ± SD. The height of the error bar = 1 SD. P < 0.05 was indicated as statistically significant. Student’s t test was carried out, and statistical significance is indicated by *P < 0.05. BUN, blood urea nitrogen; CDKL, cyclin-dependent kinase-like; Con, control; IRI, ischemia-reperfusion injury; NS, not significant; H&E, hematoxylin and eosin; Rhabdo, rhabdomyolysis; WT, wild-type.