Liebau, M. C. | Search bioRxiv

bioRxiv2025

The fibrocystin C-terminal domain inhibits Src/STAT3 signal induced cystogenesis of kidney epithelia.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by impaired function of fibrocystin/polyductin (FPC) in collecting duct epithelia resulting in cyst formation. We hypothesized that the membrane-bound C-terminal FPC domain (FPCct) is necessary to suppress cystogensis and facilitate epithelial homeostasis. In ARPKD, cystic kidney epithelia are characterized by a secretory phenotype associated with high intracellular cAMP levels and enhanced STAT3-dependent transcription. Moreover, impaired FPC function may lead to enhanced activation of Src tyrosine kinase, thereby activating STAT3 signaling and its downstream transcriptional activity. To investigate the effects of FPC loss on the cystic epithelial cell phenotype, we used an established principal-like MDCK cell line (pl-MDCK) and studied monolayers in both two and three-dimensional culture. In this in vitro model of collecting duct epithelia, FPC-deficient cells showed two-fold elevated basal cAMP levels and enhanced apical secretion leading to three-fold higher luminal pressure. Forskolin-stimulated elevation of cAMP levels triggered enhanced Src-dependent activation of STAT3 resulting in a pronounced cystic phenotype. Notably, expression of wildtype FPCct reduced both STAT3-dependent transcription and the secretory phenotype in knockout epithelial cells. Our data suggest that FPCct interacts with Src kinase at the plasma membrane, thereby reducing Src-mediated STAT3 phosphorylation and limiting STAT3-dependent transcription. Thus, FPCct appears to act like a physiological suppressor of cystogenic signaling, as found in healthy kidney epithelia, that is essential for maintaining epithelial homeostasis. Protein constructs that restore FPC C-terminal function may offer a therapeutic lead to mitigate epithelial dysfunction and slow disease progression in ARPKD.

cell biology↗

bioRxiv2024

The nephronophthisis protein GLIS2/NPHP7 is required for the DNA damage response in kidney tubular epithelial cells

Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. While nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novel Nphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2Y122X). Homozygous mice develop cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibit an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys becomes apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with the in vivo data, the knockdown of Glis2 in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of GFP-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2Y122X and a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DNA damage response, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies. Author SummaryNephronophthisis (NPH) is a rare inherited kidney disease characterized by cyst formation, fibrosis, and kidney failure at a young age. It is typically caused by mutations in genes essential for the proper function of cilia, small sensory, antenna-like cell protrusions. However, emerging evidence suggests that genome instability and impaired DNA repair may also contribute to NPH. Thus, in some cases, ciliopathies may result from defects in nuclear proteins rather than ciliary proteins. To investigate this, we generated a mouse model with a defective NPHP7/Glis2 gene using genome-editing tools. The encoded protein GLIS2 has been described to be in the cilium and the nucleus, while its detailed function remained unclear. As these mice aged, they exhibited signs of DNA damage and later developed cystofibrotic kidney disease. Consistently, we demonstrated that the GLIS2 protein interacts with key DNA repair proteins and is rapidly recruited to sites of DNA damage and repair. Thus, inefficient DNA damage repair appears to contribute to kidney disease in this mouse model. These findings underscore the crucial role of genome stability in preventing kidney disease, providing new insights into the underlying causes of NPH.

cell biology↗