Kibra knockdown inhibits the aberrant Hippo pathway, suppresses renal cyst formation and ameliorates renal fibrosis in nphp1(KO) mice.

INTRODUCTION: Nephronophthisis (NPH) is an autosomal recessive interstitial cystic kidney disease, which is the most common genetic cause of end-stage renal disease (ESRD) in childhood. The Hippo pathway is regulated by the cilium and has been suggested to be linked to NPH. The aim of the study was to investigate the involvement of Hippo pathway in the pathogenesis of nphp1 defect-associated NPH (NPH1). METHOD: Nphp1 knockout (nphp1(KO)) Madin-Darby Canine Kidney (MDCK) cells and nphp1(KO) C57BL/6J mice were generated via CRISPR gene editing strategy. The siRNAs targeting Kibra, MST1 and LATS1 were designed. An AAV9 vector was designed for Kibra knockdown. The expression and phosphorylation of core Hippo pathway molecules were evaluated. Pathological renal changes were evaluated via light microscopy respectively with haematoxylin-eosin and Masson staining. RESULTS: In nphp1(KO) MDCK cells, nphp1(KO) mice and NPH1 patients' kidneys, Kibra, p-MST1/2, p-LATS and p-YAP exhibited a notable increase in levels, with an even greater elevation observed in renal cyst cells, indicating the Hippo pathway activated in these nphp1-deficient contexts. Nphp1 re-expression reversed the Hippo pathway activation in cells, indicating that the Hippo pathway activation is related to nphp1 deficiency in vitro. Meanwhile, in vitro, MST1 knockdown downregulated LATS1 and YAP phosphorylation, LATS1 knockdown downregulated YAP phosphorylation, suggesting the activation of the canonical Hippo pathway in nphp1-deficient contexts. Knockdown of the upstream regulator Kibra inhibited the Hippo pathway activation in both nphp1(KO) MDCK cells and mice. Following Kibra knockdown, the organisation of nphp1(KO) MDCK cells became more compact, the intensity of the actin fibres increased. Besides, decreased renal fibrosis and cyst formation were observed in nphp1(KO) mice. CONCLUSIONS: The canonical Hippo pathway is aberrantly activated in nphp1-deficient conditions. Kibra may serve as a crucial upstream regulator of nphp1 deficiency-related Hippo pathway activation. Kibra upregulation and activation of the Hippo pathway are involved in the pathogenesis of NPH1. KEY POINTS: Canonical Hippo pathway activated in nphp1-deficient disease models and patients. Kibra was a key upstream molecule in regulating the activation of canonical Hippo pathway in nphp1-deficient disease models and patients and closely related to renal cyst formation and fibrosis in nphp1(KO) mice.