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Title: Effects of disease-causing mutations of podocin on podocyte biology
Author: Kuzmuk, Valeryia
ISNI:       0000 0004 7972 8159
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2019
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The slit diaphragm component podocin is the protein product of the NPHS2 gene, which is mutated in a subset of patients with autosomal recessive steroid-resistant Nephrotic Syndrome (SRNS) that manifests as early childhood onset of proteinuria, focal segmental glomerulosclerosis (FSGS) and fast progression to end-stage renal disease (ESRN). Most disease-causing mutations of podocin result in its incorrect intracellular trafficking leading to aberrant formation/function of the slit diaphragm complex. Among these, the most frequent mutation is R138Q (57.5%), which alters the intracellular trafficking of podocin to the plasma membrane (PM), leading to its retention in the endoplasmic reticulum (ER). Interestingly, the most frequent worldwide mutation (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) is also retained in the ER. Our collaborators have shown that this is due to aberrant interaction of this mutant with cytokeratin 8 (K8). Disruption of this interaction leads to the correction of the F508del-CFTR processing defect leading to its expression at the plasma membrane. Standard in vitro techniques were used to understand the effects of R138Q mutation on podocin trafficking and biology and to characterize the nature of R138Q-Keratin 8 interaction in podocin's bona fide cell type, the kidney podocyte. Furthermore, a conditional podocin knock-in mouse carrying a R140Q mutation, the mouse analogue of human R138Q, was created using doxycycline-inducible Cre-recombinase technology that allows for the study of the effects of the mutation in postnatal life and represents an ideal model for pharmacological studies. In this study, a previously unknown mechanism for the intracellular retention of the R138Q podocin mutant was identified. First, I demonstrated that Keratin 8 may physically interact with the R138Q podocin, and that ablation of K8 expression using shRNA in human R138Qexpressing cells led to the correct association of podocin with lipid raft microdomains at the plasma membrane and the recovery of the adhesive function of the mutant cells. Excitingly, interruption of this interaction in the R138Q podocin mutant podocytes with a small molecule compound also results in the correct targeting of the mutant protein to the plasma membrane and restores its function. The involvement of keratin 8 network in the misfolded protein response was further demonstrated in vivo, where a continuous 28-days treatment with a small molecule compound prevented the development of proteinuria in the NPHS2flox/R140Qmice. Together, these results show that the disruption of the K8-R138Q interaction potentially using chaperone drugs already developed in the Cystic Fibrosis (CF) field may provide a novel therapeutic target for SRNS patients bearing the R138Q mutation. I have also identified a novel binding partner of podocin, and a new gene mutated in NS.
Supervisor: Saleem, Moin ; Welsh, Gavin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available