SNP in Slit-Diaphragm Proteins is Associated with Altered Physicochemical Properties: Consequences for Impaired Glomerular Filtration Barrier Function

The glomerular filtration barrier (GFB), localized at the interface of blood and urine, is crucial for renal filtration. Slit diaphragm (SD), a specialized intercellular junction between adjacent podocytes, is a significant component of GFB. Several transmembrane proteins (nephrin, NEPH1, and TRPC6) and adapter proteins (podocin and CD2AP) are tethered together and form a highly complex SD. Non-synonymous single-nucleotide polymorphisms (nsSNPs) in SD proteins cause congenital nephrotic syndrome, characterized by massive proteinuria. Nevertheless, the physicochemical consequences of these mutations remain elusive. We performed an integrated computational analysis of 25 clinically associated nsSNPs across five core SD proteins. We considered AlphaFold-derived structures and assessed the effects of SNPs on thermodynamic stability, secondary structure, aggregation propensity, and surface hydrophobicity. ΔΔG and RMSD are physicochemical descriptors, whereas aggregation and solubility scores reflect surface remodeling. The results suggest that some SNPs are destabilizing and confer high aggregation risk (podocin-D160G, TRPC6-R895C); some elicit surface remodeling (podocin-R168H, NEPH1-S573L); and variants exert dual effects on solubility and aggregation (TRPC6-R895C, podocin-V180M, CD2AP-T374A). Overall, most amino acid substitutions in SD proteins destabilize protein folding and promote aggregation, highlighting the role of physicochemical events in podocyte injury. Although the study provides quantitative insights into how amino acid substitutions alter the behavior of SD proteins, it has limitations, including reliance on AlphaFold-derived structures and the absence of experimental validation. Collectively, this work provides insights into mutation-driven physicochemical remodeling of SD proteins and establishes a rational framework for prioritizing clinically relevant variants for experimental validation and therapeutic interventions.