Within the first funding period of RENOPROTECT, we have used the Drosophila model to understand the mechanisms in cubilin-mediated albumin reabsorption and to identify additional players operating in this process. Our results suggest that cubilin forms a protein complex with the the chloride-proton exchanger Clc-c, which corresponds to the Dent’s disease protein Clc-5. We could show that the depletion of Clc-c caused very similar phenotypes as cubilin depletion, e.g. with regard to albumin uptake and the organization of the endolysosomal pathway. The knockdown of Clc-c and Cubn also caused an increase in cortical and perivesicular actin filaments suggesting that the regulation of ion transport controls cortical actin disassociation to enable endosomal transport. Interestingly, activation of the actin-depolymerizing factor cofilin could reduce actin accumulation and thereby restore uptake of albumin (manuscript in preparation). In another Drosophila study that was recently published in Science (Jouandin, Marelja et al, 2022), we identified molecular mechanisms of how mTOR is controlled by lysosomal cystine. By identifying a new metabolic pathway relevant for the proximal tubules, this paper also contributes to the overall aim of the RENOPROTECT project.
Finally, we explored whether the combination of glomerular albuminuria and dyslipidemia could cause lipotoxic effects in the proximal tubules, as albumin might function as a vector for intracellular delivery of damaging fatty acids. For this, we treated a diabetic mouse model with a high fat diet. Interestingly, excess fat exposure caused injury in renal proximal tubular cells (PTCs), but only in those cells that could not store fat in lipid droplets. Mechanistically, we identified stress in the endoplasmic reticulum (ER) as the main cause of proximal tubular injury. ER stress was caused by elevated levels of saturated triacylglycerol precursors, reduced lipid droplet formation and, consequently, decreased membrane fluidity in the ER. The addition of monounsaturated fatty acid rescued the cytotoxic effects by normalizing membrane order and by increasing both triacylglycerol and lipid droplet formation. These results were recently published in eLife (Perez-Marti et al, 2022), emphasizing the importance of monounsaturated fatty acids for the dietary management of diabetic kidney disease by preventing lipid bilayer stress in the ER and promoting triacylglycerol and lipid droplet formation in PTCs.
