Periodic Reporting for period 2 - RENOPROTECT (Targeting tubular reabsorption for kidney protection)
Reporting period: 2022-07-01 to 2023-12-31
The rate of chronic kidney disease is on the rise worldwide. Many forms of chronic kidney disease are featured by the loss of protein into the urine (proteinuria). When the cause of proteinuria lies within the glomerulus, such as in diabetic kidney disease, then the protein overload in the tubular lumen may lead to damage of the downstream tubular cells. Particularly vulnerable are proximal tubular cells (PTCs), because these cells are specialized in protein reabsorption and have a high metabolic demand. While normal PTC metabolism is fueled by the uptake of fatty acids bound to albumin, an overload of fatty acids can lead to ER and mitochondrial stress. Genetic variants in the CUBN gene, encoding for the main albumin uptake receptor cubilin, lead to the reduction of albumin uptake and albuminuria in humans. Importantly, this condition seems to be associated with normal and, potentially, improved renal function. Here, we hypothesize that cubilin (or other protein uptake receptor) dysfunction could provide resistance against kidney diseases involving glomerular proteinuria.
To address this hypothesis, we will first study mechanisms of cubilin-mediated protein uptake in a humanized Drosophila model. Second, we will explore monoallelic CUBN expression and partial cryptic exon inclusion as two possible genetic mechanisms by which heterozygosity at the CUBN locus could promote proximal tubule fitness and tissue repair under different protein and lipid overload conditions. The final objective is to identify strategies to block tubular protein in conditions with glomerular proteinuria. Apart from genetic manipulations, this will include a novel nanoparticle delivery method. Altogether, our integrative translational approach will combine human genetics and experimental studies to explore a new mechanism of proximal tubule homeostasis that may also be applicable to other tissues. Our goal is to establish a novel paradigm for kidney protection with high relevance for the diagnosis, prognosis and treatment of kidney disease.
To address this hypothesis, we will first study mechanisms of cubilin-mediated protein uptake in a humanized Drosophila model. Second, we will explore monoallelic CUBN expression and partial cryptic exon inclusion as two possible genetic mechanisms by which heterozygosity at the CUBN locus could promote proximal tubule fitness and tissue repair under different protein and lipid overload conditions. The final objective is to identify strategies to block tubular protein in conditions with glomerular proteinuria. Apart from genetic manipulations, this will include a novel nanoparticle delivery method. Altogether, our integrative translational approach will combine human genetics and experimental studies to explore a new mechanism of proximal tubule homeostasis that may also be applicable to other tissues. Our goal is to establish a novel paradigm for kidney protection with high relevance for the diagnosis, prognosis and treatment of kidney disease.
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.
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.
We expect to generate more data on the protective effects of tubular protein uptake inhibition in glomerular proteinuria. We also hope to better understand the entire repertoire of uptake mechanisms for proteins and lipids along the proximal tubular segments.