Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases

Abstract

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered lowmolecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/ megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp:: 1 / 2 vdbp-mCherry) zebrafish line expressing in the liver the Nterminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (1 / 2 vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.

SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule

“…Studies in model organisms and congenital disorders including Donnai-Barrow syndrome, caused by mutations in the LRP2 gene coding for megalin, or Dent disease, caused by mutations in the CLCN5 gene coding for the endosomal chloride-proton exchanger ClC-5, have highlighted the critical role of megalin in the reabsorption of LMW ligands by PT cells. 24,25,30 Of note, alteration of megalin expression was also observed in patients with tenofovir-induced PT dysfunction, 31 confirming that the defect is not restricted to COVID-19. Conversely, the expression of megalin is preserved in patients presenting acute tubular injury without overt PT dysfunction, related, for example, to sepsis, aminoglycosides, or hepatorenal syndrome.…”

“…These lesions are associated with a ∼50% reduction in the apical staining for megalin in PT cells. Studies in model organisms and congenital disorders including Donnai-Barrow syndrome, caused by mutations in the LRP2 gene coding for megalin, or Dent disease, caused by mutations in the CLCN5 gene coding for the endosomal chloride-proton exchanger ClC-5, have highlighted the critical role of megalin in the reabsorption of LMW ligands by PT cells 24 , 25 , 30 . Of note, alteration of megalin expression in patients with COVID-19 was also observed in patients with tenofovir-induced PT dysfunction 31 , confirming the defect is not restricted to COVID-19.…”

A Great Catch for Investigating Inborn Errors of Metabolism—Insights Obtained from Zebrafish

“…Many of these disorders have been investigated in zebrafish due to the high evolutionary conservation of genes and proteins between zebrafish and humans. Furthermore, the model has particular advantages such as simple stainings and compound tracking, such as lysotracker, as well as specific transgenic lines that have been generated to track lysosomal processing [ 157 , 158 ].…”

New zebrafish model for monitoring proximal tubule physiology in genetic and acquired renal Fanconi syndromes

“…Recently the research group led by Chen et al 6 developed a zebrafish line that uses endogenous processes to assay for PT function without injection of a conjugated dye, expediting the ability to test many samples in a short amount of time. First, the researchers used TAL-ENs to create mutant lines for lrp2a Transmission electron microscopy of the mutants shows lrp2a mutants had fewer endocytic vesicles and clcn7 mutants had larger vesicles, providing evidence of successful mutagenesis of lrp2a (endocytosis defects) and clcn7 (lysosomal processing defects).…”

“…For example, using the Tg(lfabp:: 1 / 2 vdbp-mCherry) line in a chemical screen with commonly prescribed drugs to identify those that affect PT function would lead to a better understanding of previously unknown factors that play a role in kidney dysfunction. Also, a similar approach to the studies of Chen et al 6 to rescue tubule function by treating mutants with a large number of molecules to rescue PT perturbations would uncover novel mechanisms of kidney protection.…”