Abstract
Cell surface proteins are internalized into the cell through endocytosis and either degraded within lysosomes or recycled back to the plasma membrane. While perturbations in endosomal internalization are known to modulate renal function, it is not known whether similar alterations in recycling affect renal function. Rififylin is a known regulator of endocytic recycling with E3 ubiquitin protein ligase activity. In this study, using two genetically similar strains, the Dahl Salt-sensitive rat and an S.LEW congenic strain, which had allelic variants within a < 330 kb segment containing rififylin, we tested the hypothesis that alterations in endosomal recycling affect renal function. The congenic strain had 1.59-fold higher renal expression of rififylin. Transcriptome analysis indicated that components of both endocytosis and recycling were upregulated in the congenic strain. Transcription of Atp1a1 and cell surface content of the protein product of Atp1a1, the alpha subunit of Na+K+ATPase were increased in the proximal tubules from the congenic strain. Because rififylin does not directly regulate endocytosis and it is also a differentially expressed gene within the congenic segment, we reasoned that the observed alterations in the transcriptome of the congenic strain constitute a feedback response to the primary functional alteration of recycling caused by rififylin. To test this, recycling of transferrin was studied in isolated proximal tubules. Recycling was significantly delayed within isolated proximal tubules of the congenic strain, which also had a higher level of polyubiquitinated proteins and proteinuria compared with S. These data provide evidence to suggest that delayed endosomal recycling caused by excess of rififylin indirectly affects endocytosis, enhances intracellular protein polyubiquitination and contributes to proteinuria.
Highlights
The composition of plasma membranes of virtually all eukaryotic cells is established, maintained, and remodeled by exocytosis, endocytosis, and a process of membrane recycling facilitated by endosomes
The rat strains chosen as tools for this study were the Dahl S rat and a > 99% genetically identical strain, the S.LEW congenic strain, which has a < 330 kb of the LEW rat genome introgressed onto the genome of the S rat (Figure 1A)
Hypertension in the Dahl S rat is accompanied with proteinuria (Sustarsic et al, 1981; Sterzel et al, 1988; Garrett et al, FIGURE 4 | Quantitation of the α-1 subunit of Na+K+ATPase on the plasma membranes of cells from proximal tubules (n = 3 animals per group)
Summary
The composition of plasma membranes of virtually all eukaryotic cells is established, maintained, and remodeled by exocytosis, endocytosis, and a process of membrane recycling facilitated by endosomes. Cells are estimated to internalize their cell surface equivalent one to five times per hour (Steinman et al, 1983) This rapid removal of membrane from the cell surface is balanced by endosomal recycling pathways, which return most of the endocytosed proteins and lipids back to the plasma membrane (Maxfield and McGraw, 2004). Within the apical membranes of proximal tubule cells in the kidney, an extensive endocytic apparatus plays a key role in the reabsorption and degradation of glomerular-filtered albumin and other proteins (Marshansky et al, 2002) and in the recycling of many functionally important membrane transporters (Brown and Stow, 1996). We hypothesized that any alterations in endosomal recycling disrupts cellular homeostasis and thereby could affect renal function. The current study was designed to test whether altered endosomal recycling facilitated by a congenic segment previously mapped on rat chromosome 10 containing rififylin (Gopalakrishnan et al, 2011) can affect renal molecular and cellular physiology and thereby contribute to the extent of www.frontiersin.org
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