Hemoglobin Inhibits Uptake of Filtered Proteins by Proximal Tubule Cells: Implications for Sickle Cell Disease and Vitamin D Status

Abstract

Proximal tubule (PT) dysfunction, including tubular proteinuria, is a significant complication in sickle cell disease (SCD) that can eventually lead to chronic kidney disease. The PT is especially susceptible to cytotoxic damage, and tubular dysfunction in SCD is thought to result from prolonged exposure to hemoglobin (Hb) released from damaged red blood cells. Filtered Hb dimers are internalized into PT cells upon binding to the multiligand receptors megalin and cubilin. These receptors bind to numerous filtered proteins, including albumin and vitamin D binding protein, and are important for maintaining protein‐free urine and vitamin D homeostasis. We hypothesized that toxicity from exposure to Hb could impair PT cell endocytic function, causing tubular proteinuria. Using a fluorescence‐based protein uptake assay in a PT cell culture model, we found that concentrations of Hb predicted to enter the tubule lumen during hemolytic crisis profoundly inhibit the uptake of other megalin/cubilin ligands (albumin and vitamin D binding protein) by PT cells. These effects were independent of heme reduction state, occurred in the absence of a cytotoxic response, and appear to be due to direct competition for megalin/cubilin binding. The Glu7Val mutant that causes SCD was equally effective at inhibiting albumin uptake compared with wild type Hb. Haptoglobin restored albumin uptake in the presence of Hb, suggesting that haptoglobin binding to the Hb αβ dimer‐dimer interface interferes with Hb binding to megalin/cubilin. Using these data, we established a robust, scalable assay that enables us to screen for selective inhibitors of Hb uptake that preserve PT function. Our studies suggest that the primary cause of tubular proteinuria in SCD is impaired endocytosis of megalin/cubilin ligands due to competition from filtered Hb, rather than toxicity. Our data also suggest a potential explanation for the vitamin D deficiency commonly observed in sickle cell patients. Current studies are focused on characterizing the effects of Hb on vitamin D metabolism and signaling in PT cells.Support or Funding InformationNational Institutes of Health RO1 DK101484, RO1 DK100357, P30 DK079307, T32 DK061296, TL1 TR001858; Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute P3HVB pilot grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.