Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation

Abstract

Abnormal traffic of proteins through the glomerular capillary has an intrinsic renal toxicity possibly linked to the subsequent process of proximal tubular reabsorption. Here we investigated in vitro the effect of protein overload on proximal tubular cell production of RANTES, a nuclear factor-kappa B (NF-kappa B)-dependent chemokine with potent chemotactic activity for monocytes/macrophages and T lymphocytes. Confluent pig LLC-PK1 cells were incubated for 24 and 48 hours with Eagle's MEM plus 0.5% FCS containing bovine serum albumin (BSA, 1 to 30 mg/ml). Tumor necrosis factor-alpha (TNF-alpha; 100 U/ml) was used as a positive control. RANTES was measured in cell supernatants by ELISA. Bovine serum albumin (BSA) induced a time- and dose-dependent increase in proximal tubular cell RANTES production. Selected experiments using transwells showed that the RANTES release was predominantly basolateral. The stimulatory effect on tubular RANTES was not specific to albumin but was shared by immunoglobulin (Ig) G. We then explored the role of NF-kappa B on BSA-induced RANTES. The NF-kappa B inhibitors pyrrolidine dithiocarbamate (PDTC; 25 microM) and sodium salicylate (10 mM) significantly reduced BSA-induced RANTES production. Electrophoretic mobility shift assay of nuclear extracts of LLC-PK1 exposed to BSA revealed an intense NF-kappa B activation as early as 30 minutes in a dose-dependent fashion, which was inhibited by PDTC. Supershift analysis revealed that the protein subunits of activated NF-kappa B were p65/p65 homodimer, p65/cRel, p50/p65 heterodimers. Given its chemotactic activity, RANTES released into the interstitium might promote inflammatory cell recruitment and contribute to interstitial inflammation and renal disease progression.