PROTEIN OVERLOAD STIMULATES RANTES PRODUCTION BY PROXIMAL TUBULAR CELLS DEPENDING ON NF-KB ACTIVATION

Abnormal traffic of proteins through the glomerular capillary has an i ntrinsic renal toxicity possibly linked to the subsequent process of p roximal 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 ch emotactic activity for monocytes/macrophages and T lymphocytes. Conflu ent 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 posi tive control. RANTES was measured in cell supernatants by ELISA. Bovin e serum albumin (BSA) induced a time-and dose-dependent increase in pr oximal tubular cell RANTES production. Selected experiments using tran swells showed that the RANTES release was predominantly basolateral. T he stimulatory effect on tubular RANTES was not specific to albumin bu t 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 mu M) and sodium salicylate (10 mM) signific antly 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-depende nt fashion, which was inhibited by PDTC. Supershift analysis revealed that the protein subunits of activated NF-kappa B were p65/p65 homodim er, p65/cRel, p50/cRel and p50/p65 heterodimers. Given its chemotactic activity, RANTES released into the interstitium might promote inflamm atory cell recruitment and contribute to interstitial inflammation and renal disease progression.