Alkaloid uptake pathways in renal tubular epithelial cells from different processed products of Phellodendri chinensis Cortex

Abstract

This study aimed to investigate the absorption of alkaloids from Phellodendri chinensis Cortex (PC) by human renal tubular epithelial cells (HK-2). Cellular uptake and affinity ultrafiltration assays were employed to determine the alkaloid uptake pathway in HK-2 cells. Stemming from the hypothesis that salt-water processed PC introduces these alkaloids into the kidney at a cellular level, this research focused on different processed products of PC that are tailored for renal targeting. Utilizing the UPLC-QqQ-MS method, we quantified variations in the uptake capacity of phellodendrine, magnoflorine, jatrorrhizine, berberrubine, and berberine from raw Phellodendri chinensis Cortex (RPC), salt-water processed Phellodendri chinensis Cortex (SPC), and wine processed Phellodendri chinensis Cortex (WPC) in HK-2 cells. This study also tracked the concentration changes of these five alkaloids in HK-2 cells during the administration phase. Further, we evaluated the influence of two inhibitors on the absorption of these five alkaloids from PC and its processed products into HK-2 cells: the organic anion transporters (OATs) inhibitor-probenecid (PRO), and the organic cationic transporters (OCTs) inhibitor-tetraethylammonium chloride (TEAC). A pivotal component of this research was an investigation into the effects of PC and its processed products on the expression levels of OCT2, OAT1, and OAT3 proteins in HK-2 cells, facilitated by Western blot analysis. Finally, we appraised the binding affinity of PC's alkaloids to OCT2, OAT1, and OAT3 proteins using an ultrafiltration centrifugation technique. The uptake of different processed products of PC by HK-2 cells showed the following trend: SPC group > RPC group > WPC group. When considering inhibitor uptake in HK-2 cells, the group treated with PRO (an OATs inhibitor) demonstrated a higher uptake than the group treated with TEAC (an OCTs inhibitor). It was observed that different processed products of PC elevated the expression of OCT2 and OAT1 proteins in HK-2 cells. Specifically, both the SPC and berberrubine groups displayed enhanced expression of these proteins, with a marked increase noted for OCT2. Through affinity ultrafiltration assays, it was determined that the binding affinity of alkaloids from different processed products of PC to OCT2 and OAT1 significantly exceeded that to OAT3. These results indicate that PC-derived alkaloids are absorbed by HK-2 cells, predominantly through transport mechanisms mediated by OCT2 and OAT1, with OCT2 serving as the dominant transporter. The higher intake of alkaloids in SPC group can likely be linked to the amplified activity of kidney uptake transporters.

Keywords: Affinity ultrafiltration; Alkaloids; Phellodendri chinensis Cortex; Renal tubular epithelial cells; Salt-water processing; UPLC-QqQ-MS.