A morphological and functional comparison of proximal tubule cell lines established from human urine and kidney tissue.

Promising renal replacement therapies include the development of a bioartificial kidney using functional human kidney cell models. In this study, human conditionally immortalized proximal tubular epithelial cell (ciPTEC) lines originating from kidney tissue (ciPTEC-T1 and ciPTEC-T2) were compared to ciPTEC previously isolated from urine (ciPTEC-U). Subclones of all ciPTEC isolates formed tight cell layers on Transwell inserts as determined by transepithelial resistance, inulin diffusion, E-cadherin expression and immunocytochemisty. Extracellular matrix genes collagen I and -IV α1 were highly present in both kidney tissue derived matured cell lines (p<0.001) compared to matured ciPTEC-U, whereas matured ciPTEC-U showed a more pronounced fibronectin I and laminin 5 gene expression (p<0.01 and p<0.05, respectively). Expression of the influx carrier Organic Cation Transporter 2 (OCT-2), and the efflux pumps P-glycoprotein (P-gp), Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) were confirmed in the three cell lines using real-time PCR and Western blotting. The activities of OCT-2 and P-gp were sensitive to specific inhibition in all models (p<0.001). The highest activity of MRP4 and BCRP was demonstrated in ciPTEC-U (p<0.05). Finally, active albumin reabsorption was highest in ciPTEC-T2 (p<0.001), while Na(+)-dependent phosphate reabsorption was most abundant in ciPTEC-U (p<0.01). In conclusion, ciPTEC established from human urine or kidney tissue display comparable functional PTEC specific transporters and physiological characteristics, providing ideal human tools for bioartificial kidney development.

Inhibitory effect of a callophycin A derivative on iNOS expression via inhibition of Akt in lipopolysaccharide-stimulated RAW 264.7 cells.

In previous studies, (R)-2-isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2,3(9H)-dicarboxylate (1), a callophycin A derivative, was found to strongly inhibit nitrite production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, while (R)- or (S)-callophycin A showed only weak inhibition. We currently report additional studies to define the mechanisms underlying the inhibitory action of 1. Expression of inducible nitric oxide synthase (iNOS) was reduced at both protein and mRNA levels. Major upstream signaling molecules and transcription factors regulating iNOS expression were examined, but it was found that 1 did not affect the phosphorylated and total protein levels of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and signal transducer and activator of transcription 1 (STAT1), nor did it mediate the degradation of the inhibitor of nuclear factor-κB α-isoform (IκBα). However, starting at early time points, 1 consistently inhibited the phosphorylation of protein kinase B/Akt at serine 473. In addition, 1 suppressed the protein expression of octamer-binding transcription factor-2 (Oct-2) and the expression of microRNA 155 (miR-155). In sum, compound 1 inhibits LPS-induced nitrite production by a unique and complex mechanism. Reduction of iNOS expression is accompanied by inhibition of Akt activation, Oct-2 protein expression, and miR-155 expression.