Pendrin is an anion exchanger expressed in type B intercalated cells of the cortical collecting duct (CCD). Whether pendrin localizes to other nephron segments with intercalated cells is unknown. Moreover, whether pendrin is expressed in proximal tubule is debated. Thus the distribution of pendrin mRNA and protein expression in mouse kidney was investigated by using light and electron microscopic immunohistochemistry and quantitative real-time PCR. We observed that pendrin mRNA is expressed mainly in cortex. Within cortex, pendrin mRNA is at least fivefold higher in CCD and the connecting tubule (CNT) than in the other segments. Pendrin protein was observed in a subset of cells within the distal convoluted tubule as well as in type B and in non-A-non-B intercalated cells of the CNT and CCD. In type B intercalated cells, pendrin immunoreactivity was highest in apical cytoplasmic vesicles with little immunolabel along the apical plasma membrane. In non-A-non-B intercalated cells, intense pendrin immunoreactivity was detected along the apical plasma membrane. These differences in the subcellular distribution of pendrin immunolabel were confirmed by morphometric analysis. In conclusion, pendrin is expressed in the mouse distal convoluted tubule, CCD, and CNT along the apical plasma membrane of non-A-non-B intercalated cells and in subapical cytoplasmic vesicles of type B intercalated cells.

Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)

End-users need to be aware that IV bags made from a particular polymer by the same manufacturer may contain components or use a manufacturing process that results in a different product. Analysis of samples after freezing and thawing proved to be useful in identifying potential incompatibility of dulanermin in the IV bags.

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