Electrical properties of rabbit early distal convoluted tubule in primary culture

Abstract

Distal bright convoluted tubules (DCTb) were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The quality and the degree of polarization of the growing epithelia were assessed by indirect immunofluorescence studies using a monoclonal antibody raised against the apical membrane of the DCTb in situ. The cultured monolayers had a high hexokinase activity and a low gamma-glutamyl transferase activity compared with cultured proximal convoluted tubules. Adenosine 3',5'-cyclic monophosphate production was stimulated by calcitonin and insensitive to parathyroid hormone, vasopressin, and isoproterenol. Both 20- and 30-day-old cultures developed an apical-negative transepithelial potential of -3.1 and -22.3 mV, respectively. Amiloride reversibly reduced the voltage by 90% only when applied on the apical side of the monolayers. Phenamil (10(-8), 10(-6) M) had the same effect as amiloride. Calcitonin reversibly decreased the transepithelial voltage. These data support the hypothesis that, in the DCTb in primary culture, the transepithelial voltage is due to the presence of Na channels and that calcitonin modulates this transport pathway.

Differentiated Transport Properties of Primary Cultured Distal Nephron

“…Microdissection of tubules and subse quent culture [1] has the advantage that the tubule site of origin is known with great cer tainty, however, this technique also requires a reasonable degree of manual dexterity and the quantities of material that can be prepared for culture are limited. Mass isolation tech niques, such as immunodissection [2] or Percoll density centrifugation [3] have the advan tage of yielding large quantities of cell materi al, enabling a direct comparison of biochemi cal events with the physiological response.…”

“…Fluores cence was suppressed to background levels by incubation of the monolayer in RK1 contain ing 20 m M galactose (data not shown). The proportions of CA-ase-rich and PNA-positive cells are shown in table 1. Approximately half of the cells in the monolayer were found to be CA-ase rich.…”

“…Several alternative methods have been described for isolation of tubule fragments or single cells for primary cultures of distal nephron. Dissection of positively identified tubule fragments [ 1 ] has the advan tage of almost complete certainty of nephron site, but is laborious, requires a high degree of technical expertise and good anatomical knowledge and can provide only limited quantities of material. Mass isolation tech niques such as immunodissection [2] or den sity gradient separation of tubule fragments [3] have the advantage that large quantities of material can be isolated at any one time.…”

Calcium Homeostasis and Regulation of Calbindin-D9kby Glucocorticoids and Vitamin D as Bioactive Molecules

“…The previous reports described GR expression and distribution in the duodenum and kidney (Merot et al, 1989;Farman et al, 1991;Lee et al, 2006). In our previous study, we further employed Dex to elucidate the role of GR in the regulation of calcium-processing genes, and its differential expression in CaBP-9k KO mice (Kim et al, 2009).…”

Primary culture of collecting duct cell epithelium from neonate rabbit kidney in monolayer

“…Recently, there has been considerable interest in the use of primary cultures of renal tubule as models for the native epithelia (Horster, Fabritius & Schmolke, 1985;Bello-Reuss & Weber, 1987;Merot, Bidet, Gachot, Le Maout, Koechlin, Tauc & Poujeol, 1989). A culture of principal cells from neonate collecting duct maturation sites (Minuth, 1983(Minuth, , 1987 shows many of the differentiated properties of the adult collecting duct (CCD).…”