Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH

Abstract

Rabbit connecting tubules and cortical collecting ducts, which represent 79 +/- 5% of the calbindin-D28k-containing kidney cells, were isolated by immunodissection from the rabbit kidney superficial cortex and seeded on permeable filters. After 6 days in culture the monolayers developed a potential difference (PD) of -24 +/- 3 mV (lumen negative) and a transepithelial resistance (R) of 284 +/- 19 omega.cm2. Addition of 10(-6) M amiloride to or removal of Na+ from the mucosal side reversed the PD to +6 +/- 4 mV and concomitantly increased R to 660 +/- 122 omega.cm2. The cells developed functional parathyroid hormone (PTH) and arginine vasopressin receptors, but calcitonin receptors were absent. The monolayer actively absorbed Ca2+ against an electrochemical gradient with a rate of 121 +/- 13 nmol.h-1.cm-2. Removal of serosal Na+ inhibited Ca2+ absorption by 63 +/- 8%. Exposure to 10(-7) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for 48 h or 10(-7) M bovine PTH (bPTH)-(1-34) for 1 h, increased transcellular Ca2+ absorption by 53 +/- 13% or 24 +/- 8%, respectively. The effects of 1,25(OH)2D3 and PTH in combination were neither additive nor potentiating. In addition, the cultured cells expressed calbindin-D28k, and, after exposure to 10(-7) M 1,25(OH)2D3 for 48 h, the calbindin-D28k content increased fourfold.

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

“…The second m essenger cAMP has been frequently implicated as th e m ediator of horm one-stim ulated Ca2+ reabsorption in the distal tubule [5,24,25]. As demon strated in the present study, activation of adenylyl cyclase by forskolin indeed rapidly enhanced apical Ca2+ influx, increased [Ca2+], and stim ulated apical-tobasolateral 45Ca2+ flux.…”

“…Data of apical-to-basolateral 45Ca2+ flux experi ments were converted from cpm/sample to nmoLh^.cnr2. Basolateral-to~apical 4sCa2+ flux was negligibly small, as shown previously [5].…”

“…Rabbit kidney connecting tubule and cortical collecting duct cells, hereafter referred to as cortical collecting system cells, were isolated from New Zealand White rabbits (-0.5 kg bod y weight) by immunodissection with monoclonal antibody R2G9 and set in primary culture on either transparent (fluorescence measurements) or non transparent (45Ca2+-flux studies), permeable filters (Costar, Cambridge, MA, USA), as previously described in detail [5]. The culture m edium was a 1:1 (v/v) mixture of Dulbecco's modified Eagle's m edium (DMEM) and F-12 medium (Gibco, Paisley, UK) supplem ented with 5% (v/v) decomplemented fetal calf serum (Serva, Heidelberg, Germany), 50 (ig/ml gentamicin, 10 jil/ml of a lOOx mixture of non-essential amino acids (Gibco), 5 jug/ml insulin, 5 |ig/ml transferrin, 50 nM hydrocortisone, 70 ng/ml prostaglandin Ei; 50 nM Na2Se03 and 5 pM triiodothyronine, equilibrated with 5% C 0 2-95°/o air at 37°C.…”

Molecular Mechanisms of Primary Hypercalciuria

“…Calbindin D 28K knockout mice (126) exhibit excessive hypercalciuria when fed a high-calcium diet (127). Basolateral calcium transport occurs via both the plasma membrane Na ϩ /Ca 2ϩ exchanger (NCX) and Ca 2ϩ -ATPase (PMCA), which have been estimated to transport 70% and 30% of calcium respectively (128,129).…”

Vasopressin-stimulated Ca 2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca 2+

“…In rabbit, the CNT and cortical collecting duct (CCD) have also been implicated in active Ca2* reabsorption |2, 3,13,31,33,35], which is in contrast to the situation in the rat, where the distal convoluted tubule is the main site of Ca2* transport [2], Active Ca2* reabsorption is under the control of both 1,25-dihydroxyvitamin D 3 [1,25-(OIT)2D 3l and parathyroid hormone (PTH). I,25-(OH)2D 3 stimulates Ca2* reabsorption in a long-term fashion prob ably through increased expression of eaibindin-D28K [3, 351.…”