Renal Papillary Collecting Tubule Research Articles

Effect of sulindac on prostaglandin synthesis in human and in cultured rat renal and vascular smooth muscle cells.

To examine the hypothesis that sulindac does not inhibit renal prostaglandin (PG) synthesis, we investigated the effects of sulindac and other nonsteroidal anti-inflammatory drugs on PG synthesis in human and in cultured rat renal and vascular smooth muscle (VSM) cells. In 7 patients with chronic glomerular disease, creatinine clearance and proteinuria were not changed by sulindac but were significantly reduced by diclofenac sodium. However, urinary excretion of PGE2 was decreased by both drugs. In cultured glomerular mesangial (GM), renal papillary collecting tubule (RPCT) and VSM cells from mesenteric artery, indomethacin, tiaprofenic acid, aspirin and ibuprofen inhibited both basal and arachidonic acid (AA)-stimulated PG synthesis dose-dependently. Although sulindac sulfoxide, at the same concentrations, inhibited both basal and AA-stimulated PGE2 synthesis in RPCT cells, it was less potent to inhibit PGI2 synthesis in VSM cells or PGE2 synthesis in GM cells. Active form sulindac sulfide inhibited PG synthesis in all types of cells but its inactive form sulfone did not. We conclude that sulindac inhibits renal PG synthesis but has little effect on renal function. This may be explained by its relatively weak potency on glomerular or vascular PG synthesis inhibition possibly due to the different biotransformation of the sulfoxide to the active sulfide in these cells.

Inhibition by Phorbol Ester of Cellular Adenosine 3′,5′-Monophosphate Production and Cellular Free Calcium Mobilization in Response to Arginine Vasopressin in Rat Renal Papillary Collecting Tubule Cells in Culture*

We determined whether tumor-promoting factor phorbol ester modulates cellular cAMP production and the cellular free calcium concentration ([Ca2+]i) in response to arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cAMP production in a dose-dependent manner. A 1-h exposure to 3 x 10(-7)-3 x 10(-6) M phorbol-12-myristate-13-acetate (PMA) significantly attenuated the cAMP response to AVP (1 x 10(-9) M AVP; 474.9 +/- 24.8 vs. 368.1 +/- 22.8 fmol/microgram protein; P less than 0.01). The dose-response relation with AVP thus shifted to the right. Such an inhibition was totally reversed in the presence of 2 x 10(-6) M 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase-C. Also, 1 x 10(-7) M AVP produced an increase in [Ca2+]i from 99.4 +/- 3.3 to 200.0 +/- 8.6 nM. When cells were preexposed to 1 x 10(-6) M PMA, an increase in [Ca2+]i in response to 1 x 10(-7) M AVP was significantly diminished (75.5 +/- 4.6 to 101.4 +/- 4.3 nM). The inhibition by PMA of AVP-induced increment in [Ca2+]i was significantly attenuated in the presence of 2 x 10(-5) M H-7 compared to that in its absence. Prolonged exposure to PMA did not alter the AVP-induced increases in cAMP production and [Ca2+]i. These results indicate that phorbol ester inhibits the cellular action of AVP mediated through the activation of protein kinase-C and suggest that there is an interaction between cAMP and phosphatidylinositol systems in modulating the AVP action in renal papillary collecting tubule cells.

Vasopressin-Induced Increases in Cellular Free Calcium Concentration Measured in Single Cells of Rat Renal Papillary Collecting Tubule.

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.

Enhancement by Veratridine and 60 mM KC1 of Vasopressin-Induced 3′,5′-Cyclic Adenosine Monophosphate Production and Cellular Free Calcium Concentration in Rat Renal Papillary Collecting Tubule Cells in Culture*

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using depolarizing agents in rat renal papillary collecting tubule cells in culture. One-hour exposure of cells to veratridine enhanced AVP-induced cAMP production in a dose-dependent manner. This enhancement by veratridine of cellular cAMP production in response to AVP was totally blunted by cotreatment with 5 X 10(-4) M verapamil, 3 X 10(-3) M cobalt, or Ca2+-free medium containing 1 X 10(-3) M EGTA. These agents block cellular Ca2+ uptake by different mechanisms. Similarly, 60 mM KCl enhanced AVP-induced cAMP production, and this effect was blocked by pretreatment with verapamil, cobalt or Ca2+-free medium containing 1 X 10(-3) M EGTA. When cellular free Ca2+ concentrations [Ca2+]i were measured by the fluorescence dye fura-2, both 1 X 10(-4) M veratridine and 60 mM KCl significantly increased [Ca2+]i from 67.6 to 141.8 nM and from 74.6 to 166.2 nM, respectively. Such rises in [Ca2+]i depended on extracellular Ca2+ since the increase in [Ca2+]i was completely blocked in Ca2+-free medium or in the presence of 3 X 10(-3) M cobalt. In addition, veratridine and 60 mM KCl significantly augmented the AVP-induced increase in [Ca2+]i. The possible mechanisms by which depolarizing agents induce cellular Ca2+ mobilization include the opening of voltage-sensitive Ca2+ channels in the plasma membrane. The present results indicate that veratridine and 60 mM KCl enhance AVP-induced cAMP production and cellular free Ca2+ concentration through cellular Ca2+ uptake in renal papillary collecting tubule.

Papillary collecting tubule responsiveness to vasoactive hormones in spontaneously hypertensive rats.

Renal papillary collecting tubule (RPCT) hormone responsiveness was compared between cultured RPCT cells from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Unstimulated cells from 4-week-old SHR produced less prostaglandin E2 (PGE2) and cyclic (c)AMP than comparable cells from WKY, while cells from both stains synthesized similar amounts of PGE2 after stimulation with arachidonate, A23187 or bradykinin and similar amounts of cAMP after stimulation with vasopressin or PGE2. There was no difference in basal or stimulated levels of cyclic (c)GMP between the strains. In RPCT cells from 16-week-old rats, basal levels of cAMP, cGMP and PGE2 were significantly lower than those from 4-week-old rats, but they did not differ between the strains. These results suggest that RPCT cells of SHR and WKY at the post-weaning period may differ in the metabolism of PGE2 and cAMP. This difference may be attributed to the possible defect in arachidonate availability in SHR.

Specificity in calcium oxalate adherence to papillary epithelial cells in cultures

Attachment of microcrystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with [14C]CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a beta value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/alpha ox value (maximum micrograms of crystallites adhering to 1 cm2 of binding area) of 287 micrograms/cm2. On coincubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/alpha AP value of 93 micrograms/cm2. Microcrystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding.

Cyclic GMP accumulation by atriopeptins in cultured rat renal and vascular smooth muscle cells.

To evaluate the role of cyclic guanosine monophosphate (cGMP) in the vascular and renal action of atrial natriuretic peptide (ANP), we compared the effects of atriopeptins (APs) on cGMP accumulation in cultured cells from rat mesenteric vascular smooth muscle (VSM), glomerular mesangium (GM) and renal papillary collecting tubule (RPCT), and also evaluated the relationship between renal sodium or water excretion and urinary cGMP in AP-infused rats. Both AP I and AP III increased intracellular cGMP levels dose-dependently in all types of cells, while they did not affect intracellular cAMP levels or prostaglandin synthesis. AP III was 100 times more potent than AP I. The magnitude change in cGMP levels was largest in GM cells. The sensitivity of VSM and GM cells to AP III were greater than that of RPCT cells. There were significant positive relationships between urinary excretion of sodium or water and that of cGMP levels in AP-infused rats. These results may suggest that GM and VSM cells are the principal targets for ANP to stimulate cGMP synthesis and, possibly, to exert the renal sodium and water excretion, and also support the hypothesis that cGMP mediates the cellular action of ANP.

Calcium Oxalate Crystal Interaction with Rat Renal Inner Papillary Collecting Tubule Cells

Rat renal inner papillary collecting tubule cells (RPCT) have been isolated and maintained in primary culture. The cells have been found to be of only one type and they have maintained the characteristics of RPCT cells. The RPCT cells in culture appear as a monolayer with intermittent clumps of rounded cells.When small calcium oxalate monohydrate crystals (COM) or calcium oxalate dihydrate crystals (COD) are added to the monolayer of RPCT cells, the crystals bind on or about these clumps of rounded-up cells. The use of this system as a model for the study of crystal membrane interactions in crystalluria and urolithiasis is discussed. (J. Urol., 138: 640–643, 1987)

Kinin effects on electrogenic ion transport in primary cultures of pig renal papillary collecting tubule cells.

Confluent monolayers of pig renal papillary collecting tubule (RPCT) cells were formed on Millipore filters coated with collagen. They were clamped in Ussing-type chambers and used to measure short-circuit current (SCC). The monolayers had low potentials (0.1 mV) with the basolateral side positive. Small inward currents flowed under short-circuit conditions. Increases in SCC were obtained following addition of a number of agents. Receptors associated with SCC changes were disposed as follows: for kinins (e.g., lysyl-bradykinin) they were present on both sides of the tissue, while those for arginine vasopressin and norepinephrine were present on the basolateral side only. Epithelia responded to PGE2 added to the apical or basolateral face of the tissue; application to one side prevented the response from the contralateral side. The tissues also responded to forskolin, an activator of adenylate cyclase, with a sustained inward current that was sensitive to furosemide. Similar sustained inward currents were recorded following exposure to 8-bromoadenosine-3',5'-cyclic monophosphate (BrcAMP). Responses to kinins were attenuated by inhibition of fatty acid cyclooxygenase with either indomethacin or piroxicam or by replacing chloride with impermeant ions. If the SCC was first increased with forskolin, BrcAMP, or norepinephrine, the kinin effects on SCC were either abolished or reversed. It is concluded that kinin can cause chloride secretion in RPCT monolayers, possibly via a prostaglandin or a prostaglandin-adenylate cyclase mechanism. Secondary effects of kinin, exposed by first raising tissue cAMP levels, are not precluded.

Evidence for a role of calmodulin in cellular cAMP production in response to vasopressin, prostaglandin E2 and forskolin in cultured rat renal papillary collecting tubule cells

In the present study, the role of calmodulin in the cellular action of arginine vasopressin (AVP), prostaglandin (PG) E2 and forskolin on adenosine-3', 5'-monophosphate (cAMP) production was examined in cultured rat renal papillary collecting tubule cells. In the presence of the phosphodiesterase inhibitor, submaximal concentrations of 10(-9) M AVP, 2 X 10(-8) M PGE2 and 2.4 X 10(-7) M forskolin significantly increased cellular cAMP accumulation by 2.3, 6.0 and 8.4-fold, respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), attenuated the cellular production of cAMP in a dose-related manner in response to all three stimuli. A dose which inhibited the cellular production of cAMP by 50% (ID50) ranged from 1.6 X 10(-5) to 2.8 X 10(-5) M for trifluoperazine and from 3.5 X 10(-5) to 4.4 X 10(-5) M for W-7. Basal accumulation of cellular cAMP was also decreased by treatment with either trifluoperazine or W-7, but an effective dose was relatively higher than that which inhibited agents-stimulated cellular cAMP production. Since forskolin is recognized as activating adenylate cyclase at one step of the catalytic component and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic component, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells. Further study demonstrated the inhibition of AVP- or PGE2-induced cellular cAMP production by treatment with either a calcium-free medium or verapamil, a blocker of cellular calcium uptake, before and during the experiment. These findings suggest that an increase in cytosolic calcium, which interacts with calmodulin to form an active complex, is, at least in part, due to the increased cellular influx of calcium from the extracellular space.

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE 1, PGE 2 and PGI 2, but not PGD 2 or PGF 2α, increased intracellular cAMP concentrations. At maximal concentrations (10 −5 M tthe effects of PGE 2 plus PGI 2 (or PGE 1), but not of PGI 2 plus PGE 1, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10 −5 M PGE 2. PGs, when tested at concentrations (e.g. 10 −9 M ) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10 −5 M ), the effects of AVP plus PGE 1, PGE 2, PGI 2 or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.

Kinin-induced prostaglandin synthesis by renal papillary collecting tubule cells in culture.

Cells having morphological and histochemical properties of collecting tubules were isolated from rabbit renal papillae. Confluent monolayer cultures of these renal papillary collecting tubule (RPCT) cells formed hemicysts and adhered with morphological asymmetry to Millipore filters. Cultures of 1-day-old RPCT cells synthesized cAMP in response to arginine vasopressin (AVP) (half-maximal response to 10(-10) M), oxytocin, and parathyroid hormone (half-maximal responses at 5 X 10(-9) M) but not to adrenergic agents. After 10 days of growth (fourfold increase in cell number) RPCT cells retained the same pattern of histochemical and hormonal responses as 1-day-old cells. Hormones were tested for their influence on the release of immunoreactive prostaglandins (iPG) by RPCT cells; the major product under both basal and stimulated conditions was iPGE2. At very low concentrations (greater than or equal to 10(-10) M), bradykinin, lysyl-bradykinin, and methionyl-lysyl-bradykinin caused four- to sixfold increases in the rate of iPGE2 formation within 3 min; smaller (less than twofold) increases were observed with relatively high concentrations of epinephrine (10(-5) M), norepinephrine (10(-5) M), and angiotensin II (10(-7) M), but only after longer incubations. Significantly, neither AVP (10(-7) M) nor [deamino]AVP (10(-7) M) caused prostaglandin release by RPCT cells. Our results indicate that kinins can act directly on the collecting tubule to elicit PGE2 formation; furthermore, this effect of kinins may be natriuretic, since PGE2 has been shown to inhibit Na+ resorption by the medullary collecting tubule and thick ascending limb.