Duct Cells In Culture Research Articles

Angiopoietin-like protein 2 increases renal fibrosis by accelerating transforming growth factor-β signaling in chronic kidney disease

Renal fibrosis is a common pathological consequence of chronic kidney disease (CKD) with tissue fibrosis closely associated with chronic inflammation in numerous pathologies. However, molecular mechanisms underlying that association, particularly in the kidney, remain unclear. Here, we determine whether there is a molecular link between chronic inflammation and tissue fibrosis in CKD progression. Histological analysis of human kidneys indicated abundant expression of angiopoietin-like protein 2 (ANGPTL2) in renal tubule epithelial cells during progression of renal fibrosis. Numerous ANGPTL2-positive renal tubule epithelial cells colocalized with cells positive for transforming growth factor (TGF)-β1, a critical mediator of tissue fibrosis. Analysis of M1 collecting duct cells in culture showed that TGF-β1 increases ANGPTL2 expression by attenuating its repression through microRNA-221. Conversely, ANGPTL2 increased TGF-β1 expression through α5β1 integrin-mediated activation of extracellular signal-regulated kinase. Furthermore, ANGPTL2 deficiency in a mouse unilateral ureteral obstruction model significantly reduced renal fibrosis by decreasing TGF-β1 signal amplification in kidney. Thus, ANGPTL2 and TGF-β1 positively regulate each other as renal fibrosis progresses. Our study provides insight into molecular mechanisms underlying chronic inflammation and tissue fibrosis and identifies potential therapeutic targets for CKD treatment.

Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy

Polyomavirus-associated nephropathy (PVAN) is a significant complication after kidney transplantation, often leading to premature graft loss. In order to identify antiviral responses of the renal tubular epithelium, we studied activation of the viral DNA and the double-stranded RNA (dsRNA) sensors Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I) in allograft biopsy samples of patients with PVAN, and in human collecting duct cells in culture after stimulation by the dsRNA mimic polyriboinosinic:polyribocytidylic acid (poly(I:C)), cytokines, or infection with BK virus. Double staining using immunofluorescence for BK virus and TLR3 showed strong signals in epithelial cells of distal cortical tubules and the collecting duct. In biopsies microdissected to isolate tubulointerstitial lesions, TLR3 but not RIG-I mRNA expression was found to be increased in PVAN. Collecting duct cells in culture expressed TLR3 intracellularly, and activation of TLR3 and RIG-I by poly(I:C) enhanced expression of cytokine, chemokine, and IFN-β mRNA. This inflammatory response could be specifically blocked by siRNA to TLR3. Finally, infection of the collecting duct cells with BK virus enhanced the expression of cytokines and chemokines. This led to an efficient antiviral immune response with TLR3 and RIG-I upregulation without activation of IL-1β or components of the inflammasome pathway. Thus, PVAN activation of innate immune defense mechanisms through TLR3 is involved in the antiviral and anti-inflammatory response leading to the expression of proinflammatory cytokines and chemokines.

The balance of endogenous IL-1β and IL-1 receptor antagonist expressions in the brain regulates immunological fatigue-like behavior induced by poly I:C injection

Polyomavirus-associated nephropathy (PVAN) is a significant complication after kidney transplantation, often leading to premature graft loss. In order to identify antiviral responses of the renal tubular epithelium, we studied activation of the viral DNA and the double-stranded RNA (dsRNA) sensors Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I) in allograft biopsy samples of patients with PVAN, and in human collecting duct cells in culture after stimulation by the dsRNA mimic polyriboinosinic:polyribocytidylic acid (poly(I:C)), cytokines, or infection with BK virus. Double staining using immunofluorescence for BK virus and TLR3 showed strong signals in epithelial cells of distal cortical tubules and the collecting duct. In biopsies microdissected to isolate tubulointerstitial lesions, TLR3 but not RIG-I mRNA expression was found to be increased in PVAN. Collecting duct cells in culture expressed TLR3 intracellularly, and activation of TLR3 and RIG-I by poly(I:C) enhanced expression of cytokine, chemokine, and IFN-β mRNA. This inflammatory response could be specifically blocked by siRNA to TLR3. Finally, infection of the collecting duct cells with BK virus enhanced the expression of cytokines and chemokines. This led to an efficient antiviral immune response with TLR3 and RIG-I upregulation without activation of IL-1β or components of the inflammasome pathway. Thus, PVAN activation of innate immune defense mechanisms through TLR3 is involved in the antiviral and anti-inflammatory response leading to the expression of proinflammatory cytokines and chemokines.

Differentiation of Pancreatic β Cells from Human Pancreatic Duct Cells Derived from a Partial Pancreas Tissue

Background: Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. This has led to a search for alternative sources of transplantable insulin-producing cells including pancreatic duct cells. We aimed to establish in vitro culture of pancreatic duct cells from a partial pancreas tissue in human, which could be harnessed to differentiate into pancreatic β cells.Methods: We isolated pancreatic duct cells from small pieces of pancreas tissue (1~3 g) derived from non-diabetic humans (n = 8) undergoing pancreatic surgery due to cancer. Pancreas tissue was finely minced after injection of collagenase P into the parenchyma. The mince was incubated in a shaking water bath at 37℃ for 25 min and passed through a 150 μm mesh. The released cells were recovered, washed, and plated in a dish containing CMRL culture medium with serum.Results: Isolated pancreatic cells grew in monolayer and became confluent in 1~2 wks showing typical epithelial cobblestone morphology. Immunochemistry demonstrated that ~90% of the cultured cells were cytokeratin7-positive duct cells. To induce β cell differentiation, the cells were incubated in DMEM/F12 culture medium without serum. In addition, treatment with Matrigel overlay, exendin-4, cholera toxin or forskolin was done. Though β cell differentiation was found by immunostaining and RT-PCR, the differentiation efficiency was very low. Over-expression of neurogenin-3 by recombinant adenovirus did not increase β cell differentiation of the cultured duct cells significantly. Conclusion: We established in vitro culture of pancreatic duct cells from a partial pancreas tissue in human, which differentiate into pancreatic cells. However, a strategy to optimize β cell differentiation in this model is needed. (J Kor Diabetes Assoc 31:236~242, 2007)

Clusterin induces differentiation of pancreatic duct cells into insulin-secreting cells

We recently reported that expression of the gene encoding clusterin (Clu) is upregulated in the regenerating pancreas, particularly in tissues undergoing differentiation. This led us to propose that clusterin participates in the cytodifferentiation of pancreatic tissue, particularly the endocrine islet cells. The aim of this study was to investigate whether clusterin induces the differentiation of duct-lining cells into insulin-secreting cells. We isolated ductal tissue from rat pancreas and cultured it to develop epithelial cell explants for transfection of the Clu cDNA as well as for treatment of clusterin protein. The number of newly differentiated insulin cells increased 6.9-fold upon Clu overexpression compared with controls. Ins1 mRNA and peptide levels were also increased. Furthermore, glucose-stimulated insulin secretion was observed in the differentiated insulin cells. These cells were immunoreactive for insulin and C-peptide, but negative for other islet hormones and for cytokeratin-20, which indicates a fully differentiated state. Insulin cell differentiation was also increased in a dose-dependent manner by treating duct cells in culture with clusterin, indicating a growth-factor-like action of clusterin in insulin cell differentiation. These results suggest that clusterin can be considered as a potential morphogenic factor that promotes differentiation of pancreatic beta cells.

Clusterin Expression in the Early Process of Pancreas Regeneration in the Pancreatectomized Rat

We have previously reported upregulation of clusterin at the time of islet cell regeneration after beta-cell injury. This led us to speculate that clusterin might be involved in the neogenic regeneration of the pancreas. Clusterin expression was examined throughout the process of pancreatic neogenesis in pancreatectomized rats. For in vitro analysis, duct cells were isolated from the rat pancreas and clusterin cDNA was transfected for its overexpression. Clusterin and its mRNA increased significantly in the early phase of regeneration, particularly at 1-3 days after pancreatectomy. Clusterin was transiently expressed in the differentiating acinar cells but faded afterwards. Interestingly, these clusterin cells were negative for PCNA (proliferating cell nuclear antigen), whereas most epithelial cells in ductules in the regenerating tissue showed extensive proliferative activity. Clusterin expression was also detected in some endocrine cells of the regenerating tissue. Transfection of clusterin cDNA into primary cultured duct cells resulted in a 2.5-fold increase in cell proliferation and induced transformation of non-differentiated duct cells into differentiated cells displaying cytokeratin immunoreactivity. Taken together, these results suggest that clusterin may play essential roles in the neogenic regeneration of pancreatic tissue by stimulating proliferation and differentiation of duct cells.

Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model

The proliferative capacity of adult pancreatic islet cells is limited, although the formation of new islets from cells associated with the ductal epithelium is achievable even in the adult gland. Understanding the mechanism whereby proliferation and subsequent differentiation of putative precursor cells leads the appearance of new islets, i.e., islet neogenesis, may be important as a modality for treatment of both Type I and type II diabetes, in which there is an absolute or relative deficiency of insulin. It appears that certain genes and their protein products are essential to the initiation of the initial step in the pathway. We have shown that partial obstruction of the hamster pancreas is able to reverse streptozotocin-induced diabetes more than 50% of the time. An extract, termed ilotropin, prepared from obstructed pancreata, also reverses the diabetes, whereas extracts of control non-obstructed pancreata do not. Ilotropin contains a protein that is heat and acid stable with MW around 20-45 kDa that is capable of stimulating the proliferation of isolated duct cells in culture. Using mRNA and a differential display technique, 20 genes were found to be expressed in the partially obstructed (regenerating), but not the non-obstructed (non-regenerating) pancreas. One of these islet neogenesis-associated proteins (INGAP) proved to be unique to the obstructed pancreas, and a peptide contained within the sequence was capable of stimulating the proliferation of ductal cells in culture. INGAP was found to be expressed early in the neogenic process before the onset of ductal cell proliferation, and was capable of stimulating tritiated thymidine uptake into protodifferentiated epithelial cells, compatible with the notion that it might be involved in initiating the process of islet neogenesis.

Targeting of CFTR protein is linked to the polarization of human pancreatic duct cells in culture

A relationship between targeting of the protein CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and cellular polarization has been observed in various types of epithelial cells. However, there are no reports on this in human exocrine pancreatic cells, which are functionally altered in patients with cystic fibrosis. The expression of CFTR and its targeting to apical plasma membranes was investigated during growth and polarization of human ductal pancreatic cancerous Capan-1 cells. Despite their neoplastic origin, the cancerous pancreatic duct cells of the Capan-1 line secrete Cl - and HCO 3 - ions. We showed by electron microscopy, impregnation of cells with tannin and freeze-fracture that these cells become polarized during growth in culture, and are joined by tight junctions. The expression of CFTR and the various stages in its anchorage to membranes was followed using a specific polyclonal antibody, ECL-885, directed against a synthetic peptide mimicking one of the extracellular loops of CFTR. Qualitative and quantitative confocal microscopic studies showed that: ( i) the expression of CFTR was constant during growth, irrespective of cellular conformation, ( ii) the number of cells presenting CFTR anchored to membranes increased with time in culture, ( iii) the rise in membrane-bound CFTR-immunoreactivity accompanied the polarization of the cells, ( iv) CFTR anchored to plasma membranes was distributed regularly over the surface of non-polarized cells, but was localized only at the apical membranes of the polarized cells. Moreover, patch-clamp studies indicated the presence of few Cl - cAMP-dependent conductance CFTR channels on unpolarized cells, and a larger number of CFTR channels on the apical plasma membranes of polarized cells. These results indicated that the anchorage of a functional CFTR to the plasma membrane is progressive and occurs in step with polarization of these human pancreatic duct cells in culture. We suggest that the targeting of CFTR to the apical membranes is directly linked to the process of cellular polarization.

Regulation of ion content in primary cultures from reabsorptive ducts of human sweat glands studied by X-ray microanalysis.

X-ray microanalysis was used to investigate whether cAMP- and/or Ca2+-activated regulation of chloride and potassium efflux is expressed in primary cultures of sweat gland duct cells. The effects of extracellular UTP and ATP on the duct cells, and the signalling system involved in the response to ATP was also studied. Primary cultures from duct cells of human sweat glands responded to 1 microM carbachol, 2 microM of the Ca2+ ionophore A23187, or 5 mM 8-bromo-cAMP stimulation for 5 min, resulting in a decrease in cellular Cl and K concentrations. 50 microM 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), a Cl- channel blocker, can inhibit the decrease in Cl concentration induced by cAMP. Extracellular (200 microM) ATP caused a decrease of Cl and K in cultured duct cells, while (200 microM and 2 mM) UTP was ineffective. Both the phosphoinositidase C inhibitor U73122 (10 microM) and the absence of extracellular Ca2+ abolished the ATP-induced decrease in Cl and K content. Alloxan (1.25 mM), an adenylate cyclase inhibitor, had an inhibitory effect on the response to ATP. The decrease in K, but not in Cl, content in the cells elicited by ATP was blocked by prior incubation with 100 ng/ml pertussis toxin, indicating the coupling of ATP to pertussis toxin-sensitive G-proteins. In conclusion, both Ca2+- and cAMP-dependent Cl- permeability is present in primary cultures from duct cells of human sweat gland. The response to ATP can be mediated both by Ca2+- and by cAMP-dependent pathways, and is coupled to pertussis toxin-sensitive G-proteins.

Nephrotoxicity of amphotericin B is attenuated by solubilizing with lipid emulsion.

Nephrotoxicity is the major adverse effect of conventional amphotericin B (AMB/D), often limiting administration of full dosage. The new liposomal amphotericin B seems to be less toxic. The new liposomal amphotericin B seems to be less toxic. In this study, it is proposed that solubilizing the standard AMB/D preparation with 10% lipid emulsion will attenuate nephrotoxicity. Rats were injected with either AMB/D (Fungizone), AMB, AMB/D plus lipid emulsion (AMB/D/LE), or sodium deoxycholate (D). Renal function studies were performed on day 5. To assess a direct tubular toxic effect, isolated rat proximal tubule suspensions and inner medullary collecting duct cells in culture were exposed to AMB/D, AMB, AMB/D/LE, liposomal amphotericin B, and D for 60 min in normoxia. Lactate dehydrogenase (LDH) release was assessed as an index of cell injury. Creatinine clearance (ml/min per 100 g) averaged 0.79 +/- 0.04 in control rats, 0.29 +/- 0.09 in AMB rats (P < 0.001 versus control), 0.38 +/- 0.04 in AMB/D rats, 0.46 +/- 0.05 in D rats, and 0.78 +/- 0.03 in AMB/LE rats. Renal blood flow (ml/min per 100 g) was 3.45 +/- 0.31 in control, 1.29 +/- 0.28 in AMB, 1.42 +/- 0.23 in AMB/D, 3.03 +/- 0.39 in D, and 2.71 +/- 0.21 in AMB/D/LE rats. The fractional excretion of potassium (%) was 27.3 +/- 1.18 in control rats, 61.6 +/- 7.00 in AMB/D rats, 58.4 +/- 15.32 in AMB rats, and 37.9 +/- 2.06 in AMB/D/LE rats. LDH release (%) in proximal tubules incubated with AMB/D and D was 43.6 +/- 3.39 and 58.6 +/- 4.20, respectively. Addition of lipid emulsion decreased LDH release: 21.6 +/- 1.22 for AMB/D/LE and 26.4 +/- 3.03 for deoxycholate plus lipid emulsion. AMB did not demonstrate any toxic effect in proximal tubule suspensions. D was not toxic to inner medullary collecting duct cells at 0.16 mg/ml, whereas D at a higher dose and AMB induced a significant LDH release. Addition of lipid emulsion did not affect the antifungal activity as assessed by the Etest method. In conclusion, an alternative way of administering standard AMB with reduced nephrotoxicity is proposed.

Epidermal growth factor and fluid and electrolyte balance in the rat.

Epidermal growth factor (EGF) has been shown to induce a renal diuresis and natriuresis in sheep and stimulate prostaglandin synthesis from inner rat medullary collecting duct cells in culture. The aims of our study were 1) to investigate whether the renal effects of intravenous infusion of EGF were species specific and 2) to determine the mechanism of these effects by studying the interaction between EGF and indomethacin (a prostaglandin synthase inhibitor) in the conscious rat. Sprague-Dawley rats received intravenous infusions of either 0.9% saline or 0.2 or 2.0 micrograms EGF.kg-1.h-1 over a 6-day period after an initial baseline period. Infusion of 2.0 micrograms EGF.kg-1.h-1 caused an increase in urine volume (baseline: 5.5 +/- 0.2 ml to day 5: 9.0 +/- 0.4 ml, P < 0.01) and corresponding polydipsia, but not natriuresis. Administration of indomethacin with 2.0 micrograms EGF.kg-1.h-1 attenuated (P < 0.05) the diuretic (day 5 EGF + vehicle: 12.2 +/- 1.1 ml vs. EGF + indomethacin: 8.7 +/- 0.9 ml) and polydipsic effects of EGF. These studies demonstrate that intravenous infusion of EGF causes a diuretic effect in rats without natriuresis and that prostaglandins play a role in the diuretic effect of EGF in the rat.

Role of H(+)-K(+)-ATPase in pHi regulation in inner medullary collecting duct cells in culture.

Studies in inner medullary collecting duct (IMCD) cells in primary culture have proposed two mechanisms for Na(+)-independent hydrogen ion transport: an H(+)-adenosinetriphosphatase (H(+)-ATPase) and an H(+)-K(+)-ATPase. In the present study, we have employed two sources of IMCD cells, cells in primary culture derived from the terminal papilla of the Munich-Wistar rat (IMCDp) and an established murine cell line (mIMCD-3), to define the predominant mechanism(s) of Na(+)-independent intracellular pH (pHi) recovery in the IMCD. In confluent monolayers of IMCDp and mIMCD-3 cells, pHi was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) following addition and withdrawal of NH4Cl. Removal of K+ completely abolished Na(+)-independent pHi recovery in both IMCDp (delta pHi/min = 0.039 +/- 0.006 to 0.005 +/- 0.003; P < 0.001) and in mIMCD-3 (delta pHi/min = 0.055 +/- 0.009 to -0.003 +/- 0.002; P < 0.001) cells, respectively. In mIMCD-3 cells, K(+)-dependent pHi recovery was abolished by either of two specific inhibitors of the H(+)-K(+)-ATPase, Sch-28080 (5 or 10 microM) or A-80915A (10 microM). In contrast, bafilomycin A1 (2.5 and 10 nM), an inhibitor of the H(+)-ATPase, failed to attenuate K(+)-dependent pHi recovery. Moreover, sequence verified mouse gastric and colonic alpha-H(+)-K(+)-ATPase probes hybridized to total RNA from mIMCD-3 cells. Based on these findings, we conclude that Na(+)-independent pHi recovery from an acid load in both IMCDp and mIMCD-3 cells in critically dependent on extracellular K(+)-That K(+)-dependent pHi recovery was inhibited by both Sch-28080 and A-80915A but not by bafilomycin A1 suggests that the predominant mechanism by which Na(+)-independent pHi recovery is accomplished in IMCD is through the H(+)-K(+)-ATPase. Expression of both gastric and colonic alpha-H(+)-K(+)-ATPase mRNA in mIMCD-3 cells suggests that one or both of these H(+)-K(+)-ATPases may be responsible for proton secretion in the IMCD.

Metabolism and activation of pancreas specific nitrosamines by pancreatic ductal cells in culture.

Metabolism of 14C labeled N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitrosobis(2-hydroxypropyl)amine (BHP) by pancreatic duct cells in culture involves the following two pathways: reduction or oxidation reactions at the beta-carbon which result in the inter-conversion of these nitrosamines and activation reactions which result in the decomposition of the nitrosamine, the evolution of 14CO2 and the labeling of macromolecules. Reduction of BOP to HPOP seems to contribute significantly to the metabolism of the former nitrosamine by pancreatic duct cells, however, redox reactions at the beta-carbon of HPOP or BHP are not extensive. In terms of DNA damage, all three nitrosamines yield methyl and hydroxypropyl adducts. As expected, HPOP and BHP yield higher levels of O6-hydroxypropylguanine than BOP, while the latter yields higher levels of O6-methylguanine. There is no correlation between the ability of these nitrosamines to alkylate duct cell DNA in vitro and their carcinogenic potency in vivo. Concentrations of DNA adducts induced by pancreas specific nitrosamines (PSNs) in cultured duct cells at concentrations comparable to those found in the pancreatic juice of animals treated with BOP, are almost an order of magnitude lower than those induced in the pancreas of such animals. Discrepancies between in vitro and in vivo formation of active metabolites and DNA adducts may be attributed to the decline of the cells' ability to activate PSNs during culturing. In the same vein, the ductal cell may not be the main source of active metabolites targeting its DNA in the animal model.

Cellular responses to steroids in the enhancement of Na+ transport by rat collecting duct cells in culture. Differences between glucocorticoid and mineralocorticoid hormones.

It has recently been discovered that both mineralocorticoid (MC) and glucocorticoid (GC) hormones can stimulate electrogenic Na+ absorption by mammalian collecting duct cells in culture. In primary cultures of rat inner medullary collecting duct (IMCD) cells, 24-h incubation with either MC or GC agonist stimulates Na+ transport approximately threefold. We have now determined that the effects were not additive, but the time courses were different. As aldosterone is known to stimulate citrate synthase, Na+/K+ ATPase activity, and ouabain binding in cortical collecting duct principal cells, we determined the effects of steroids on these parameters in IMCD cells. MC and GC agonists both produced a small increase in citrate synthase activity. There was no increase in Na+/K+ ATPase activity but specific ouabain binding was increased more than two-fold by either agonist. To determine the role of apical Na+ entry in the steroid-induced effects, the Na+ channel inhibitor, benzamil, was used. Benzamil did not alter the stimulation of citrate synthase activity by either steroid. In contrast, GC stimulation of ouabain binding was prevented by benzamil, whereas MC stimulation was not. We conclude that there are differences in the way that MC and GC hormones produce an increased Na+ transport. Both appear to produce translocation (or activation) of pumps into the basolateral membrane. GC stimulation of pump translocation requires increased Na+ entry whereas MC stimulation does not.

Carbonic anhydrase II gene expression in mouse pancreatic duct cells.

Our goal is to create a transgenic mouse model for human pancreatic duct cell adenocarcinoma using the promoter/enhancer region of the carbonic anhydrase (CA) II gene to drive the expression of SV-40 T-antigen in pancreatic duct cells. This requires that the CA II gene be expressed in mouse pancreatic duct cells and not in other pancreatic cells, as has already been shown to be the case in the human and guinea pig pancreas. We have shown with an enzyme histochemical assay that mouse pancreatic duct cells contain CA activity in both intact pancreas and cultured interlobular duct epithelium. In addition, CA activity was detected with a biochemical assay in homogenates of cultured duct epithelium. The specific activity of duct cells was 2.75-fold greater than in whole pancreas, suggesting that a substantial amount of total pancreatic CA activity is contributed by duct cells. At least some of the CA in cultured duct cells was inferred to be CA II by Northern blot analysis of RNA extracted from the cells. The concentration of CA II mRNA in the cultured duct cells was substantially greater than in whole pancreas and would appear to account for the majority, if not all, of the CA II in the mouse pancreas.

Stimulation of DNA synthesis in pancreatic duct cells by gastrointestinal hormones: interaction with other growth factors.

Pancreatic duct cells of the Syrian hamster were grown as monolayers on thin layers of type I collagen coated onto microporous membranes. The effects of a number of potential trophic factors were tested by their ability to increase [3H]thymidine incorporation into cellular DNA. To measure the effect of growth factors, cells were subjected to a period of growth factor depletion to induce a state of partial quiescence in DNA synthesis. Cells responded with a significant increase in thymidine incorporation after the addition of epidermal growth factor (EGF) alone or a growth factor mixture containing EGF plus insulin, transferrin, selenium, linoleic acid, bovine pituitary extract, triiodothyronine, and dexamethasone. When the serum substitute, Nu Serum IV (5%, vol/vol), was added to this mixture, addition of several gastrointestinal (GI) hormones including secretin, vasoactive intestinal polypeptide (VIP), bombesin, and gastrin caused significant increases in thymidine incorporation at concentrations of 0.01-1 microM. At 1 microM, these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) greater than bombesin (153%) greater than VIP (138%) greater than gastrin (126%). Cholecystokinin octapeptide, a known trophic factor for pancreatic acinar cells, did not cause significant increases in thymidine incorporation in cultured duct cells. These results suggest that pancreatic duct cells possess receptors for a number of GI hormones and respond to the trophic effects of hormones known to stimulate pancreatic growth in vivo.

PH dependence of inhibition of arginine vasopressin-induced adenosine 3',5'-monophosphate production by cellular sodium depletion in rat renal inner medullary collecting duct cells in culture.

The present study was undertaken to determine whether the change in cellular Na+ concentration ( [Na+]i) or cellular pH (pHi) is essential for the modulation by Na+/H+ antiporter of the cellular action of arginine vasopressin (AVP) in renal inner medullary collecting duct cells in culture. Extracellular Na+ depletion promptly decreased [Na+]i from 15.8 to 5.4 mM (P less than 0.01), which was closely related to the decrease in pHi (7.19 to 6.97; P less than 0.01). In the presence of 0.5 mM 3-isobutyl-1-methylxanthine, AVP increased cellular cAMP production in a dose-dependent manner. This was significantly blunted in the Na(+)-depleted cells (1 nM AVP; 481.9 vs. 341.0 fmol/micrograms protein; P less than 0.01). When cells were incubated with the Na(+)-depleted medium containing 25 mM NaHCO3, [Na+]i decreased promptly, but the pHi remained unchanged. Under this condition, the AVP-induced increase in cellular cAMP production was not altered (1 nM AVP; 390.9 vs. 334.8 fmol/micrograms protein). Also, after the Na(+)-depleted cells were incubated in 20 mM NH4Cl, which promptly normalized pHi despite the decreased [Na+]i, the response of cAMP production to AVP was restored. Amiloride (1 x 10(-5)-1 x 10(-3) M), which blocks the Na+/H+ exchange, decreased pHi and AVP- and forskolin-induced cAMP production in a dose-dependent manner. These results indicate that the decrease in [Na+]i promptly inhibits AVP-induced cAMP production mediated through the reduction in pHi in renal inner medullary collecting duct cells.

PH dependence of inhibition of arginine vasopressin-induced adenosine 3',5'-monophosphate production by cellular sodium depletion in rat renal inner medullary collecting duct cells in culture

pH dependence of inhibition of arginine vasopressin-induced adenosine 3',5'-monophosphate production by cellular sodium depletion in rat renal inner medullary collecting duct cells in culture

Endothelin synthesis by rat inner medullary collecting duct cells.

Endothelin has been shown to affect a broad range of renal functions, including rat inner medullary collecting duct Na/K ATPase activity, renin release, renal blood flow, and glomerular filtration rate. The source of endothelin in the kidney has been assumed to be endothelial cells. However, the inner medulla contains the highest concentration of immunoreactive endothelin in the kidney. Additionally, MDCK cells, a distal tubule-like cell line, synthesize endothelin. In order to determine if primary renal tubule cells release endothelin, supernatants collected from rat inner medullary collecting duct cells in culture were tested for endothelin-1 detected by specific radioimmunoassay. Inner medullary collecting duct cells produced endothelin-1 in a time-dependent manner, releasing 1,016.7 +/- 60.1 pg of endothelin-1 per mg/cell protein/24 h. Inner medullary collecting duct cells expressed a 2.2-kilobase mRNA on blot hybridization with rat prepro endothelin-1 cDNA. Vasopressin, thrombin, bradykinin, and epinephrine did not affect endothelin-1 release. These data demonstrate endothelin-1 production by inner medullary collecting duct cells and suggest a possible autocrine role for the peptide.

Crystal-Cell Interactions: Crystal Binding to Rat Renal Papillary Tip Collecting Duct Cells in Culture

Retention of stone crystallites by urothelium is clearly one of the prime requisites for urinary stone disease. Studies in the literature as early as 1937 have highlighted that the initiation of renal calculi followed the formation of subepithelial calcified plaques in the renal pelvis. The renal papilla is one of the primary sites for crystal fixation and stone maturation. We have developed an in vitro model system for the study of kidney stone crystal retention to tubular epithelium using rat renal papillary collecting tubule (RPCT) cells in primary culture. We have qualitatively and quantitatively analyzed the binding of preformed calcium oxalate monohydrate (COM), hydroxyapatite (HA), and uric acid (UA) crystals to RPCT cells. Our goal was to determine if three common urinary stone crystals evidenced different crystal-cell binding characteristics. Also, since these crystals are frequently observed admixed in stones, we have studied the inhibitive binding characteristics of these crystals with RPCT cells. The RPCT cells in culture grow both as the typical polygonal cells in monolayer and as clumps of aggregated cells. The cells in the aggregates are viable epithelial cells that have lost their attachment to the basement membrane, resulting in the exposure of surface molecules that would not normally be present unless the cells were damaged or if there was a loss of intercellular tight junctions. COM, HA, and UA crystals all preferentially bound to the aggregated cells and all exhibited similar saturable binding patterns.(ABSTRACT TRUNCATED AT 250 WORDS)