Risk of malignancy in resected cystic tumors of the pancreas < or =3 cm in size: is it safe to observe asymptomatic patients? A multi-institutional report.

Recent international consensus guidelines propose that cystic pancreatic tumors less than 3 cm in size in asymptomatic patients with no radiographic features concerning for malignancy are safe to observe; however, there is little published data to support this recommendation. The purpose of this study was to determine the prevalence of malignancy in this group of patients using pancreatic resection databases from five high-volume pancreatic centers to assess the appropriateness of these guidelines. All pancreatic resections performed for cystic neoplasms < or =3 cm in size were evaluated over the time period of 1998-2006. One hundred sixty-six cases were identified, and the clinical, radiographic, and pathological data were reviewed. The correlation with age, gender, and symptoms (abdominal pain, nausea and vomiting, jaundice, presence of pancreatitis, unexplained weight loss, and anorexia), radiographic features suggestive of malignancy by either computed tomography, magnetic resonance imaging, or endoscopic ultrasound (presence of solid component, lymphadenopathy, or dilated main pancreatic duct or common bile duct), and the presence of malignancy was assessed using univariate and multivariate analysis. Among the 166 pancreatic resections for cystic pancreatic tumors < or =3 cm, 135 cases were benign [38 serous cystadenomas, 35 mucinous cystic neoplasms, 60 intraductal papillary mucinous neoplasms (IPMN), 1 cystic papillary tumor, and 1 cystic islet cell tumor], whereas 31 cases were malignant (14 mucinous cystic adenocarcinomas and 13 invasive carcinomas and 4 in situ carcinomas arising in the setting of IPMN). A greater incidence of cystic neoplasms was seen in female patients (99/166, 60%). Gender was a predictor of malignant pathology, with male patients having a higher incidence of malignancy (19/67, 28%) compared to female patients (12/99, 12%; p < 0.02). Older age was associated with malignancy (mean age 67 years in patients with malignant disease vs 62 years in patients with benign lesions (p < 0.05). A majority of the patients with malignancy were symptomatic (28/31, 90%). Symptoms that correlated with malignancy included jaundice (p < 0.001), weight loss (p < 0.003), and anorexia (p < 0.05). Radiographic features that correlated with malignancy were presence of a solid component (p < 0.0001), main pancreatic duct dilation (p = 0.002), common bile duct dilation (p < 0.001), and lymphadenopathy (p < 0.002). Twenty-seven of 31(87%) patients with malignant lesions had at least one radiographic feature concerning for malignancy. Forty-five patients (27%) were identified as having asymptomatic cystic neoplasms. All but three (6.6%) of the patients in this group had benign disease. Of the patients that had no symptoms and no radiographic features, 1 out of 30 (3.3%) had malignancy (carcinoma in situ arising in a side branch IPMN). Malignancy in cystic neoplasms < or =3 cm in size was associated with older age, male gender, presence of symptoms (jaundice, weight loss, and anorexia), and presence of concerning radiographic features (solid component, main pancreatic duct dilation, common bile duct dilation, and lymphadenopathy). Among asymptomatic patients that displayed no discernable radiographic features suggestive of malignancy who underwent resection, the incidence of occult malignancy was 3.3%. This study suggests that a group of patients with small cystic pancreatic neoplasms who have low risk of malignancy can be identified, and selective resection of these lesions may be appropriate.

Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazide-sensitive, electrically neutral transport system.

The urinary bladder of the winter flounder absorbs NaCl by a process independent of the transepithelial voltage. In contrast to most other epithelia which have a neutral NaCl-absorptive system, the flounder bladder has a high transepithelial resistance. This feature simplifies analysis of the cellular transport system because the rate of ion transfer through the paracellular pathway is rather low. Experiments were designed to distinguish among three possible mechanisms of neutral NaCl absorption: (a) Na/K/2Cl cotransport; (b) parallel Na/H and Cl/OH exchange; (c) and simple NaCl cotransport. A clear interdependency of Na and Cl for net absorption was demonstrated. NaCl absorption was not dependent on mucosal K and was minimally sensitive to loop diuretics. Thus a Na/K/2Cl transport system was unlikely. The mechanism was not parallel exchange as evidenced by insensitivity to amiloride and to 4,4'-diisothiocyano-2,2'-disulfonic stilbene, an inhibitor of anion exchange. In addition, inhibitors of carbonic anhydrase had no effect. Net absorption was almost completely abolished by hydrochlorothiazide (0.1 mM). Its action was rapid, reversible, and effective only from the mucosal surface. Metolazone, a structurally dissimilar diuretic in the benzothiadiazide class had qualitatively similar actions. The mechanism of NaCl absorption in this tissue appears to be a simple interdependent process. Its inhibition by thiazide diuretics appears to be a unique feature. The flounder bladder may be a model for NaCl absorption in the distal renal tubule.

Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibitions of the medullary portion.

Prostaglandins are present in large quantities in the kidney and have been shown to directly affect transepithelial transport. The present studies were designed to examine whether prostaglandin E2 could affect chloride transport across the thick ascending limb of Henle. Isolated segments of the cortical and medullary thick ascending limb of Henle were perfused in vitro and the transepithelial voltage and net chloride flux were measured. Exposure of the medullary thick ascending limb to 2 microM prostaglandin E2 resulted in a fall in net chloride transport of 40--50% with a concomitant fall in voltage. In contrast, net chloride transport in the cortical thick ascending limb was not affected by prostaglandin E2. Under similar conditions, the medullary thick ascending limb possessed twice the capacity to transport chloride than did the cortical thick ascending limb. The results suggest that endogenous renal prostaglandins may play a modulating role in the addition of salt to the renal medullary interstitium and may, under some circumstances, by chloruretic.

Consequences of potassium recycling in the renal medulla. Effects of ion transport by the medullary thick ascending limb of Henle's loop.

The consequences of K recycling and accumulation in the renal medulla were examined by measuring the effect of elevated K concentration on ion transport by the medullary thick ascending limb of Henle's loop. Perfused and bathed in vitro, thick limbs from both mouse and rabbit displayed a graded, reversible reduction of transepithelial voltage after increasing K concentration from 5 to 10, 15, or 25 mM. The effect was reproducible whether osmolality was 328 or 445 mosmol/kg H2O, and whether K replaced Na or choline. Net chloride absorption and transepithelial voltage were reduced by almost 90% when ambient K concentration was 25 mM. When either lumen or bath K was increased to 25 mM, net Na absorption was reduced. There was spontaneous net K absorption when perfusate and bath K concentration was 5 mM. Analysis of transepithelial K transfer after imposition of chemical gradients demonstrated rectification in the absorptive direction. Absorption of K by this segment provides a means to maintain high medullary interstitial concentration. Accumulation of K in the outer medulla, by reducing NaCl absorption, would increase volume flow through the loop of Henle and increase Na and water delivery to the distal nephron. K recycling thus might provide optimum conditions for K secretion by the distal nephron.

Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule.

This study was designed to examine whether prostaglandin E2 can directly affect sodium transport across isolated perfused rabbit renal collecting tubules. Changes in transepithelial potential and isotopic sodium fluxes in response to peritubular prostaglandin E2 were measured. In addition, changes in transepithelial potential of the outer medullary collecting tubule in response to prostaglandin E2 were also measured. With few exceptions, all rabbits received 5 mg/day desoxycorticosterone acetate for 4-11 days before experimentation. The results of the experiments show that: (a) prostaglandin E2 inhibits the negative transepithelial potential in the cortical collecting tubule as well as the outer medullary collecting tubule; (b) prostaglandin E2 inhibits net sodium transport out of the lumen by inhibiting efflux while backflux is unaffected; (c) prostaglandin E2 produces this inhibition within 15 min, and the effects are dose dependent and reversible. These results suggest that prostaglandin E2 may modulate sodium transport in vivo and may contribute to the final regulation of sodium excretion.

Inhibition of cortical collecting tubule chloride transport by organic acids.

Cl self-exchange by the rabbit cortical collecting tubule (CCT) occurs via an apical anion exchanger in series with a basolateral Cl conductance. We studied the effects of organic acids on CCT Cl self-exchange. We found no evidence for transport of acid anions by the self-exchange system. Rather, Cl self-exchange was inhibited by a variety of organic acids. The degree of inhibition correlated with the chloroform/water partition coefficient and was enhanced by lowering pH, indicating inhibition by the lipid-soluble, protonated species. Inhibition by the representative acid iso-butyrate was dose-dependent and showed sidedness (basolateral greater than apical). Iso-butyrate also reversibly reduced transepithelial conductance without altering K permeability, suggesting inhibition of the principal cell basolateral Cl conductance. Because small organic compounds with similar lipid solubilities but no carboxyl group had no effect, both the carboxyl group and the lipid-solubility of organic acids appear to be important. The results are consistent with blockade of chloride channels by organic acids.

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.

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.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

Cortical and papillary micropuncture examination of chloride transport in segments of the rat kidney during inhibition of prostaglandin production. Possible role for prostaglandins in the chloruresis of acute volume expansion.

Prostaglandins have been postulated to participate in the regulation of salt excretion during acute volume expansion. The present papillary and cortical micropuncture studies were designed to examine the effect of prostaglandin synthesis inhibitors on segmental chloride transport during hydropenia (with and without meclofenamate) and 10% volume expansion (with and without both meclofenamate and indomethacin). Both inhibitors significantly decreased the urinary excretion rate of prostaglandins E(2) and F(2alpha). Clearance studies on the intact right kidney demonstrated no effect of either agent on glomerular filtration rate, but a significant reduction in chloride excretion during hydropenia and volume expansion was observed. To assess the specific site(s) of enhanced chloride reabsorption, absolute and fractional chloride delivery was measured in the late proximal tubule, thin descending limb of Henle, and the early and late distal tubules. In addition, the fraction of filtered chloride remaining at the base and tip of the papillary collecting duct was compared to that fraction remaining at the superficial late distal tubule. During hydropenia, meclofenamate had no effect on fractional chloride delivery out of the superficial late distal tubule or the juxtamedullary thin descending limb of Henle, but significantly reduced the fraction of chloride delivered to the base of the papillary collecting duct. During volume expansion, neither meclofenamate nor indomethacin had an effect on absolute chloride delivery out of the proximal tubule or the thin descending limb of Henle. However, absolute chloride delivery to the early distal tubule was significantly reduced, and was associated with a decrease in fractional chloride reabsorption in this segment. Furthermore, the fraction of chloride delivered to the base of the collecting duct was significantly reduced. Fractional reabsorption along the terminal 1 mm of the collecting duct was not altered by either meclofenamate or indomethacin. These results suggest that inhibitors of prostaglandin synthesis result in an increase in chloride reabsorption in the superficial loop of Henle, and in segments between the superficial late distal tubule and the base of the collecting duct. The results are consistent with the view that prostaglandins inhibit chloride transport in the thick ascending limb of Henle, and/or the cortical and outer medullary collecting tubule.

Diffuse intimal fibromuscular dysplasia with multiorgan failure.

A woman presented with a rapid onset of hypertension, angina pectoris, peripheral vascular disease, renal involvement, and a large liver cyst. Surgical removal of the liver cyst precipitated renal and liver failure and a terminal arrhythmia. At autopsy, there was intimal fibromuscular dysplasia involving the arteries to the heart, liver, kidneys, and intestines and evidence of recent infarction of the intestines, kidney, and liver. This case illustrates that intimal fibromuscular dysplasia (FMD) can be a diffuse and rapidly progressive disease. Some treatments currently being evaluated for preventing restenosis following angioplasty may find use in treating this uncommon disease.

Candidate genes in the regulation of Na+ transport by inner medullary collecting duct cells from Dahl rats.

Recently, we reported that primary cultures of inner medullary collecting duct cells from Dahl salt-sensitive (S) rats absorb more Na+ than do cells cultured from Dahl salt-resistant (R) rats. To begin to evaluate the molecular basis for this difference, we selected four candidate gene products that on the basis of their physiology and genetics could participate in regulation of Na+ transport by these cells. During 24-hour exposure, inhibitors of the cytochrome P450 enzymes had no effect on Na+ transport by either S or R monolayers. Twenty-four-hour exposure to NG-monomethyl-L-arginine (0.5 mmol/L), a nonspecific inhibitor of NO synthase, also had no effect on Na+ transport by either S or R monolayers. Neither atrial natriuretic peptide 1-28 (100 nmol/L) nor 8-Br-cyclic GMP (100 micromol/L) had any short-term effect on Na+ transport by either S or R monolayers. 18-Hydroxy-11-deoxycorticosterone (100 nmol/L), an adrenocorticoid hormone that is produced in greater amounts in S rats, stimulated Na+ transport by both S and R monolayers via the mineralocorticoid receptor; however, its effect was less potent than aldosterone. Congenic rats in which the R isoform of the 11beta-hydroxylase gene was bred onto the S background had monolayers that transported Na+ at a rate similar to the S rats. These results demonstrate that neither cytochrome P450 genes, NO synthase genes, the atrial natriuretic peptide receptor gene, nor the 11beta-hydroxylase gene is a likely candidate to explain the difference in Na+ transport between S and R inner medullary collecting duct monolayers in primary culture.

Genetic characterization of the "new" Harlan Sprague Dawley Dahl salt-sensitive rats.

In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S(HSD)). To evaluate the genetic integrity and salt sensitivity of thse new S(HSD), we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S(HSD), 16 "authentic" SS/Jr from the inbred colony of John Rapp (S(Rapp)), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R(HSD)), and (genotyping only) 6 known "contaminated" Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S(Rapp) and S* and 18 were polymorphic between S(Rapp) and R(Rapp), but none of the 22 markers revealed polymorphisms between S(Rapp) and the new S(HSD). The phenotyping studies showed that during an ultra-low salt diet, mean arterial pressure was higher (P < .05) in both authentic S(Rapp) (129 +/- 2 mm Hg; mean +/- SE) and new S(HSD) (120 +/- 2 mm Hg) than in R(HSD) (93 +/- 1 mm Hg). A high salt diet increased mean arterial pressure in every S(HSD) and S(Rapp). Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P < 0.05) greater in authentic S(Rapp) (+51 +/- 3 mm Hg) than in new S(HSD) (+39 +/- 3 mm Hg). In addition, salt-induced mortality was significantly greater in S(Rapp) (62.5%) than S(HSD) (8.3%) after 8 weeks (P < 0.01). S(HSD) were genotypically indistinguishable from S(Rapp), had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S(HSD) supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S(HSD) compared with S(Rapp). This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S(HSD) and S(Rapp) may now constitute distinct substrains of Dahl SS/Jr.

The structure of the rat amiloride-sensitive epithelial sodium channel gamma subunit gene and functional analysis of its promoter.

Prolonged dietary Na+ depletion and chronic administration of adrenal steroids increase steady-state mRNA levels of the gamma subunit of the epithelial sodium channel (gammaENaC) in rat colon. This increase correlates with a marked increase in transepithelial Na+ transport and is thought to occur via transcriptional regulation. To begin to evaluate these mechanisms in detail, we determined the organization of the rat gammaENaC gene. A rat genomic library was screened and overlapping lambda clones that together span the gene (approximately 36 kb) and contain at least 1 kb of 5' flanking genomic DNA were isolated. As in the human gene, the rat gammaENaC gene contains 13 exons and a CpG island at the 5' end of the gene. A single transcription start site was identified in rat kidney by nuclease protection assay defining a 5' untranslated region of 126 nt. The translation initiation codon was identified within the second exon and the entire 3' UTR (approximately 1 kb) was within the last exon. 800 bp of 5' flanking sequence, as well as the 3.4 kb first intron, were sequenced and analyzed for transcriptional regulatory motifs. Analogous to the human gammaENaC gene [Thomas, C.P., Doggett, N.A., Fisher, R., Stokes J.B., 1996. Genomic organization and the 5' flanking region of the gamma subunit of the human amiloride-sensitive epithelial sodium channel. J. Biol. Chem. 271, 26 062--26 066], two GC boxes were seen at -30 and -61 to the transcription start site. In addition, putative AP-1, AP-2, CRE, Sp1 and GATA-1 and GRE motifs were identified elsewhere in the 5' flanking region or the first intron. Two mammalian-wide interspersed repeats and a rodent-specific B1 repeat were also identified within the first intron. Fragments containing the putative GRE motifs coupled to luciferase did not confer a glucocorticoid-stimulated response in two cell lines that contained a functional glucocorticoid receptor. However, a 76 nt rat gammaENaC 5' flanking fragment (-76 to +68) directed expression of luciferase in the epithelial cell lines H441 and FRTL5, suggesting that this minimal region that contained both GC boxes was sufficient for promoter activity.

SGK is a primary glucocorticoid-induced gene in the human.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.