Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.

Polycystic kidney disease is characterized by the progressive enlargement of kidneys due to expanding fluid-filled cysts with the rate of renal volume increase held to be a marker of disease progression. Magnetic resonance imaging (MRI) is used to monitor changes in renal volume in patients with polycystic kidney disease; however, it has not been effectively used in mice to monitor changes in kidney volume during drug treatment studies. We used a powerful 9.4-T horizontal bore magnetic resonance scanner to track changes in kidney volume in pcy/pcy mice, an ortholog of nephronophthisis type 3. Mice were sequentially scanned from 4 to 30 weeks of age and kidney volumes determined from high-resolution images. Kidney volume and maximal cross-sectional surface area correlated positively with kidney weight and the histologic determination of surface area. The increase in kidney volume was exponential up to 20 weeks of age, after which there was a plateau consistent with the replacement of normal parenchyma by fibrotic tissue. Our study demonstrates that MRI accurately determines the rate of kidney volume increase in mice with polycystic kidney disease and hence may be useful in assessing the effectiveness of therapeutic agents to slow disease progression.

Calcium channel inhibition accelerates polycystic kidney disease progression in the Cy/+ rat.

In polycystic kidney disease, abnormal epithelial cell proliferation is the main factor leading to cyst formation and kidney enlargement. Cyclic AMP (cAMP) is mitogenic in cystic but antimitogenic in normal human kidney cells, which is due to reduced steady-state intracellular calcium levels in cystic compared to the normal cells. Inhibition of intracellular calcium entry with channel blockers, such as verapamil, induced cAMP-dependent cell proliferation in normal renal cells. To determine if calcium channel blockers have a similar effect on cell proliferation in vivo, Cy/+ rats, a model of dominant polycystic kidney disease, were treated with verapamil. Kidney weight and cyst index were elevated in verapamil-treated Cy/+ rats. This was associated with increased cell proliferation and apoptosis, elevated expression, and phosphorylation of B-Raf with stimulation of the mitogen-activated protein kinase MEK/ERK (mitogen-activated protein kinase kinase/extracellular-regulated kinase) pathway. Verapamil had no effect on kidney morphology or B-Raf stimulation in wild-type rats. We conclude that treatment of Cy/+ rats with calcium channel blockers increases activity of the B-Raf/MEK/ERK pathway accelerating cyst growth in the presence of endogenous cAMP, thus exacerbating renal cystic disease.

cAMP-dependent fluid secretion in rat inner medullary collecting ducts.

We used an unambiguous in vitro method to determine if inner medullary collecting ducts (IMCD) have intrinsic capacities to absorb and secrete solutes and fluid in an isotonic medium. IMCD(1), IMCD(2), and IMCD(3) were dissected from kidneys of young Sprague-Dawley rats. 8-Bromo-3',5'-cyclic monophosphate (8-BrcAMP) stimulated lumen formation and progressive dilation in all IMCD subsegments; lumen formation was greatest in IMCD(1.) Benzamil potentiated the rate of lumen expansion in response to 8-BrcAMP. Fluid entered tubule lumens by transcellular secretion rather than simple translocation of intracellular fluid. Secreted lumen solutes were osmometrically active. Inhibition of protein kinase A with H-89 and Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate blocked fluid secretion. The rate of lumen expansion was reduced by the selective addition of ouabain, barium, diphenyl-2-carboxylate, bumetanide, glybenclamide, or DIDS, or reduction of extracellular Cl(-). We conclude that IMCD absorb and secrete electrolytes and fluid in vitro and that secretion is accelerated by cAMP. We suggest that salt and fluid secretion by the terminal portions of the renal collecting system may have a role in modulating the composition and volume of the final urine.

cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway.

BACKGROUND: : Cellular proliferation is a key factor in the enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). We determined the extent to which adenosine 3':5'-cyclic monophosphate (cAMP) may regulate the in vitro proliferation of cyst epithelial cells derived from human ADPKD cysts. METHODS: : Epithelial cells from cysts of individuals with ADPKD and from normal human kidney cortex (HKC) of individuals without ADPKD were cultured. The effects of agonists and inhibitors on the rate of cellular proliferation and the activation of extracellular signal-regulated kinase (ERK1/2) were determined. RESULTS: : 8-Br-cAMP (100 micromol/L) stimulated the proliferation of cells from eight different ADPKD subjects to 99.0% above baseline; proliferation was inhibited by protein kinase A (PKA) antagonists H-89 (97%) and Rp-cAMP (90%). Forskolin (10 micromol/L), which activates adenylyl cyclase, increased proliferation 124%, and receptor-mediated agonists arginine vasopressin, desmopressin, secretin, vasoactive intestinal polypeptide, and prostaglandin E2 stimulated proliferation 54.2, 56.3, 46.7, 37.1, and 48.3%, respectively. The mitogen extracellular kinase (MEK) inhibitor PD98059 completely inhibited ADPKD cell proliferation in response to cAMP agonists, but genistein, a receptor tyrosine kinase inhibitor, did not block cAMP-dependent proliferation. cAMP agonists increased the activity of ERK above control levels within five minutes. In contrast to ADPKD, proliferation and ERK activity of cells derived from normal HKC were not stimulated by cAMP agonists, although electrogenic Cl- secretion was increased by these agonists in both ADPKD and HKC cell monolayers. CONCLUSIONS: : We conclude that cAMP agonists stimulate the proliferation of ADPKD but not HKC epithelial cells through PKA activation of the ERK pathway at a locus distal to receptor tyrosine kinase. We suggest that the adenylyl cyclase signaling pathway may have a unique role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular proliferation and transepithelial solute and fluid secretion.

A synthetic channel-forming peptide induces Cl(-) secretion: modulation by Ca(2+)-dependent K(+) channels.

A synthetic Cl(-) channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl(-) and fluid secretion. The sequence of the core peptide, M2GlyR, corresponds to the second membrane-spanning region of the glycine receptor, a domain thought to line the pore of the ligand-gated Cl(-) channel. Using a pharmacological approach, we show that the flux of Cl(-) through the artificial Cl(-) channel can be regulated by modulating basolateral K(+) efflux through Ca(2+)-dependent K(+) channels. Application of C-K4-M2GlyR to the apical surface of monolayers composed of human colonic cells of the T84 cell line generated a sustained increase in short-circuit current (I(SC)) and caused net fluid secretion. The current was inhibited by the application of clotrimazole, a non-specific inhibitor of K(+) channels, and charybdotoxin, a potent inhibitor of Ca(2+)-dependent K(+) channels. Direct activation of these channels with 1-ethyl-2-benzimidazolinone (1-EBIO) greatly amplified the Cl(-) secretory current induced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBIO on I(SC) was significantly greater than the sum of the individual effects of the two compounds and was independent of cAMP. Treatment with 1-EBIO also increased the magnitude of fluid secretion induced by the peptide. The cooperative action of C-K4-M2GlyR and 1-EBIO on I(SC) was attenuated by Cl(-) transport inhibitors, by removing Cl(-) from the bathing solution and by basolateral treatment with K(+) channel blockers. These results indicate that apical membrane insertion of Cl(-) channel-forming peptides such as C-K4-M2GlyR and direct activation of basolateral K(+) channels with benzimidazolones may coordinate the apical Cl(-) conductance and the basolateral K(+) conductance, thereby providing a pharmacological approach to modulating Cl(-) and fluid secretion by human epithelia deficient in cystic fibrosis transmembrane conductance regulator Cl(-) channels.

Epithelial transport in polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD), the genetic defect results in the slow growth of a multitude of epithelial cysts within the renal parenchyma. Cysts originate within the glomeruli and all tubular structures, and their growth is the result of proliferation of incompletely differentiated epithelial cells and the accumulation of fluid within the cysts. The majority of cysts disconnect from tubular structures as they grow but still accumulate fluid within the lumen. The fluid accumulation is the result of secretion of fluid driven by active transepithelial Cl- secretion. Proliferation of the cells and fluid secretion are activated by agonists of the cAMP signaling pathway. The transport mechanisms involved include the cystic fibrosis transmembrane conductance regulator (CFTR) present in the apical membrane of the cystic cells and a bumetanide-sensitive transporter located in the basolateral membrane. A lipid factor, called cyst activating factor, has been found in the cystic fluid. Cyst activating factor stimulates cAMP production, proliferation, and fluid secretion by cultured renal epithelial cells and also is a chemotactic agent. Cysts also appear in the intrahepatic biliary tree in ADPKD. Normal ductal cells secrete Cl- and HCO3-. The cystic ductal cell also secretes Cl-, but HCO3- secretion is diminished, probably as the result of a lower population of Cl-/HCO3- exchangers in the apical membrane as compared with the normal cells. Some segments of the normal renal tubule are also capable of utilizing CFTR to secrete Cl-, particularly the inner medullary collecting duct. The ability of Madin-Darby canine kidney cells and normal human kidney cortex cells to form cysts in culture and to secrete fluid and the functional similarities between these incompletely differentiated, proliferative cells and developing cells in the intestinal crypt and in the fetal lung have led us to suggest that Cl- and fluid secretion may be a common property of at least some renal epithelial cells in an intermediate stage of development. The genetic defect in ADPKD may not directly affect membrane transport mechanisms but rather may arrest the development of certain renal epithelial cells in an incompletely differentiated, proliferative stage.

A synthetic peptide derived from glycine-gated Cl- channel induces transepithelial Cl- and fluid secretion.

M2GlyR is a synthetic 23-amino acid peptide that mimics the second membrane-spanning region of the alpha-subunit of the postsynaptic glycine receptor. This peptide has been shown to form an anion-selective channel in phospholipid bilayers. We have investigated the possibility that the peptide may incorporate into the apical membrane of secretory epithelia and induce the secretion of Cl- and water. We improved the solubility of this peptide by adding four lysine residues to the carboxy terminus, C-K4-M2GlyR, and assayed its channel-forming activity using a subculture of Madin-Darby canine kidney (MDCK) cells. The addition of 100 microM C-K4-M2GlyR to the apical surface of MDCK monolayers significantly increased short-circuit current (Ise), hyperpolarized transepithelial potential difference, and induced fluid secretion. The increase in Ise was inhibited by 100 microM bumetanide and by Cl- channel inhibitors. The effectiveness of the channel blockers followed the sequence niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoate > diphenylamine-2-carboxylate (DPC) > glibenclamide. The effect of the peptide was not inhibited by 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid. Removing Cl from the bathing solutions also inhibited the effect of the peptide. The Cl- efflux pathway induced by C-K4-M2GlyR differs from the native pathway activated by the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin. First, intracellular cAMP levels were unaffected. Second, the concentration of DPC required to inhibit the effect of the peptide was much lower than that needed to block the forskolin response (100 microM vs. 3 mM). These results support the hypothesis that the synthetic peptide C-K4-M2GlyR can from Cl -selective channels in the apical membrane of secretory epithelial cells and can induce sustained transepithelial secretion of Cl- and fluid.

Chloride and fluid secretion by cultured human polycystic kidney cells.

Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3',5'-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended within a polymerized collagen matrix and stimulated to form cysts. Individual cultured cysts were placed in a chamber on the stage of an inverted microscope equipped with epifluorescent and video analysis attachments. The rate of fluid secretion, cell volume and changes in intracellular Cl- were measured. In the absence of secretagogues, fluid was absorbed from the cyst cavity (-2.36 +/- 0.64 nl/min/cm2 inner surface area). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-1-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 +/- 1.28 nl/min/cm2). Bumetanide reversibly reduced fluid secretion to 0.95 +/- 0.60 nl/min/cm2. Cell volume rapidly decreased by 7.5 +/- 0.9% with the initiation of secretion and bumetanide caused an additional loss (4.2 +/- 1.0%). Furosemide had a similar effect on forskolin-induced fluid secretion. Cellular chloride concentration was monitored with the use of the indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ). Removal of Cl- from the bath reduced intracellular [Cl-] (MEQ fluorescence increased by 11.4 +/- 2.3%). In cysts pretreated with furosemide to prevent Cl- entry, the application of forskolin caused a decrease in Cl- concentration (MEQ fluorescence increased by 9.3 +/- 2.6%). Using monolayers of cultured ADPKD cells, grown on permeant supports, we compared the changes in short circuit current (ISC) induced by forskolin in the presence and absence of external Cl-. Forskolin increased ISC (from 8.9 +/- 2.7 to 10.6 +/- 2.7 microA/cm2) in the presence of Cl-, but did not significantly affect ISC in its absence. These data indicate that cultured ADPKD cells can direct fluid transport in either the absorptive or the secretory direction, and that cAMP stimulates secretion and this secretion is accompanied by a net loss of cell solute. Inhibition of secretion by bumetanide or furosemide caused an additional loss of cell solute, including Cl-. The ionic transepithelial current induced by forskolin is dependent on the presence of Cl-. These data support the thesis that chloride secretion drives fluid secretion by cultured ADPKD cells.

Anion secretion drives fluid secretion by monolayers of cultured human polycystic cells.

We have investigated the hypothesis that active anion transport drives fluid secretion by the cystic epithelium in autosomal dominant polycystic kidney disease (ADPKD). We prepared monolayers of a primary culture derived from cystic tissue removed from ADPKD patients. The monolayers were grown on permeant supports, and fluid secretion was initiated by forskolin. The results were compared with those obtained with monolayers of Madin-Darby canine kidney (MDCK) cells, known to secrete Cl-. In the absence of the agonist, ADPKD monolayers absorbed fluid (0.20 +/- 0.02 microliter.cm surface area-2.h-1). Forskolin reversed this to secretion (0.60 +/- 0.03 microliter.cm-2.h-1). Control MDCK monolayers did not transport fluid in either direction, but forskolin induced secretion (0.48 +/- 0.03 microliter.cm-2.h-1). The electrical properties of the monolayers were monitored in Ussing chambers. Forskolin increased the transepithelial potential difference (Vte) of ADPKD monolayers (-0.9 +/- 0.1 to -1.1 +/- 0.1 mV) and the short-circuit current (Isc) (6.6 +/- 0.7 to 9.2 +/- 0.8 microA/cm2). The transepithelial resistance (Rte) fell (156 +/- 9 to 138 +/- 10 omega.cm2). Similar results were obtained with MDCK monolayers. The polarity of Vte and the direction of the Isc are compatible with the hypothesis that active secretion of anion drives fluid secretion. Basolateral application of the Na-K-2Cl cotransporter, bumetanide, reduced forskolin-stimulated fluid secretion by ADPKD monolayers (0.56 +/- 0.05 to 0.28 +/- 0.03), depolarized Vte, and inhibited Isc without affecting Rte. Apical application of the Cl- channel blocker, diphenylamine-2-carboxylate, also inhibited fluid secretion by ADPKD monolayers (0.65 +/- 0.03 to 0.27 +/- 0.02 microliter.cm-2.h-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of cell volume and membrane potential changes to fluid secretion in a model of renal cysts.

Renal tubular epithelia ordinarily absorb NaCl and water, although recent evidence indicates that renal cysts secrete fluid. We have utilized the experimental advantages offered by cultured cysts, formed in a collagen matrix by propagating Madin-Darby canine kidney cells, to investigate the mechanisms involved in fluid secretion by this renal epithelium. The rate of fluid transport (adduced from changes in cavity volume), cell volume and changes in membrane potential were measured simultaneously in isolated cysts. Under basal conditions, cysts absorbed fluid (-0.83 +/- 0.34 x 10(-6) ml/min/cm2 cavity surface area, N = 23). AVP and IBMX changed the direction of net fluid transport to secretion (4.24 +/- 0.49 x 10(-6) ml/min/cm2). Cell volume initially fell 7.4 +/- 0.5% and remained stable thereafter as secretion continued. Membrane electrical potential (bis-oxonol epifluorescence) hyperpolarized in 13 cysts and depolarized in 6, the mean change was 1.9 +/- 3.1%. Fluid secretion was abolished by 0.1 mM ouabain. Secretion was not affected by 0.1 mM DIDS and cell pH (bis-carboxyethyl-carboxyfluorescein epifluorescence) was not altered by the induction of secretion, suggesting that secretion is not dependent on Cl-HCO3 exchange. Barium, in the presence of AVP and IBMX, depolarized the cell membrane potential (bis-oxonol fluorescence increased 22.3 +/- 0.03%), reversed secretion to absorption (from 3.21 +/- 0.93 to -1.52 +/- 0.61 x 10(-6) ml/min/cm2), and increased cell volume 2.7 +/- 0.5%. Bumetanide (100 microM) reduced fluid secretion from 4.49 +/- 1.23 to -0.75 +/- 0.55 x 10(-6) ml/min/cm2, further reduced cell volume 4.4 +/- 1.2% and hyperpolarized the membranes (bis-oxonol fluorescence fell 24.3 +/- 5.0%). In the absence of AVP and IBMX bumetanide had no effect on fluid transport, cell volume or membrane potentials. We conclude that AVP reversed the direction of fluid transport in these cultured renal epithelial cysts from absorption to secretion by stimulating a coordinated interaction of basolateral and apical K, Cl and Na transport mechanisms.

Cellular electrolyte and volume changes induced by acidosis in the rabbit proximal straight tubule.

Cellular acidosis induced either by high Pco2 or by low HCO3- concentrations has been shown to cause cell swelling in isolated, lumen-collapsed, S2 segments of the rabbit proximal tubule (Sullivan et al., Am J Physiol 1990; 258: F831-F839). The swelling is not followed by a volume regulatory response. The ionic basis of the swelling has been investigated by measurement of the cellular K+, Na+, and Cl- content (electron probe) and HCO3- concentration (pH-sensitive fluorescent dye). Cell content of K+, Na+, and Cl- was expressed as a ratio to P content. Exposure to 15% CO2 increased K/P from 0.98 to 1.16, Cl/P from 0.14 to 0.20, and Na/P from 0.09 to 0.11. Cell (HCO3-) increased from 22 to 32 mM. Reduction in bath (HCO3-) from 25 to 5 mM reduced cell (HCO3-) from 24 to 8 mM and increased K/P from 0.75 to 0.90. Na/P fell from 0.13 to 0.09, and Cl/P fell from 0.15 to 0.12. Thus, swelling resulting from acidosis induced by high CO2 was accompanied by an accumulation of K+, Cl-, and HCO3-; that resulting from acidosis induced by a fall in (HCO3-) was combined with an accumulation of K+ and an unidentified anion. To determine if the swelling induced by a fall in pH might be coupled with depolarization of the basolateral membrane, the effect of 1 mM barium was tested. Barium caused cell volume to increase 10.2%. Cell pH rose from 7.38 to 7.56, K/P increased from 0.63 to 0.73, Na/P did not change, and Cl/P rose from 0.17 to 0.20. Cell (HCO3-) increased 10.4 mM. When the pH of the barium-treated tissue was reduced to 7.02 by raising Pco2, additional cell swelling and accumulation of K+ occurred. The effect on cell volume of a reduction of bath (HCO3-) from 25 to 5 mM at constant bath pH was determined. Cell pH was not altered. Cell volume decreased 3% initially and then returned to the control level. When the bath (HCO3-) was restored to 25 mM, cell volume increased 3.9% and then returned to the baseline. Thus, volume regulation was not impaired. It was concluded that a fall in cell pH induces swelling, and this is coupled with an accumulation of K+. This is probably the result of a pH effect on barium-sensitive and barium-insensitive K+ conductance pathways. The nature of the anions that balance the gain in K+ depends on the means used to induce acidosis.

Effect of cellular acidosis on cell volume in S2 segments of renal proximal tubules.

The presence of pH-sensitive transport mechanisms in the basolateral membrane of proximal tubular cells suggests that cell volume and its regulation may be sensitive to changes in cell pH. We have measured the response of cell pH and cell volume to changes in the acid-base composition of solutions bathing isolated, lumen-collapsed, proximal S2 tubular segments taken from the rabbit kidney. Cell pH was determined by measurement of the fluorescence emission of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Cell volume was calculated from measurements of tubular diameter. An increase in CO2 from 5 to 15% reduced cell pH 0.30 units and raised cell bicarbonate concentration ([HCO3]) 10 mM. Cell volume rose to 108.6% of control in 4 min. A decrease in bath [HCO3] from 25 to 5 mM reduced cell pH 0.41 units and cell [HCO3] by 15 mM. Cell volume gradually increased to 105.7% at 8 min. The rate of the regulatory volume decrease after cell swelling on exposure to a 160 mosM solution was determined in the presence of 5 and 15% CO2. The latter reduced the maximum fractional rate of recovery of volume from 0.18 to 0.11 min-1 but did not affect the extent of regulation. We conclude that acidosis causes cell swelling and reduces the rate of volume regulation in response to hypotonic media.

The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus.

Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.