Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney.
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Because of the availability of knockout mouse models to examine renal transport mechanisms, it has become increasingly important to describe the cellular distribution of major renal transporters in mice. We have used immunocytochemistry and freeze-fracture electron microscopy to compare the renal distribution of aquaporin-4 (AQP4) with that previously described in rat. In rat kidney AQP4 is present exclusively in basolateral membranes of collecting duct principal cells. In mice, however, AQP4 was also detected by immunocytochemistry in basolateral membranes of proximal tubule S3 segments, and not detected in S1 and S2 segments of proximal tubule. Freeze-fracture electron microscopy revealed orthogonal arrays of intramembrane particles (OAPs) on the basolateral membranes of the S3 segment. In AQP4-knockout mice, immunostaining was absent and OAPs were found neither in collecting ducts nor in the S3 segment of the proximal tubule. The urinary concentrating capacity after deletion of both AQP1 and AQP4 was further reduced compared with that of AQP1 or AQP4 null mice, suggesting an additive effect of AQP1 and AQP4 in the concentrating mechanism. The functional significance of the apparent species-dependent expression of AQP4 in proximal tubules is unknown, but may relate to physiological differences between rats and mice. (+info)
Ras signaling in the inner medullary cell response to urea and NaCl.
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The small guanine nucleotide-binding protein Ras, activated by peptide mitogens and other stimuli, regulates downstream signaling events to influence transcription. The role of Ras in solute signaling to gene regulation was investigated in the murine inner medullary collecting duct (mIMCD3) cell line. Urea treatment (100-200 mM), but not sham treatment, increased Ras activation 124% at 2 min; the effect of NaCl did not achieve statistical significance. To determine the contribution of Ras activation to urea-inducible signal transduction, mIMCD3 cells were stably transfected with an expression plasmid encoding a dominant negative-acting N17Ras mutant driven by a dexamethasone-inducible (murine mammary tumor virus) promoter. After 24 h of induction, selected cell lines exhibited sufficient N17Ras overexpression to abolish epidermal growth factor- and hypotonicity-mediated signaling to extracellular signal-regulated kinase (ERK) phosphorylation, as determined by immunoblotting. Conditional N17Ras overexpression inhibited urea- and NaCl-inducible ERK phosphorylation by 40-50%, but only at 15 min, and not 5 min, of treatment. N17Ras induction, however, almost completely inhibited urea-inducible Egr-1 transcription, as quantitated by luciferase reporter gene assay, but failed to influence tonicity-inducible (TonE-mediated) transcription. N17Ras overexpression also blocked urea-inducible expression of the transcription factor Gadd153 but did not influence osmotic or urea-inducible apoptosis. In addition, urea treatment induced recruitment of the Ras activator Sos to the plasma membrane. Taken together, these observations suggest a role for Ras signaling in the IMCD cell response to urea stress. (+info)
Ca2+ and cAMP-activated Cl- conductances mediate Cl- secretion in a mouse renal inner medullary collecting duct cell line.
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1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD. (+info)
Vasopressin regulates endothelin-B receptor in rat inner medullary collecting duct.
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Previous studies have shown that endothelin (ET) antagonizes the actions of arginine vasopressin (AVP) in the renal collecting ducts. On the other hand, the effects of AVP on ET function within the collecting ducts of the kidney have not been investigated extensively. Using isolated inner medullary collecting ducts (IMCD), we examined the possibility that a decrease in ET(B) receptor mRNA accompanied AVP-induced downregulation of ET(B) receptors. Binding studies revealed that overnight incubation of rat IMCD cells with AVP significantly reduced the maximal binding capacity (B(max)) of ET. Activation of adenylate cyclase by forskolin decreased the total ET(B) receptor density by approximately 42% but did not affect the density of ET(A) receptors. The Rp diastereoisomer of adenosine 3', 5'-cyclic monophosphothionate, Rp-cAMPS (a specific inhibitor of protein kinase A), blocked the AVP-induced reduction in ET receptor density. Using competitive PCR method, we also observed downregulation of ET(B) receptor mRNA in IMCD treated with AVP. Additional studies were done with IMCD to determine whether AVP inhibited the ET-induced accumulation of cGMP. We saw a reduction in ET-induced cGMP accumulation when IMCD was incubated overnight with AVP. This inhibition of ET-induced accumulation of cGMP was blocked by Rp-cAMPS. These results suggest that AVP regulates ET(B) receptor expression in IMCD. (+info)
Mechanisms of inactivation of the action of aldosterone on collecting duct by TGF-beta.
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The purpose of these experiments was to investigate the mechanisms whereby transforming growth factor-beta (TGF-beta) antagonizes the action of adrenocorticoid hormones on Na(+) transport by the rat inner medullary collecting duct in primary culture. Steroid hormones 1) increased Na(+) transport by three- to fourfold, 2) increased the maximum capacity of the Na(+)-K(+) pump by 30-50%, 3) increased the steady-state levels of the alpha(1)-subunit of the Na(+)-K(+)-ATPase by approximately 30%, and 4) increased the steady-state levels of the alpha-subunit of the rat epithelial Na(+) channel (alpha-rENaC) by nearly fourfold. TGF-beta blocked the effects of steroids on the increase in Na(+) transport and the stimulation of the Na(+)-K(+)-ATPase and pump capacity. However, there was no effect of TGF-beta on the steroid-induced increase in mRNA levels of alpha-rENaC. The effects of TGF-beta were not secondary to the decrease in Na(+) transport per se, inasmuch as benzamil inhibited the increase in Na(+) transport but did not block the increase in pump capacity or Na(+)-K(+)-ATPase mRNA. The results indicate that TGF-beta does not inactivate the steroid receptor or its translocation to the nucleus. Rather, they indicate complex pathways involving interruption of the enhancement of pump activity and activation/inactivation of pathways distal to the steroid-induced increase in the transcription of alpha-rENaC. (+info)
Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer.
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Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0. 021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer. (+info)
ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells.
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1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane. (+info)
Collecting duct (Na+/K+)-ATPase activity is correlated with urinary sodium excretion in rat nephrotic syndromes.
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In puromycin aminonucleoside (PAN)-treated nephrotic rats, sodium retention is associated with increased (Na+/K+)-ATPase activity in the cortical collecting ducts (CCD). This study was undertaken to determine whether stimulation of (Na+/K+)-ATPase in the CCD is a feature of other experimental nephrotic syndromes, whether it might be responsible for renal sodium retention, and whether it is mediated by increased plasma vasopressin levels or activation of calcineurin. For this purpose, the time courses of urinary excretion of sodium and protein, sodium balance, ascites, and (Na+/K+)-ATPase activities in microdissected CCD were studied in rats with PAN or adriamycin nephrosis or HgCl2 nephropathy. The roles of vasopressin and calcineurin in PAN nephrosis were evaluated by measuring these parameters in Brattleboro rats and in rats treated with cyclosporin or tacrolimus. Despite different patterns of changes in urinary sodium and protein excretion in the three nephrotic syndrome models, there was a linear relationship between CCD (Na+/K+)-ATPase activities and sodium excretion in all three cases. The results also indicated that there was no correlation between proteinuria and sodium retention, but ascites was present only when proteinuria was associated with marked reduction of sodium excretion. Finally, the lack of vasopressin in Brattleboro rats or the inhibition of calcineurin by administration of either cyclosporin or tacrolimus did not prevent development of the nephrotic syndrome in PAN-treated rats or stimulation of CCD (Na+/K+)-ATPase. It is concluded that stimulation of Na(+/K+)-ATPase in the CCD of nephrotic rats might be responsible for sodium retention and that this phenomenon is independent of proteinuria and vasopressin and calcineurin activities. (+info)