Cellular localization of P2Y(2) purinoceptor in rat renal inner medulla and lung.
(73/1605)
Physiological and pharmacological studies have demonstrated that extracellular ATP, acting through P2Y(2) purinoceptor, modulates water permeability of renal medullary collecting duct cells and the secretion of ions, mucin, and surfactant phospholipids by respiratory epithelia. Here we provide direct molecular evidence for the expression of P2Y(2) purinoceptor in these cells. RT-PCR confirmed P2Y(2) purinoceptor mRNA expression in rat lung and kidney and demonstrated expression in renal collecting ducts. Northern analysis showed that both lung and kidney express one 3.6-kb P2Y(2) purinoceptor mRNA transcript. Immunoblots using peptide-derived polyclonal antibody to P2Y(2) purinoceptor showed that inner medullary collecting ducts (IMCD) express two distinct and specific products (47 and 105 kDa) and account for the majority of the receptor expression in inner medulla, whereas the 105-kDa form is predominant in lung. Immunoperoxidase labeling on cryosections showed localization of receptor protein in the apical and basolateral domains of IMCD principal cells and in the secretory cells (Clara cells and goblet cells) of the terminal respiratory bronchioles. (+info)
PI3K signaling in the murine kidney inner medullary cell response to urea.
(74/1605)
Growth factors and other stimuli increase the activity of phosphatidylinositol-3 kinase (PI3K), an SH2 domain-containing lipid kinase. In the murine kidney inner medullary mIMCD3 cell line, urea (200 mM) increased PI3K activity in a time-dependent fashion as measured by immune complex kinase assay. The PI3K effector, Akt, was also activated by urea as measured by anti-phospho-Akt immunoblotting. In addition, the Akt (and PI3K) effector, p70 S6 kinase, was activated by urea treatment in a PI3K-dependent fashion. PI3K inhibition potentiated the proapoptotic effect of hypertonic and urea stress. Urea treatment also induced the tyrosine phosphorylation of Shc and the recruitment to Shc of Grb2. Coexistence of activated Shc and PI3K in a macromolecular complex was suggested by the increase in PI3K activity evident in anti-Shc immunoprecipitates prepared from urea-treated cells. Taken together, these data suggest that PI3K may regulate physiological events in the renal medullary cell response to urea stress and that an upstream tyrosine kinase conferring activation of both PI3K and Shc may govern urea signaling in these cells. (+info)
Nuclear localization and transactivating capacities of the papillary renal cell carcinoma-associated TFE3 and PRCC (fusion) proteins.
(75/1605)
The papillary renal cell carcinoma-associated t(X;1)(p11;q21) leads to fusion of the transcription factor TFE3 gene on the X-chromosome to a novel gene, PRCC, on chromosome 1. As a result, two putative fusion proteins are formed: PRCCTFE3, which contains all known domains for DNA binding, dimerization, and transactivation of the TFE3 protein, and the reciprocal product TFE3PRCC. Upon transfection into COS cells, both wild type and fusion proteins were found to be located in the nucleus. When comparing the transactivating capacities of these (fusion) proteins, significant differences were noted. PRCCTFE3 acted as a threefold better transactivator than wild type TFE3 both in a TFE3-specific and in a general (Zebra) reporter assay. In addition, PRCC and the two fusion proteins were found to be potent transactivators in the Zebra reporter assay. We propose that, as a result of the (X;1) translocation, fusion of the N-terminal PRCC sequences to TFE3 alters the transactivation capacity of the transcription factor thus leading to aberrant gene regulation and, ultimately, tumor formation. (+info)
Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla.
(76/1605)
Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized. (+info)
Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells.
(77/1605)
We investigated the effects of hyperosmolality on survival and proliferation of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. High NaCl and/or urea (but not glycerol) reduces the number of viable cells, as measured with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550-1,000 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G(2)M phase of the cell cycle within 8 h, as measured by flow cytometry. Up to 600 mosmol/kg the effect is only transient, and by 12 h at 550 mosmol/kg the effect reverses and most cells are in G(1). Flow cytometry with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the S phase of the cell cycle slows, depending on the concentrations of NaCl and/or urea, and that the duration of G(2)M increases greatly (from 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Addition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more also induces apoptosis, as demonstrated by characteristic electron microscopic morphological changes, appearance of a subdiploid peak in flow cytometry, and caspase-3 activation. The number of cells with subdiploid DNA and activated caspase-3 peaks at 8-12 h. Caspase-3 activation occurs in all phases of the cell cycle, but to a disproportionate degree in G(0)/G(1) and S phases. We conclude that elevated NaCl and/or urea reduces the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G(2)M and G(1), and by inducing apoptotic cell death. (+info)
Interstitial water and solute recovery by inner medullary vasa recta.
(78/1605)
A recent model of volume and solute microvascular exchange in the renal medulla was extended by simulating the deposition of NaCl, urea, and water into the medullary interstitium from the loops of Henle and collecting ducts with generation rates that undergo spatial variation within the inner medullary interstitium. To build an exponential osmolality gradient in the inner medulla, as suggested by Koepsell et al. (H. Koepsell, W. E. A. P. Nicholson, W. Kriz, and H. J. Hohling. Pflugers Arch. 350: 167-184, 1974), the ratio of the interstitial area-weighted generation rate of small solutes to that of water must increase along the corticomedullary axis. We satisfied this condition either by holding the area-weighted generation rate of water constant while increasing that of NaCl and urea or by reducing the input rate of water with medullary depth. The latter case, in particular, yielded higher solute concentrations at the papillary tip. Assuming that the fraction of the filtered load recovered by inner medullary vasa recta for water, NaCl, and urea is 1%, 1%, and 40%, respectively, papillary tip osmolality is 1,470 mosmol/kgH(2)O when urea generation and NaCl generation per unit volume of interstitium increase exponentially and linearly, respectively. The inner medullary osmolar gradient also increases further when 1) medullary blood flow is reduced, 2) hydraulic conductivity of descending vasa recta (DVR) is lowered, and 3) vasa recta permeability to NaCl and urea is maximized. The coupling between water and small solute transport, resulting from aquaporin-1-mediated transcellular flux in DVR, also enhances tip osmolality. (+info)
Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats.
(79/1605)
Experiments were designed to elucidate the role of endothelin B receptors (ET(B)) on arterial pressure and renal function in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy and were treated with either DOCA and salt (0.9% NaCl to drink) or placebo. DOCA-salt rats given the ET(B)-selective antagonist, A-192621, for 1 wk (10 mg. kg(-1). day(-1) in the food) had significantly greater systolic arterial pressure compared with untreated DOCA-salt rats (208 +/- 7 vs. 182 +/- 4 mmHg) whereas pressure in placebo rats was unchanged. In DOCA-salt, but not placebo rats, A-192621 significantly decreased sodium and water excretion along with parallel decreases in food and water intake. To determine whether the response in DOCA-salt rats was due to increased expression of ET(B) receptors, endothelin receptor binding was performed by using membranes from renal medulla. Maximum binding (B(max)) of [(125)I]ET-1, [(125)I]ET-3, and [(125)I]IRL-1620 increased from 227 +/- 42, 146 +/- 28, and 21 +/- 1 fmol/mg protein, respectively, in placebo rats to 335 +/- 27, 300 +/- 38, and 61 +/- 6 fmol/mg protein, respectively, in DOCA-salt hypertensive rats. The fraction of receptors that are the ET(B) subtype was significantly increased in DOCA-salt (0.88 +/- 0.07) compared with placebo (0.64 +/- 0.01). The difference between [(125)I]ET-3 and [(125)I]IRL-1620 binding is consistent with possible ET(B) receptor subtypes in the kidney. These results indicate that ET(B) receptors in the renal medulla are up-regulated in the DOCA-salt hypertensive rat and may serve to maintain a lower arterial pressure by promoting salt and water excretion. (+info)
Ras signaling in the inner medullary cell response to urea and NaCl.
(80/1605)
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)