Flow induces cytosolic Ca 2ϩ increases ([Ca 2ϩ ] i ) in intact renal tubules, but the mechanism is elusive. Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger auto-and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flowinduced [Ca 2ϩ ] i response in the renal tubule involves mechanically stimulated nucleotide release. This study investigated (1) the expression of P2 receptors in mouse medullary thick ascending limb (mTAL) using P2Y 2 receptor knockout (KO) mice, (2) whether flow increases induce [Ca 2ϩ ] i elevations in mTAL, and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor. [Ca 2ϩ ] i was imaged in perfused mTAL with fura-2 or fluo-4. It is shown that luminal and basolateral P2Y 2 receptors are the main purinergic receptor in this segment. Moreover, the data suggest presence of basolateral P2X receptors. Increases of tubular flow were imposed by promptly rising the inflow pressure, which triggered a marked increase of [Ca 2ϩ ] i . This [Ca 2ϩ ] i response was significantly reduced in P2Y 2 receptor KO tubules (fura-2 ratio increase WT 0.44 Ϯ 0.09 [n ϭ 28] versus KO 0.16 Ϯ 0.04 [n ϭ 13]). Furthermore, the flow response was greatly inhibited with luminal and basolateral scavenging of extracellular ATP (apyrase 7.5 U/ml) or blockage of P2 receptors (suramin 300 M). The flow response could still be elicited in the absence of extracellular Ca 2ϩ . These results strongly suggest that increase of tubular flow elevates [Ca 2ϩ ] i in intact renal epithelia. This flow response is caused by release of bilateral nucleotides and subsequent activation of P2 receptors.
The electroneutral Na + -dependent HCO 3 − transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up-regulated during NH 4 + -induced metabolic acidosis. Here we used in vitro perfusion and BCECF video-imaging of mTAL tubules to investigate functional localization and regulation of Na + -dependent HCO 3 − influx during NH 4 + -induced metabolic acidosis. Tubule acidification was induced by removing luminal Na + (∆pH i : 0.88 ± 0.11 pH units, n = 10). Subsequently the basolateral perfusion solution was changed to CO 2 /HCO 3 − buffer with and without Na + . Basolateral Na + -H + exchange function was inhibited with amiloride. Na + -dependent HCO 3 − influx was determined by calculating initial base flux of Na + -mediated re-alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min −1 mm −1 . A 2.4-fold increase of base flux to 21.8 ± 3.2 pmol min −1 mm −1 was measured in NH 4 + -treated animals (11 days, n = 11). Na + -dependent re-alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n = 10). In addition, Na + -dependent HCO 3 − influx was of similar magnitude in chloride-free medium and also up-regulated after NH 4 + loading. Na + -dependent HCO 3 − influx was not inhibited by 400 µm DIDS. A strong up-regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT-PCR analysis revealed no evidence for the Na + -dependent HCO 3 − transporter NBC4 or the two Na + -dependent CI − /HCO 3 − exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS-insensitive NBCn1. Function and protein are strongly up-regulated during NH 4 + -induced metabolic acidosis. We suggest that NBCn1-mediated basolateral HCO 3 − influx is important for basolateral NH 3 exit and thus NH 4 + excretion by means of setting pH i to a more alkaline value.
Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.
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