The mechanisms and molecules involved in renal tubular flow sensing have become a vital and exciting research area. It is the astonishing link between the central cilium found to be a flow-sensing organelle and the ‘defective cilium’ in different genetic diseases leading to the heterogeneous group of renal cystic diseases that drive this field. Defective tubular flow-sensing is postulated as causing the development of polycystic kidney disease (1). An increase in flow over the apical surface of cultured renal epithelia cells (MDCK) induces an increase in cytosolic calcium ([Ca²⁺]_i). This ‘flow response’ is dependent on the presence of the central cilium (2,3). The mechanism of flow-induced [Ca²⁺]_i increases in renal epithelia remains elusive, but is postulated to involve polycystin 2 (TRPP2)-mediated influx of Ca²⁺ over the luminal membrane (4). At the same time extensive evidence indicates that in numerous biological systems including epithelia mechanical alterations promote ‘non-lytic’ release of nucleotides like ATP and trigger auto- and paracrine stimulation of the family of P2 receptors with subsequent increases of [Ca²⁺]_i. We therefore hypothesized that the flow-response in the renal tubule may involve mechanically-stimulated release of nucleotides. This study therefore first investigated the expression patter of luminal and basolateral P2 receptors in mouse medullary thick ascending limb (mTAL) using the P2Y2 receptor knock-out mouse. Secondly, we investigated, if a flow-induced [Ca²⁺]_i elevation can also be found in the isolated perfused intact nephron. Thirdly, it was studied, if this flow-response is affected in mice deplete of the main purinergic receptor in the mTAL. In vitro perfusion of isolated mTAL segments and fura-2 imaging were used to record nucleotide-stimulated [Ca²⁺]_i signals. The results identify a luminal and basolateral P2Y2 receptor as the main ATP/UTP stimulated P2 receptor in this segment. Additional functional evidence suggests the presence of a basolateral P2X receptor. Subsequently, we measured mTAL [Ca²⁺]_i following changes in pressure/flow in P2Y2 receptor KO mice and their WT littermates. Pressure changes were imposed by the prompt raising of the hydrostatic pressure gradient by 80 cmH2O for 3 min. Pressure increases led to acute distension of the tubule in both WT and KO mice (by 26 and 22%, respectively). In either genotype, the increase in tubular diameter was followed by an increase in [Ca²⁺]_i, as indicated by the Fura-2 ratio. This flow response was significantly larger in WT vs. KO tubules (ratio increase 0.49 ± 0.16 (n=15) vs. 0.16 ± 0.04 (n=16)). The addition of 300 µM basolateral suramin reduced the flow response in KO mice further by 65% (to 0.06 ± 0.07 (n=7)). A flow response could be elicited in the absence of bilateral Ca²⁺. In MDCK cells ATP scavenging by bilateral apyrase (5U/ml) significantly reduced the flow response by 42%. We also report the observation that isolated perfused mTAL tubules from P2Y2 wild-type mice display a lively [Ca²⁺]_i oscillatory behaviour. [Ca²⁺]_i oscillations were visible in video-microscopy as spontaneous ‘blinking’ (increase in fura-2 ratio) randomly distributed over the entire length of the tubule. A systematic analysis was undertaken to quantify spontaneous oscillatory events in mTAL of P2Y2 WT and KO mice. Spontaneous, transient [Ca²⁺]_i increases (events) above a chosen threshold of >0.1 ratio units were measured in multiple regions (20 x 25 µm) of interest of the entire tubule. In total, 1036 events with a mean frequency of one event every 6.2 min was measured in P2Y2 WT mice (n=18 tubules, total observation time: 253 min). The mean amplitude and duration of the events was 0.27 ± 0.01 ratio units and 42.2 ± 1.3 s, respectively. In sharp contrast spontaneous [Ca²⁺]_i oscillation were nearly absent in P2Y2 KO mice. A total of 138 events with a mean frequency of one event per 31.3 min were measured. Their amplitude was reduced to 0.15 ± 0.01 with no change in their duration (42.5 ± 2.9 s) (n=11 tubules, total observation time: 166 min). These data strongly indicate that the flow-induced [Ca²⁺]_i increase in the intact renal tuble (mTAL) and cultured renal epithelial cells involves a mechanical-stimulated release of nucleotides and subsequent activation of P2 receptors. These data also indicate that spontaneous [Ca²⁺]_i oscillations in mouse mTAL require luminal and/or basolateral P2Y2 receptors and further support a mechanically stimulated, flow-dependent release of nucleotides and subsequent activation of P2 receptors.