Therefore, immunofluorescence studies were performed, using mouse kidney sections to localize -tubulin, a ubiquitous component of primary cilia. 20 nl/min (80 mM [NaCl]) rapidly produced significant elevations in cytosolic Ca2+concentration ([Ca2+]i) in AA smooth muscle cells [evidenced by changes in fluo-4 intensity (F); F/F0= 1.45 0.11] and AA vasoconstriction. Complete removal of the cTAL around the MD plaque and application of laminar flow through a perfusion pipette directly to the MD apical surface essentially produced the same results even when low (10 mM) or zero NaCl solutions were used. Acetylated -tubulin immunohistochemistry identified the presence of primary cilia in mouse MD cells. Under no flow conditions, bending MD cilia directly with a micropipette rapidly caused significant [Ca2+]ielevations in AA smooth muscle cells (fluo-4 F/F0: 1.60 0.12) and vasoconstriction. P2 receptor blockade with suramin significantly reduced the flow-induced TGF, whereas scavenging superoxide with tempol did not. In conclusion, MD cells are equipped with a tubular flow-sensing mechanism that may contribute to MD cell function and TGF. Keywords:tubuloglomerular feedback, primary cilium, mechanosensor, juxtaglomerular apparatus macula densa(MD) cells in the cortical thick ascending limb (cTAL) are the sensory element of the juxtaglomerular apparatus (JGA) and play an important role in the control of renal blood flow, glomerular filtration rate, and renin release. MD cells are strategically positioned DMT1 blocker 1 in the JGA such that their apical membrane is exposed to the tubular fluid, whereas their basilar aspects are in contact with the effector cells of the JGA, namely the renin-producing juxtaglomerular granular cells and the contractile cells in the extraglomerular mesangium and the afferent arteriole (AA). Consistent with this anatomic localization, MD cells can detect alterations in tubular fluid characteristics (for example, ionic composition) and generate and release chemical mediators that act on JGA effectors in a paracrine fashion. According to the prevailing paradigm, elevations in tubular NaCl concentration ([NaCl]) at the MD trigger basolateral ATP release from these cells, which directly or through its breakdown to adenosine cause AA vasoconstriction and reductions in glomerular filtration rate [tubuloglomerular feedback (TGF)] (6,7,14,22,35,39,40). Identification of the luminal characteristic(s) that is sensed by the MD was the topic of intense research more than 25 years ago conducted by several laboratories, and the results were long debated (1,2,4,22,35). In particular, variables such as tubular fluid [NaCl], flow rate, and osmolality were considered as top candidates for the characteristic that can trigger TGF (1,2,4,22,35). Briggs et al. (4) and Schnermann and Briggs (35) found that most experimental maneuvers that cause TGF ultimately translate into alterations in tubular DMT1 blocker 1 fluid [NaCl] at the MD, which has prevailed as the key luminal signal for TGF. However, increases in the distal microperfusion rate of a variety of electrolyte (including chloride-free) and nonelectrolyte solutions were demonstrated to elicit TGF responses (1,21), suggesting the role of increased fluid flow per se. These earlier reports also suggest that there may be more than one tubular signal that MD cells can detect. Support for this view includes, for example, the recent study by our laboratory on the localization of the succinate receptor GPR91 at the apical membrane of MD cells that can sense alterations in local tissue metabolism (41). In past years, changes in tubular fluid flow rate and the flow-sensing primary cilia and their associations with ATP release received much attention (10,20,2831). Primary cilia are solitary, immotile organelles that sense extracellular chemical and mechanical signals and transduce them into cellular responses. Primary cilia are well-established DMT1 blocker 1 fluid flow Mouse monoclonal to KID sensors in many organs and cell types, including renal epithelia (10,20,2831). Bending of cilia in most cell types can trigger elevations in cytosolic Ca2+concentration ([Ca2+]i) via the function of ciliary membrane ion channels and associated proteins, including polycystins (30). Interestingly, earlier electron microscopy work by several laboratories demonstrated the presence of primary cilia in MD cells (11,12,38,43), suggesting direct flow sensing by these cells. Flow-induced ATP release from MD cells is intriguing since ATP release is integral in TGF (3,6,14,27). A recent study from our laboratory found that elevations in tubular fluid flow rate trigger an ATP-dependent calcium wave that propagates in the JGA and beyond (27). Also, a number of special MD and JGA dissection techniques (3,14,2527) and their experimental applications that allow the precise manipulation of the.