lease from juxtaglomerular granular cells is considered the ratelimiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 M isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release. multiphoton excitation; fluorescence microscopy; juxtaglomerular apparatus; renin activity; quinacrine THE JUXTAGLOMERULAR APPARATUS (JGA) represents a major structural component of the renin-angiotensin system (RAS) and is one of the most important regulatory sites of renal salt and water conservation and blood pressure maintenance. Release of renin from juxtaglomerular granular cells in the terminal afferent arteriole is considered the rate-limiting step of RAS activation and is precisely controlled by several mechanisms (16,21,24). Reductions in renal perfusion pressure, activation of the sympathetic nervous system, local hormones, and reductions in macula densa salt transport result in increases in circulating and interstitial renin levels that lead to enhanced generation of ANG I with subsequent conversion to ANG II (23,24). ANG II, one of the most potent hypertensive autacoids and the major product of the RAS, exerts its effects on renal vascular and tubular structures, as well as on other organs.

Abstract-Angiotensin (Ang) II directly stimulates epithelial sodium channel activity in the rabbit cortical collecting duct.Because Ang I and converting enzyme analogues might be present in the distal nephron, this raises the possibility of intraluminal generation of Ang II. Conversion of Ang I to Ang II was monitored by Ang II-dependent changes in intracellular sodium concentration as a reflection of sodium transport across the apical membrane. This involved imaging-based fluorescence microscopy with sodium-binding benzofuran isophthalate in isolated, perfused, cortical collecting-duct segments from rabbit kidney. Principal and intercalated cells were differentiated by rhodamineconjugated peanut lectin. Control principal cell intracellular sodium concentration, during perfusion with 25 mmol/L NaCl and zero sodium in the bath plus monensin (10 Ϫ5 mol/L) averaged 5.8Ϯ0.14 mmol/L (nϭ156). The increase in intracellular sodium concentration, when luminal NaCl was increased from 25 to 150 mmol/L, was elevated by 3.5-fold in the presence of intraluminal Ang I (10 Ϫ6 mol/L). Also, the effects of Ang I on sodium transport were not significantly different from the effects of Ang II (10 Ϫ9 mol/L). Ang I was used in micromolar concentrations to ensure that there was sufficient substrate available for conversion to Ang II. Inhibition of the angiotensin-converting enzyme with captopril reduced the stimulatory effect of Ang I. These results suggest that intraluminal conversion of Ang I to Ang II can occur in the cortical collecting duct, resulting in enhanced apical sodium entry.

. Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells.

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk Tg737 ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three-to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three-to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca 2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Ciliumcompetent monolayers responded to flow with an increase in cell Ca 2+ derived from both extracellular and intracellular stores. This flow-induced Ca 2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca 2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca 2+ . Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na + ) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.Purinergic Signalling (...

One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway.

TGFbeta-induced alphaSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial-mesenchymal transdifferentiation.

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium-chloride-potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process.

Macula densa cells couple renal haemodynamics, glomerular filtration and renin release with tubular fluid salt and water reabsorption. These cells detect changes in tubular fluid composition through a complex of intracellular signalling events that are mediated by membrane transport pathways. Increases in luminal fluid sodium chloride concentration result in alterations in cell sodium chloride concentration, cytosolic calcium, cell pH, basolateral membrane depolarization and cell volume. Macula densa signalling then involves the production and release of specific paracrine signalling molecules at their basolateral membrane. Upon moderate increases in luminal sodium chloride concentration macula densa cells release increasing amounts of ATP and decreasing amounts of prostaglandin E(2), thereby increasing afferent arteriolar tone and decreasing the release of renin from granular cells. On the other hand, further increases in luminal concentration stimulate the release of nitric oxide, which serve to prevent excessive tubuloglomerular feedback vasoconstriction. Paracrine signalling by the macula densa cells therefore controls juxtaglomerular function, renal vascular resistance and participates in the regulation of renin release.