Neuronally differentiated macula densa cells regulate tissue remodeling and regeneration in the kidney.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue-regenerative therapeutic strategies.

Impairments of retinal hemodynamics and oxygen metrics in ocular hypertension-induced ischemia-reperfusion.

Ischemia-reperfusion (I/R) is an established model for retinal neurodegeneration. However, there is limited knowledge of retinal physiological metrics and their relationships to retinal function and morphology in the I/R model. The purpose of the study was to test the hypotheses that retinal hemodynamic and oxygen metrics are impaired and associated with visual dysfunction, retinal thinning, and retinal ganglion cell (RGC) loss due to I/R injury. Intraocular pressure (IOP) was increased in one eye of 10 rats for 90 min followed by reperfusion. Fellow eyes served as controls. After one week of reperfusion, multimodal imaging was performed to quantify total retinal blood flow (TRBF) and retinal vascular oxygen contents. Retinal oxygen delivery (DO2) and metabolism (MO2) were calculated. Pattern-evoked electroretinography (PERG) and optical coherence tomography were performed to measure RGC function and retinal thicknesses, respectively. RGCs were counted from retina whole mounts. After one week of reperfusion, TRBF was lower in study eyes than in control eyes (p < 0.0003). Similarly, DO2 and MO2 were reduced in study eyes compared to control eyes (p < 0.003). PERG amplitude, TRT, IRT, ORT, and RGCs were also lower in study eyes (p ≤ 0.01). DO2 and MO2 were correlated with PERG amplitude, TRT, IRT, and ORT (r ≥ 0.6, p ≤ 0.005). The findings improve knowledge of physiological metrics affected by I/R injury and have the potential for identifying biomarkers of injury and outcomes for evaluating experimental treatments.

Retinal Vascular Physiology Biomarkers in a 5XFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder that affects the brain and retina and lacks reliable biomarkers for early diagnosis. As amyloid beta (Aß) manifestations emerge prior to clinical symptoms and plaques of amyloid may cause vascular damage, identification of retinal vascular biomarkers may improve knowledge of AD pathophysiology and potentially serve as therapeutic targets. The purpose of the current study was to test the hypothesis that retinal hemodynamic and oxygen metrics are altered in 5XFAD mice. METHODS: Thirty-two male mice were evaluated at 3 months of age: sixteen 5XFAD transgenic and sixteen wild-type mice. Spectral-domain optical coherence tomography, vascular oxygen tension, and blood flow imaging were performed in one eye of each mouse. After imaging, the imaged and fellow retinal tissues were submitted for histological sectioning and amyloid protein analysis, respectively. Protein analysis was also performed on the brain tissues. RESULTS: Retinal physiological changes in venous diameter and blood velocity, arterial and venous oxygen contents, coupled with anatomical alterations in the thickness of retinal cell layers were detected in 5XFAD mice. Moreover, an increase in Aß42 levels in both the retina and brain tissues was observed in 5XFAD mice. Significant changes in retinal oxygen delivery, metabolism, or extraction fraction were not detected. Based on compiled data from both groups, arterial oxygen content was inversely related to venous blood velocity and nerve fiber/ganglion cell layer thickness. CONCLUSIONS: Concurrent alterations in retinal hemodynamic and oxygen metrics, thickness, and tissue Aß42 protein levels in 5XFAD mice at 3 months of age corresponded to previously reported findings in human AD. Overall, these results suggest that this mouse model can be utilized for studying pathophysiology of AD and evaluating potential therapies.

Flexible ultrasound-induced retinal stimulating piezo-arrays for biomimetic visual prostheses.

Electronic visual prostheses, or biomimetic eyes, have shown the feasibility of restoring functional vision in the blind through electrical pulses to initiate neural responses artificially. However, existing visual prostheses predominantly use wired connections or electromagnetic waves for powering and data telemetry, which raises safety concerns or couples inefficiently to miniaturized implant units. Here, we present a flexible ultrasound-induced retinal stimulating piezo-array that can offer an alternative wireless artificial retinal prosthesis approach for evoking visual percepts in blind individuals. The device integrates a two-dimensional piezo-array with 32-pixel stimulating electrodes in a flexible printed circuit board. Each piezo-element can be ultrasonically and individually activated, thus, spatially reconfigurable electronic patterns can be dynamically applied via programmable ultrasound beamlines. As a proof of concept, we demonstrate the ultrasound-induced pattern reconstruction in ex vivo murine retinal tissue, showing the potential of this approach to restore functional, life-enhancing vision in people living with blindness.

The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing.

Podocyte calcium (Ca2+) signaling plays important roles in the (patho)physiology of the glomerular filtration barrier. Overactivation of podocyte transient receptor potential canonical (TRPC) channels including TRPC6 and purinergic signaling via P2 receptors that are known mechanosensors can increase podocyte intracellular Ca2+ levels ([Ca2+]i) and cause cell injury, proteinuria and glomerular disease including in diabetes. However, important mechanistic details of the trigger and activation of these pathways in vivo in the intact glomerular environment are lacking. Here we show direct visual evidence that podocytes can sense mechanical overload (increased glomerular capillary pressure) and metabolic alterations (increased plasma glucose) via TRPC6 and purinergic receptors including P2Y2. Multiphoton microscopy of podocyte [Ca2+]i was performed in vivo using wild-type and TRPC6 or P2Y2 knockout (KO) mice expressing the calcium reporter GCaMP3/5 only in podocytes and in vitro using freshly dissected microperfused glomeruli. Single-nephron intra-glomerular capillary pressure elevations induced by obstructing the efferent arteriole lumen with laser-induced microthrombus in vivo and by a micropipette in vitro triggered >2-fold increases in podocyte [Ca2+]i. These responses were blocked in TRPC6 and P2Y2 KO mice. Acute elevations of plasma glucose caused >4-fold increases in podocyte [Ca2+]i that were abolished by pharmacological inhibition of TRPC6 or P2 receptors using SAR7334 or suramin treatment, respectively. This study established the role of Ca2+ signaling via TRPC6 channels and P2 receptors in mechanical and metabolic sensing of podocytes in vivo, which are promising therapeutic targets in conditions with high intra-glomerular capillary pressure and plasma glucose, such as diabetic and hypertensive nephropathy.

Correction to: Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy.

The original version of this chapter was inadvertently published without a proper acknowledgement. The authors informed to insert the following acknowledgement in this chapter.

Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy.

Intravital multiphoton microscopy of the kidney is a powerful technique to study alterations in tissue morphology and function simultaneously in the living animal and represents a dynamic and developing research tool in the field. Recent technological advances include serial intravital multiphoton microscopy of the same kidney regions over several weeks and combined with ex vivo histology for cellular biomarker expression of the same cells, which had been subject to serial imaging before. Thus, serial intravital multiphoton microscopy followed by ex vivo histology provides unique tools to perform long-term cell fate tracing of the same renal cells during physiological and pathophysiological conditions, thereby allowing the detection of structural changes of the same renal cells over time. Examples include renal cell migration and proliferation while linking these events to local functional alterations and eventually to the expression of distinct cellular biomarkers. Here, we provide a detailed step-by-step protocol to facilitate serial intravital multiphoton microscopy for long-term in vivo tracking of renal cells and subsequent ex vivo histology for immunohistological staining of the same cells in the fixed tissue.

Alterations in Retinal Oxygen Delivery, Metabolism, and Extraction Fraction During Bilateral Common Carotid Artery Occlusion in Rats.

Purpose: The purpose of the current study was to investigate alterations in retinal oxygen delivery, metabolism, and extraction fraction and elucidate their relationships in an experimental model of retinal ischemia. Methods: We subjected 14 rats to permanent bilateral common carotid artery occlusion using clamp or suture ligation, or they underwent sham procedure. Within 30 minutes of the procedure, phosphorescence lifetime imaging was performed to measure retinal vascular oxygen tension and derive arterial and venous oxygen contents, and arteriovenous oxygen content difference. Fluorescent microsphere and red-free retinal imaging were performed to measure total retinal blood flow. Retinal oxygen delivery rate (DO2), oxygen metabolism rate (MO2), and oxygen extraction fraction (OEF) were calculated. Results: DO2 and MO2 were lower in ligation and clamp groups compared to the sham group, and also lower in the ligation group compared to the clamp group (P ≤ 0.05). OEF was higher in the ligation group compared to clamp and sham groups (P ≤ 0.03). The relationships of MO2 and OEF with DO2 were mathematically modeled by exponential functions. With moderate DO2 reductions, OEF increased while MO2 minimally decreased. Under severe DO2 reductions, OEF reached a maximum value and subsequently MO2 decreased with DO2. Conclusions: The findings improve knowledge of mechanisms that can maintain MO2 and may clarify the pathophysiology of retinal ischemic injury.

Advances in Renal Cell Imaging.

A great variety of cell imaging technologies are used routinely every day for the investigation of kidney cell types in applications ranging from basic science research to drug development and pharmacology, clinical nephrology, and pathology. Quantitative visualization of the identity, density, and fate of both resident and nonresident cells in the kidney, and imaging-based analysis of their altered function, (patho)biology, metabolism, and signaling in disease conditions, can help to better define pathomechanism-based disease subgroups, identify critical cells and structures that play a role in the pathogenesis, critically needed biomarkers of disease progression, and cell and molecular pathways as targets for novel therapies. Overall, renal cell imaging has great potential for improving the precision of diagnostic and treatment paradigms for individual acute kidney injury or chronic kidney disease patients or patient populations. This review highlights and provides examples for some of the recently developed renal cell optical imaging approaches, mainly intravital multiphoton fluorescence microscopy, and the new knowledge they provide for our better understanding of renal pathologies.

Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment.

The development of podocyte injury and albuminuria in various glomerular pathologies is still incompletely understood due to technical limitations in studying the glomerular filtration barrier (GFB) in real-time. We aimed to directly visualize the early morphological and functional changes of the GFB during the development of focal segmental glomerulosclerosis (FSGS) using a combination of transmission electron microscopy (TEM) and in vivo multiphoton microscopy (MPM) in the rat puromycin aminonucleoside (PAN) model. We hypothesized that this combined TEM + MPM experimental approach would provide a major technical improvement that would benefit our mechanistic understanding of podocyte detachment. Male Sprague-Dawley (for TEM) or Munich-Wistar-Frömter (for MPM) rats were given a single dose of 100-150 mg/kg body weight PAN i.p. and were either sacrificed and the kidneys processed for TEM or surgically instrumented for in vivo MPM imaging at various times 2-14 days after PAN administration. Both techniques demonstrated hypertrophy and cystic dilatations of the subpodocyte space that developed as early as 2-3 days after PAN. Adhesions of the visceral epithelium to the parietal Bowman's capsule (synechiae) appeared at days 8-10. TEM provided unmatched resolution of podocyte foot process remodeling, while MPM revealed the rapid dynamics of pseudocyst filling, emptying, and rupture, as well as endothelial and podocyte injury, misdirected filtration, and podocyte shedding. Due to the complementary advantages of TEM and MPM, this combined approach can provide an unusally comprehensive and dynamic portrayal of the alterations in podocyte morphology and function during FSGS development. The results advance our understanding of the role and importance of the various cell types, hemodynamics, and mechanical forces in the development of glomerular pathology.

Laminar flow downregulates Notch activity to promote lymphatic sprouting.

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow-induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow-induced lymphatic sprouting.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Educating for diversity: an evaluation of a sexuality diversity workshop to address secondary school bullying.

OBJECTIVE: To evaluate the potential of a 60-minute sexuality diversity workshop to address bullying in secondary schools. METHODS: Students completed pre- and post-workshop questionnaires. Descriptive statistics were used to summarise results with pre- to immediate post-workshop changes compared using t-tests. Thematic analysis was used to analyse open-ended questionnaire responses. RESULTS: We had 229 students (mean age 13.7 years) attending 10 workshops participate in the study. Three-quarters of students thought the workshop would reduce bullying in schools, and over 95% of the participants thought that other secondary schools should offer the workshop. There was a significant increase in valuing (p < 0.001) and understanding (p < 0.001) sexuality-diverse individuals (e.g. lesbian, gay and bisexual people), between the pre- and post-workshop results. School climates were largely perceived to be 'hard' and included 'bullying/mocking' of sexuality-diverse students; however, many individual students reported a desire to be supportive of their sexuality-diverse peers. CONCLUSIONS: Sexuality-based bullying is commonplace in secondary schools. This form of bullying is associated with depression and suicide attempts. Reducing sexuality-based bullying is very likely to have a positive impact on the mental health of young people. Brief workshops, as a part of a wider suite of interventions, have some potential to create safer school environments.

Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice.

This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

Local pH domains regulate NHE3-mediated Na⁺ reabsorption in the renal proximal tubule.

The proximal tubule Na(+)/H(+) exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na(+) reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution.

Can kidney regeneration be visualized?

BACKGROUND: Various cell types, including podocytes and parietal epithelial cells, play important roles in the development and progression of glomerular kidney diseases, albuminuria, and glomerulosclerosis. Besides their role in renal pathologies, glomerular cells have emerging new functions in endogenous repair mechanisms. A better understanding of the dynamics of the glomerular environment and cellular composition in an intact living kidney is critically important for the development of new regenerative therapeutic strategies for kidney diseases. However, progress in this field has been hampered by the lack of in vivo research tools. SUMMARY: This review summarizes the current state-of-the-art in the application of the unique intravital imaging technology of multiphoton fluorescence microscopy for the dynamic visualization of glomerular structure and function over time in the intact, living kidney. Recently, this imaging approach in combination with transgenic mouse models allowed tracking of the fate of individual glomerular cells in vivo over several days and depicted the highly dynamic nature of the glomerular environment, particularly in disease conditions. KEY MESSAGES: The technology is ready and available for future intravital imaging studies investigating new glomerular regenerative approaches in animal models.

Intravital imaging of podocyte calcium in glomerular injury and disease.

Intracellular calcium ([Ca²âº]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²âº]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²âº]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²âº]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²âº]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²âº]i waves along capillary loops. [Ca²âº]i wave propagation was ameliorated by inhibitors of purinergic [Ca²âº]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²âº]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²âº]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²âº]i in glomerular pathology and suggest that purinergic [Ca²âº]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

A novel source of cultured podocytes.

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.

Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct.

ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(-/-) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)]i in wild type CCDs. This response was blunted in Cx30(-/-) CCDs ([Ca(2+)]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)]i oscillations in free-flowing CDs of wild type but not Cx30(-/-) mice. The [Ca(2+)]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.