Experimental Models of SARS-CoV-2 Infection: Possible Platforms to Study COVID-19 Pathogenesis and Potential Treatments.

In December 2019, a novel coronavirus crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a worldwide pandemic. Development of effective clinical interventions, including vaccines and antiviral drugs that could prevent or limit theburden or transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health priority. It is thus of utmost importance to assess possible therapeutic strategies against SARS-CoV-2 using experimental models that recapitulate aspects of the human disease. Here, we review available models currently being developed and used to study SARS-CoV-2 infection and highlight their application to screen potential therapeutic approaches, including repurposed antiviral drugs and vaccines. Each identified model provides a valuable insight into SARS-CoV-2 cellular tropism, replication kinetics, and cell damage that could ultimately enhance understanding of SARS-CoV-2 pathogenesis and protective immunity.

Importance and implications of exosomes in nephrology and urology.

Exosomes are extracellular vesicles that are formed by two invaginations of the plasma membrane and can be released by all eukaryotic cells. Because of their bioactive contents, including nucleic acids and proteins, exosomes can activate a variety of functions in their recipient cells. Due to the plethora of physiological and pathophysiological functions, exosomes have received a lot of attention from researchers over the past few years. However, there is still no consensus regarding isolation and characterization protocols of exosomes and their subtypes. This heterogeneity poses a lot of methodical challenges but also offers new clinical opportunities simultaneously. So far, exosome-based research is still mostly limited to preclinical experiments and early-stage clinical trials since the translation of experimental findings remains difficult. Exosomes could potentially play an important role as future diagnostic and prognostic agents and might also be part of the development of new treatment strategies. Therefore, they have previously been investigated in a variety of nephrological and urological conditions such as acute kidney injury or prostate cancer.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

Opposing MMP-9 Expression in Mesenchymal Stromal Cells and Head and Neck Tumor Cells after Direct 2D and 3D Co-Culture.

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Modified Borggreve-Van Nes-Winkelmann rotationplasty for surgery in developing countries.

BACKGROUND: Amputation is still the most common therapy for patients suffering from osteosarcoma in Myanmar, despite the fact that limb salvage surgery e.g. Borggreve-Van Nes-Winkelmann rotationplasty for malignant tumors located within the distal femur or proximal tibia is the current state-of-the-art reconstructive procedure. A safe and reliable operation technique is crucial in order to perform a complex surgical procedure like the rotationplasty in lower-middle income economies with limited infrastructure and resources. The authors present seven cases of patients with osteosarcomas that received a Borggreve-Van Nes-Winkelmann rotationplasty with an evaluation of the procedures focusing on safety and sustainability. METHODS: From 2019 until 2020, seven young patients with osteosarcomas of the distal femur or proximal tibia were treated with Borggreve-Van Nes-Winkelmann rotationplasties in the Orthopaedic Hospital in Mandalay, Myanmar. As modification of the standard procedure the dissection and subsequent clamping of the femoral artery in order to minimize blood loss as well as the formation of an adipocutaneous flap that minimizes swelling and decreases the pressure on the vessels were successfully performed. This modified procedure resembles a safe and simplified surgical technique that is feasible under the circumstances of lower-middle income economies with good outcomes. RESULTS: All patients showed good functional and aesthetic results. One of the seven patients needed secondary wound closure due to wound dehiscence. CONCLUSIONS: A simplified and safe operation technique for the performance of the Van Nes-Borggreve rotationplasty was adapted to the given constraints in lower-middle income economies and proved to be successful. Trial registration All patients approved to participate in the study and have given consent to publication.

Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model.

BACKGROUND: The process of angiogenesis is a key element for tumor growth and proliferation and therefore one of the determining factors for aggressiveness and malignancy. A better understanding of the underlying processes of tumor induced angiogenesis is crucial for superior cancer treatment. Furthermore, the PeriCam perfusion speckle imager (PSI) system high resolution (HR) model by PERIMED presents a noninvasive method for semi-quantitative measurement of blood perfusion, based on laser speckle contrast analysis (LASCA). Aim of the present study was to utilize the chick chorioallantoic membrane (CAM) model as an in-ovo-tumor-model which enables rapid neovascularization of tumors while allowing real-time observation of the microcirculation via LASCA. METHODS: Fertilized chicken eggs were grafted with embryonal/alveolar rhabdomyosarcoma cells or primary sarcoma tumors. The blood perfusion was measured before and after tumor growth using LASCA. The procedure is accelerated and simplified through the integrated PIMSoft software which provides real-time graphs and color-coded images during the measurement. RESULTS: Sarcoma cells and primary sarcoma tumors exhibited satisfactory growth processes on the CAM. LASCA visualized microcirculation accurately and enabled an extensive investigation of the angiogenic potential of sarcoma cells on the CAM. We were able to show that sarcoma cells and primary sarcoma tumors induced larger quantities of neovasculature on the CAM than the controls. CONCLUSIONS: The utilization of LASCA for the investigation of tumor angiogenesis within the CAM model appears to be a highly beneficial, cost-efficient and easily practicable procedure. The proposed model can be used as a drug-screening model for individualized cancer therapy, especially with regards to anti-angiogenic agents.

pH sensing in skin tumors: Methods to study the involvement of GPCRs, acid-sensing ion channels and transient receptor potential vanilloid channels.

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.

Ion channels in sarcoma: pathophysiology and treatment options.

Sarcomas are characterized by aggressive growth and a high metastasis potentially leading in most cases to a lethal outcome. These malignant tumors of the connective tissue have a high heterogeneity with numerous genetic mutations resulting in more than 100 types of sarcoma that can be grouped into two main kinds: soft tissue sarcoma and bone sarcoma. Sarcomas are often diagnosed at late disease stage, whereas a guaranteed diagnosis of the sarcoma type is fundamental for successful therapy. However, there is no appropriate therapy available. Therefore, the need for new therapies, which prolong survival and improve quality of life, is high. In the last two decades, the role of ion channels in cancer has emerged. Ion channels seem to be an ideal target for anti-tumor therapies. However, different cancer types have their own altered ion channel pattern, and the knowledge about the tumor-associated ion channel expression is fundamental. Here, we focus on the role of different ion channels in sarcoma, their pathophysiology, and possible treatment options.

The degenerin region of the human bile acid-sensitive ion channel (BASIC) is involved in channel inhibition by calcium and activation by bile acids.

The bile acid-sensitive ion channel (BASIC) is a member of the ENaC/degenerin family of ion channels. It is activated by bile acids and inhibited by extracellular Ca2+. The aim of this study was to explore the molecular mechanisms mediating these effects. The modulation of BASIC function by extracellular Ca2+ and tauro-deoxycholic acid (t-DCA) was studied in Xenopus laevis oocytes heterologously expressing human BASIC using the two-electrode voltage-clamp and outside-out patch-clamp techniques. Substitution of aspartate D444 to alanine or cysteine in the degenerin region of BASIC, a region known to be critically involved in channel gating, resulted in a substantial reduction of BASIC Ca2+ sensitivity. Moreover, mutating D444 or the neighboring alanine (A443) to cysteine significantly reduced the t-DCA-mediated BASIC stimulation. A combined molecular docking/simulation approach demonstrated that t-DCA may temporarily form hydrogen bonds with several amino acid residues including D444 in the outer vestibule of the BASIC pore or in the inter-subunit space. By these interactions, t-DCA may stabilize the open state of the channel. Indeed, single-channel recordings provided evidence that t-DCA activates BASIC by stabilizing the open state of the channel, whereas extracellular Ca2+ inhibits BASIC by stabilizing its closed state. In conclusion, our results highlight the potential role of the degenerin region as a critical regulatory site involved in the functional interaction of Ca2+ and t-DCA with BASIC.

Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome.

Volume retention in nephrotic syndrome has been linked to activation of the epithelial sodium channel (ENaC) by proteolysis of its γ-subunit following urinary excretion of serine proteases such as plasmin. Here we tested whether pharmacological inhibition of urinary serine protease activity might protect from ENaC activation and volume retention in nephrotic syndrome. Urine from both nephrotic mice (induced by doxorubicin injection) and nephrotic patients exhibited high aprotinin-sensitive serine protease activity. Treatment of nephrotic mice with the serine protease inhibitor aprotinin by means of subcutaneous sustained-release pellets normalized urinary serine protease activity and prevented sodium retention, as did treatment with the ENaC inhibitor amiloride. In the kidney cortex from nephrotic mice, immunofluorescence revealed increased apical γ-ENaC staining, normalized by aprotinin treatment. In Xenopus laevis oocytes heterologously expressing murine ENaC, aprotinin had no direct inhibitory effect on channel activity but prevented proteolytic channel activation. Thus, our study shows that volume retention in experimental nephrotic syndrome is related to proteolytic ENaC activation by proteasuria and can be prevented by treatment with aprotinin. Hence, inhibition of urinary serine protease activity might become a therapeutic approach to treat patients with nephrotic-range proteinuria.

Activation of the Human Epithelial Sodium Channel (ENaC) by Bile Acids Involves the Degenerin Site.

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin ion channel family, which also includes the bile acid-sensitive ion channel (BASIC). So far little is known about the effects of bile acids on ENaC function. ENaC is probably a heterotrimer consisting of three well characterized subunits (αßγ). In humans, but not in mice and rats, an additional δ-subunit exists. The aim of this study was to investigate the effects of chenodeoxycholic, cholic, and deoxycholic acid in unconjugated (CDCA, CA, and DCA) and tauro-conjugated (t-CDCA, t-CA, t-DCA) form on human ENaC in its αßγ- and δßγ-configuration. We demonstrated that tauro-conjugated bile acids significantly stimulate ENaC in the αßγ- and in the δßγ-configuration. In contrast, non-conjugated bile acids have a robust stimulatory effect only on δßγENaC. Bile acids stimulate ENaC-mediated currents by increasing the open probability of active channels without recruiting additional near-silent channels known to be activated by proteases. Stimulation of ENaC activity by bile acids is accompanied by a significant reduction of the single-channel current amplitude, indicating an interaction of bile acids with a region close to the channel pore. Analysis of the known ASIC1 (acid-sensing ion channel) crystal structure suggested that bile acids may bind to the pore region at the degenerin site of ENaC. Substitution of a single amino acid residue within the degenerin region of ßENaC (N521C or N521A) significantly reduced the stimulatory effect of bile acids on ENaC, suggesting that this site is critical for the functional interaction of bile acids with the channel.

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain.

Proteases that cleave protease-activated receptor-2 (PAR(2)) at Arg(36)↓Ser(37) reveal a tethered ligand that binds to the cleaved receptor. PAR(2) activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR(2) at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR(2)-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR(2), causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR(2) at Ala(66)↓Ser(67) and Ser(67)↓Val(68). Elastase stimulated PAR(2)-dependent cAMP accumulation and ERK1/2 activation, but not Ca(2+) mobilization, in KNRK cells. Elastase induced PAR(2) coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK(2)) or ß-arrestin to PAR(2), consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR(2)-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR(2)-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR(2)- and TRPV4-mediated influx of extracellular Ca(2+) in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR(2)- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR(2) causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR(2) as a mediator of protease-driven inflammation and pain.

Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites.

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4.

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R(36)↓S(37) and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E(56)↓T(57), which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca(2+), activate ERK1/2, recruit ß-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.

Sensitisation of TRPV4 by PAR2 is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase.

Proteolytic activation of protease-activated receptor 2 (PAR2) may represent a major mechanism of regulating the transient receptor potential vanilloid 4 (TRPV4) non-selective cation channel in pathophysiological conditions associated with protease activation (e.g. during inflammation). To provide electrophysiological evidence for PAR2-mediated TRPV4 regulation, we characterised the properties of human TRPV4 heterologously expressed in Xenopus laevis oocytes in the presence and absence of co-expressed human PAR2. In outside-out patches from TRPV4 expressing oocytes, we detected single-channel activity typical for TRPV4 with a single-channel conductance of about 100 pS for outward and 55 pS for inward currents. The synthetic TRPV4 activator GSK1016790A stimulated TRPV4 mainly by converting previously silent channels into active channels with an open probability of nearly one. In oocytes co-expressing TRPV4 and PAR2, PAR2 activation by trypsin or by specific PAR2 agonist SLIGRL-NH2 potentiated the GSK1016790A-stimulated TRPV4 whole-cell currents several fold, indicative of channel sensitisation. Pre-incubation of oocytes with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM did not reduce the stimulatory effect of PAR2 activation on TRPV4, which indicates that the effect is independent of intracellular calcium signalling. Neutrophil elastase, a biased agonist of PAR2 that does not induce intracellular calcium signalling, also caused a PAR2-dependent sensitisation of TRPV4. The Rho-kinase inhibitor Y27362 abolished elastase-stimulated sensitisation of TRPV4, which indicates that Rho-kinase signalling plays a critical role in PAR2-mediated TRPV4 sensitisation by the biased agonist neutrophil elastase. During acute inflammation, neutrophil elastase may sensitise TRPV4 by a mechanism involving biased agonism of PAR2 and activation of Rho-kinase.

The bile acid receptor TGR5 activates the TRPA1 channel to induce itch in mice.

BACKGROUND & AIMS: Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice. METHODS: Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice. RESULTS: TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gßγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice. CONCLUSIONS: BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels.

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.

Pharmacological and electrophysiological characterization of the human bile acid-sensitive ion channel (hBASIC).

The human bile acid-sensitive ion channel (hBASIC) is a cation channel of the degenerin/epithelial Na(+) channel gene family that is expressed in the intestinal tract and can be activated by bile acids. Here, we show that in addition to its sensitivity for bile acids, hBASIC shares further key features with its rat ortholog: it is blocked by extracellular divalent cations, is inhibited by micromolar concentrations of the diarylamidine diminazene, and activated by millimolar concentrations of flufenamic acid. Furthermore, we demonstrate that two major bile acids present in human bile, chenodeoxycholic acid and deoxycholic acid, activate hBASIC in a synergistic manner. In addition, we determined the single-channel properties of hBASIC in outside-out patch clamp recordings, revealing a single-channel conductance of about 11 pS and a high Na(+) selectivity. Deoxycholic acid activates hBASIC in patch clamp recordings mainly by reducing the single-channel closed time. In summary, we provide a thorough functional characterization of hBASIC.

Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.