Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography.

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities. In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to -25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions ('in air' or 'in bulk CPA'). For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 ('scaffold in a bulk CPA') vs. 7.71 ± 0.43 ('bulk CPA', P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 ('scaffold in a bulk CPA') vs. 7.68 ± 0.34 ('bulk CPA', P < 0.0001)). At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen 'in air' were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 µg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively). Moreover, when frozen 'in air' in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend. The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

Activity-dependent conformational transitions of the insulin receptor-related receptor.

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

Insulin Receptor-Related Receptor Regulates the Rate of Early Development in Xenopus laevis.

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

Testing Meldonium: Assessing Soviet pragmatic alternatives to the randomized controlled trial.

BACKGROUND/AIMS: Current research largely tends to ignore the drug-testing model that was developed in the "Second World" as an explicit alternative to the randomized controlled trial. This system can be described as "socialist pharmapolitics," accounting for the specific features of state socialism that influenced the development and testing of experimental drugs. The clinical trials model employed in the "Second World" was heavily influenced by the Soviet Union, which was by far the most influential player in the socialist bloc during the Cold War. Based on extensive archival research, this article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It accounts for the divergences between the official model prescribed by the Soviet authorities and the messy realities of healthcare practice. It further outlines different factors that ultimately shaped how clinical trials were organized in Soviet institutions "on the ground." Accordingly, this article presents a "real-life" history of "socialist pharmapolitics" and outlines the problems that this system faced in practice. METHODS: Archival research was conducted at the Russian State Archive of Scientific and Technical Documentation in Moscow. Archival files include scientific, technical, and registration documentation such as biochemical, pharmacological, and clinical descriptions of the experimental drug Meldonium, letters between various hospitals, research institutes and the Soviet regulatory body, as well as 26 reports of completed clinical trials. Manual content analysis was used for the interpretation of results. RESULTS: This article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It demonstrates some key differences from the randomized controlled trial model. This article also highlights some of the discrepancies between the model that was officially prescribed by the Soviet authorities and the realities of experimental drug testing in the Soviet Union in the late 1980s and early 1990s. In particular, it notes some elements of randomization, double-blinding, and the use of placebo that were present in Meldonium trials despite being formally denounced by Soviet bioethics. CONCLUSION: The Soviet model for testing experimental drugs differed from the Western one substantially in a number of respects. This difference was not only proclaimed officially by the Soviet authorities, but was for the most part enforced in clinical trials in practice. At the same time, our research demonstrates that there were important differences between the official model and the clinical realities on the ground.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage.

Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell-cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.

The dimeric ectodomain of the alkali-sensing insulin receptor-related receptor (ectoIRR) has a droplike shape.

Insulin receptor-related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid-base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

с-Met receptor can be activated by extracellular alkaline medium.

Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.

Toward a Molecular Reorganization Energy-Based Analysis of Third-Order Nonlinear Optical Properties of Polymethine Dyes and J-Aggregates.

This work demonstrates the feasibility and applicability of the theory of extended multiphonon electron transitions for the description of nonlinear optical properties of polymethine dyes using quantum chemistry and model calculations. The transformation of a strong one-photon absorption band in dye monomers to a weak two-photon absorption band is rationalized from the electron-nuclear resonance condition. The power law fitting of the results of quantum chemical computations of nonlinear optical properties allows the predicting of the shift of the corresponding Egorov-like resonance curve to the shortest dye in the vinylogous series of dye monomers. The results presented provide an insight and guide for the rational molecular design and application of polymethine dyes.

Organ Preservation into the 2020s: The Era of Dynamic Intervention.

Organ preservation has been of major importance ever since transplantation developed into a global clinical activity. The relatively simple procedures were developed on a basic comprehension of low-temperature biology as related to organs outside the body. In the past decade, there has been a significant increase in knowledge of the sequelae of effects in preserved organs, and how dynamic intervention by perfusion can be used to mitigate injury and improve the quality of the donated organs. The present review focuses on (1) new information about the cell and molecular events impacting on ischemia/reperfusion injury during organ preservation, (2) strategies which use varied compositions and additives in organ preservation solutions to deal with these, (3) clear definitions of the developing protocols for dynamic organ perfusion preservation, (4) information on how the choice of perfusion solutions can impact on desired attributes of dynamic organ perfusion, and (5) summary and future horizons.

Autophosphorylation of Orphan Receptor ERBB2 Can Be Induced by Extracellular Treatment with Mildly Alkaline Media.

ErbB2 is an oncogene receptor tyrosine kinase linked to breast cancer. It is a member of the epidermal growth factor receptor (EGFR) minifamily. ErbB2 is currently viewed as an orphan receptor since, by itself, it does not bind EGF-like ligands and can be activated only when overexpressed in malignant cells or complexed with ErbB3, another member of the EGFR minifamily. Here, we report that ErbB2 can be activated by extracellular application of mildly alkaline (pH 8⁻9) media to ErbB2-transfected cells. We also show that the activation of the ErbB2 receptor by alkali is dose-dependent and buffer-independent. The endogenous ErbB2 receptor of A431 cell line can also undergo alkali-dependent autophosphorylation. Thus, we describe a novel ligand-independent mechanism of ErbB2 receptor activation.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer.

Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron-sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from Density Functional Theory (DFT) calculations. The distal cubane FeS-cluster has a three-cysteine and one-histidine coordination [Fe4S4](His)(Cys)3 first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and an all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe4S4](Cys)4 case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment, which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.

Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor.

The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646-716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

Introduction: History of the Adhesion GPCR Field.

Development of the aGPCR scientific field based on PubMed-listed research articles and selected key findings Since the discovery of adhesion G-protein-coupled receptors (aGPCRs) 20 years ago, reverse genetics approaches have dominated the elucidation of their function and work mechanisms. Seminal findings in this field comprise the description of aGPCRs as seven-transmembrane (7TM) molecules with an extended extracellular region, the identification of matricellular ligands that bind to distinct protein folds at the N-terminus, the clarification of an autoproteolytic cleavage event at a juxtamembranous GPCR proteolysis site (GPS), the elucidation of the crystal structure of the GPCR autoproteolysis-inducing (GAIN) domain that embeds the GPS and connects the receptor fragments, the demonstration that a short N-terminal sequence of the seven-transmembrane (7TM) region can serve as a tethered agonist, and, recently, the notification that aGPCRs can serve as mechanosensors. We here discuss how these discoveries have moved forward aGPCR research and, finally, linked the field to the GPCR field. We argue that crucial questions remain to be addressed before we can fully appreciate the biological nature of these fascinating receptors.

Control of Adhesion GPCR Function Through Proteolytic Processing.

Proteolytic processing events in adhesion GPCRs. aGPCRs can undergo multiple autoproteolytic (red asterisks) and proteolytic processing events by exogenous proteases (yellow asterisks) that may be involved in signaling events of the receptors. Proteolytic processing is an unusual property of adhesion family G protein-coupled receptors (aGPCRs) that was observed upon their cloning and biochemical characterization.Ever since, much effort has been dedicated to delineate the mechanisms and requirements for cleavage events in the control of aGPCR function. Most notably, all aGPCRs possess a juxtamembrane protein fold, the GPCR autoproteolysis-inducing (GAIN) domain, which operates as an autoprotease for many aGPCR homologs investigated thus far. Analysis of its autoproteolytic reaction, the consequences for receptor fate and function, and the allocation of physiological effects to this peculiar feature of aGPCRs has occupied the experimental agenda of the aGPCR field and shaped our current understanding of the signaling properties and cell biological effects of aGPCRs. Interestingly, individual aGPCRs may undergo additional proteolytic steps, one of them resulting in shedding of the entire ectodomain that is secreted and can function independently. Here, we summarize the current state of knowledge on GAIN domain-mediated and GAIN domain-independent aGPCR cleavage events and their significance for the pharmacological and cellular actions of aGPCRs. Further, we compare and contrast the proteolytic profile of aGPCRs with known signaling routes that are governed through proteolysis of surface molecules such as the Notch and ephrin pathways.

Molecular Reorganization Energy as a Key Determinant of J-Band Formation in J-Aggregates of Polymethine Dyes.

This work demonstrates the feasibility of the theory of extended multiphonon electron transitions for the description of optical spectra of polymethine dyes and J-aggregates using quantum chemistry. The formation of a strong absorption band in dye monomers and a J-band in their aggregates is uniformly explained from the electron-nuclear resonance condition. The redistribution of optical band intensities among cyanine dyes with the solvent is also explained. The dependence of the possibility of J-band formation in aggregates on the dye structure is successfully predicted. The results presented provide an insight for rational molecular design and application of polymethine dyes.

Structural determinants of the insulin receptor-related receptor activation by alkali.

IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline medium and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1-333) or L2 domain (residues 334-462) or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domain replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted toward alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points.

Insulin receptor-related receptor as an extracellular pH sensor involved in the regulation of acid-base balance.

Recent studies of insulin receptor-related receptor (IRR) revealed its unusual property to activate upon extracellular application of mildly alkaline media, pH>7.9. The activation of IRR with hydroxyl anion has typical features of ligand-receptor interaction; it is specific, dose-dependent, involves the IRR extracellular domain and is accompanied by a major conformational change. IRR is a member of the insulin receptor minifamily and has been long viewed as an orphan receptor tyrosine kinase since no peptide or protein agonist of IRR was found. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin-like growth factor receptors in amphibia. The latter two cannot be activated by alkali. Another major difference between them is that unlike ubiquitously expressed insulin and insulin-like growth factor receptors, IRR is found in specific sets of cells of only some tissues, most of them being exposed to extracorporeal liquids of extreme pH. In particular, largest concentrations of IRR are in beta-intercalated cells of the kidneys. The primary physiological function of these cells is to excrete excessive alkali as bicarbonate into urine. When IRR is removed genetically, animals loose the property to excrete bicarbonate upon experimentally induced alkalosis. In this review, we will discuss the available in vitro and in vivo data that support the hypothesis of IRR role as a physiological alkali sensor that regulates acid-base balance. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.