R. Tyler Miller

Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome

The extracellular Ca(2+)-sensing receptor (CaSR) plays an essential role in extracellular Ca(2+) homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca(2+) and ERK activity, showed a dramatic reduction in the EC(50) for extracellular Ca(2+) compared with the wild-type and a loss-of-function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.

Mechanisms of mechanical signaling in development and disease

The responses of cells to chemical signals are relatively well characterized and understood. Cells also respond to mechanical signals in the form of externally applied force and forces generated by cell–matrix and cell–cell contacts. Many features of cell function that are generally considered to be under the control of chemical stimuli, such as motility, proliferation, differentiation and survival, can also be altered by changes in the stiffness of the substrate to which the cells are adhered, even when their chemical environment remains unchanged. Many examples from clinical and whole animal studies have shown that changes in tissue stiffness are related to specific disease characteristics and that efforts to restore normal tissue mechanics have the potential to reverse or prevent cell dysfunction and disease. How cells detect stiffness is largely unknown, but the cellular structures that measure stiffness and the general principles by which they work are beginning to be revealed. This Commentary highlights selected recent reports of mechanical signaling during disease development, discusses open questions regarding the physical mechanisms by which cells sense stiffness, and examines the relationship between studies in vitro on flat substrates and the more complex three-dimensional setting in vivo.

Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver

BACKGROUND & AIMS Hepatic ammonium metabolism is critical for maintenance of normal health. Three mammalian members of an ammonium transporter family have recently been identified: Rh A glycoprotein (RhAG), Rh B glycoprotein (RhBG), and Rh C glycoprotein (RhCG). This study examined which of these are expressed in the mouse liver and in which cells they are expressed. METHODS Normal Balb/c mice were used. Messenger RNA (mRNA) expression was detected using either conventional or real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using immunoblot analysis and either immunohistochemical or immunofluorescent microscopy. RESULTS We confirmed hepatic RhBG mRNA expression using real-time RT-PCR. Immunoblot analysis identified expression of a approximately 45-kilodalton protein. Immunohistochemical and immunofluorescent microscopy identified basolateral RhBG immunoreactivity in 1-2 cell layers of hepatocytes surrounding central veins. No immunoreactivity was identified in periportal or midzonal hepatocytes. Perivenous hepatocyte-specific expression was confirmed by colocalization with glutamine synthetase. A second ammonium transporter, RhCG, was expressed but at substantially lower levels. Real-time RT-PCR quantified hepatic RhCG mRNA expression at approximately 0.4% of RhBG mRNA expression. Immunoblot analysis confirmed RhCG protein expression, and immunofluorescence microscopy identified RhCG expression in bile duct epithelia. In contrast to RhBG and RhCG, RhAG mRNA was not identified by RT-PCR. CONCLUSIONS RhBG and RhCG are expressed by the mouse liver. Basolateral RhBG is expressed by perivenous hepatocytes, where it may mediate ammonium uptake, and RhCG immunoreactivity is present in bile duct epithelial cells, where it may contribute to ammonium secretion into bile fluid.

Absence of filamin A prevents cells from responding to stiffness gradients on gels coated with collagen but not fibronectin.

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.

The Ca2+-sensing Receptor Activates Cytosolic Phospholipase A2 via a Gqα-dependent ERK-independent Pathway

The Ca2+-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A2(PLA2) by the Ca2+-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA2activity was attributable to cytosolic PLA2(cPLA2) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA2. No CaR-stimulated cPLA2 activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca2+(Ca2+ i ), whereas inhibition of phospholipase C (PLC) with U73122 completely inhibited CaR-stimulated PLC and cPLA2 activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of Gprotein Signaling) protein that inhibits Gαqactivity. CaR-stimulated cPLA2 activity was inhibited 80% by chelation of extracellular Ca2+ and depletion of intracellular Ca2+ with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca2+, calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEKK97R, had no effect on CaR-stimulated cPLA2 activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA2 via a Gαq, PLC, Ca2+-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.

Parallel Activation of Phosphatidylinositol 4-Kinase and Phospholipase C by the Extracellular Calcium-sensing Receptor

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that regulates physiological processes including Ca2+ metabolism, Na+, Cl−, K+, and H20 balance, and the growth of some epithelial cells through diverse signaling pathways. Although many effects of CaR are mediated by the heterotrimeric G proteins Gαq and Gαi, not all signaling pathways regulated by CaR have been identified. We used human embryonic kidney (HEK)-293 cells that stably express human CaR to study the regulation of inositol lipid metabolism by CaR. The nonfunctional mutant CaRR796W was used as a negative control. We found that CaR regulates phosphatidylinositol (PI) 4-kinase, the first step in inositol lipid biosynthesis. In cells pretreated with U73122 to inhibit phospholipase C activation and to block the degradation of PI 4,5-bisphosphate to form [3H]inositol trisphosphate (IP3), CaR stimulated the accumulation of [3H]PI monophosphate (PIP). Additionally, wortmannin, an inhibitor of both PI 3-kinase and type III PI 4-kinase, blocked CaR-stimulated accumulation of [3H]PIP and inhibited [3H]IP3 production. CaR-stimulated inositol lipid synthesis was attributable to PI 4-kinase and not PI 3-kinase because CaR did not activate Akt, a downstream target of PI 3-kinase. CaR associates with PI 4-kinase based on the findings that CaR and the 110-kDa PI 4-kinase β can be co-immunoprecipitated with antibodies against either CaR or PI 4-kinase. The PI-4 kinase in co-immunoprecipitates with anti-CaR antibody was activated in Ca2+-stimulated HEK-293 cells, which stably express the wild type CaR. Pertussis toxin did not affect the formation of [3H]IP3 or the rise in intracellular Ca2+ (Handlogten, M. E., Huang, C. F., Shiraishi, N., Awata, H., and Miller, R. T. (2001) J. Biol. Chem.276, 13941–13948). RGS4, an accelerator of GTPase activity of members of the Gαi and Gαq families, attenuated the CaR-stimulated PLC activation and IP3 accumulation, which is mediated by Gαq, but did not inhibit CaR-stimulated [3H]PIP formation. In HEK-293 cells, which express wild type CaR, Rho was enriched in immune complexes co-immunoprecipitated with the anti-CaR antibody. C3 toxin, an inhibitor of Rho, also inhibited the CaR-stimulated [3H]IP3production but did not lead to CaR-stimulated [3H]PIP formation, reflecting inhibition of PI 4-kinase. Taken together, our data demonstrate that CaR stimulates PI 4-kinase, the first step in inositol lipid biosynthesis conversion of PI to PI 4-P by Rho-dependent and Gαq- and Gαi-independent pathways.

The calcium-sensing receptor and its interacting proteins

•  Introduction •  Signalling by the Ca receptor •  Distinct effects of angiotensin II and Ca receptors •  Receptor activity modifying proteins (RAMPS) •  Filamin •  Potassium channels •  Other CaR‐interacting proteins •  Conclusions

Biophysical properties of normal and diseased renal glomeruli

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Youngs modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Youngs moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Youngs modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.

Calcium-sensing Receptor Decreases Cell Surface Expression of the Inwardly Rectifying K+ Channel Kir4.1

The Ca2+-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K+ channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K+ conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gαq reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gαi and Gαq. Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gαq on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K+ channel, an apical membrane distal nephron K+ channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gαq and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.

Silencing of filamin A gene expression inhibits Ca2+‐sensing receptor signaling

Filamin plays an important role in actin cytoskeleton organization, membrane stabilization, and anchoring of transmembrane proteins. Using short interfering RNA (siRNA) to selectively target the filamin A gene and silence its expression, we studied the role of filamin A in G protein coupled receptor (GPCR) signaling. Silencing of filamin A protein expression was determined by immunoblotting and immunofluorescence. Functional consequences of filamin A gene silencing were measured by studying its role in MAPK signaling pathways activated by the Ca2+‐sensing receptor. This work defines filamin A involvement in GPCR signaling pathways and describes an additional method for studying its function.