Search

Background: The kidney is the source of sKlotho and kidney-specific loss of Klotho leads to a phenotype resembling the premature multiorgan failure phenotype in Klotho-hypomorphic mice ( kl/kl mice). Klotho and the Ca-sensing receptor (CaSR) are highly expressed in the distal convoluted tubule (DCT). The physiologic mechanisms that regulate sKlotho levels are unknown., Methods: We measured sKlotho in WT and tubule-specific CaSR -/- (TS-CaSR -/- ) mice treated with calcimimetics, alkali, or acid, and Klotho shed from minced mouse kidneys, and from HEK-293 cells expressing the CaSR and Klotho, in response to calcimimetics, calcilytics, alkalotic and acidic pH, and ADAM protease inhibitors. The CaSR, Klotho, and ADAM10 were imaged in mouse kidneys and cell expression systems using confocal microscopy., Results: The CaSR, Klotho, and ADAM10 colocalize on the basolateral membrane of the DCT. Calcimimetics and HCO 3 increase serum sKlotho levels in WT but not in CaSR -/- mice, and acidic pH suppresses sKlotho levels in WT mice. In minced kidneys and cultured cells, CaSR activation with high Ca, calcimimetics, or alkali increase shed Klotho levels via ADAM10, as demonstrated using the ADAM10 inhibitor GI254023X and siRNA. In cultured cells, the CaSR, Klotho, and ADAM10 form cell surface aggregates that disperse after CaSR activation., Conclusions: We identify a novel physiologic mechanism for regulation of sKlotho levels by the renal CaSR-ADAM10-Klotho pathway. We show that CaSR activators, including alkali, increase renal CaSR-stimulated Klotho shedding and predict that this mechanism is relevant to the effects of acidosis and alkali therapy on CKD progression., (Copyright © 2021 by the American Society of Nephrology.)

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear. Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury. Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes. Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time., (Copyright © 2018 by the American Society of Nephrology.)

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.

Cell injury frequently occurs in the setting of tissue destruction and inflammation and is associated with a rise in intracellular calcium (Cai) and increased NO production. The mechanisms that trigger rises in Cai and NO during cell injury are not fully defined, but they may involve activation of G protein-coupled receptors for substances such as bradykinin, Ang II, thromboxane, and thrombin. These receptors act through G proteins from different families that have distinct functions. Receptors for bradykinin and Ang II act through members of the G alpha i and G alpha q families, whereas receptors for thrombin and thromboxane act through members of the G alpha i, G alpha q, and G alpha 12/13 families. These G proteins cooperate to regulate Cai and NO in epithelial cells through distinct mechanisms. In a number of experimental settings, activators of the adenylyl cyclase system reduce the severity of cell injury. To understand the mechanisms by which G protein-dependent signaling systems may contribute to cell injury and to define the role of adenylyl cyclase in ameliorating cell injury, the effects of adenylyl cyclase on bradykinin-stimulated Ca influx and NO in cultured renal epithelial cells that stably overexpress G alpha q and G alpha 13 were studied. This system allowed for the separation of different components of the signals initiated by receptors for thromboxane and thrombin. G alpha 13 increased bradykinin-stimulated Ca influx by a mechanism that depends on NO and cGMP. The increased Ca influx was blocked by inhibitors of NO synthase and guanylyl cyclase and by activation of adenylyl cyclase. NO production was inhibited by activators of cAMP-dependent protein kinase, which indicated that cAMP blocks Ca influx by inhibiting NO production. Expression of G alpha q, the G protein that regulates phospholipase C, also increased bradykinin-stimulated Ca influx, but by an NO, cGMP-independent mechanism that was insensitive to inhibition by adenylyl cyclase. The authors conclude that Ca influx is modulated by NO-dependent and independent mechanisms, and that to the extent that increased NO production contributes to increased Ca influx and cell injury, cell injury may be reduced by agents that activate adenylyl cyclase.