Intensive renal support in critically ill patients with acute kidney injury did not decrease mortality, improve recovery of kidney function, or reduce the rate of nonrenal organ failure as compared with less-intensive therapy involving a defined dose of intermittent hemodialysis three times per week and continuous renal-replacement therapy at 20 ml per kilogram per hour. (ClinicalTrials.gov number, NCT00076219.)

The macrophage fusion receptor (MFR), also called P84/BIT/SIRP␣/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophagemacrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.Osteoclasts and giant cells are characterized by multinucleation and a powerful ability to resorb the substrate onto which they adhere. Although osteoclasts and giant cells play an important role in bone remodeling and immune defense, respectively, they are also associated with osteoporosis, granulomatous diseases, and tumors.Multinucleation appears to endow macrophages with the capacity to digest and resorb extracellular infectious agents, foreign material, and other components that are too large to be internalized, such as bone. This resorption occurs in an "extracellular lysosomal compartment" sealed off between the multinucleated cell and its target substrate (reviewed in Ref.1). The plasma membrane that faces that extracellular domain is highly ruffled and specialized. Multinucleation gives macrophages added resorptive capacity, in part by making available a large excess of plasma membrane.Understanding the mechanism by which macrophages differentiate into osteoclasts and multinucleated giant cells is of extreme importance. One of the key steps in the differentiation of osteoclasts and giant cells is the fusion mechanism of their mononucleated precursor cells. It is assumed that both osteoclasts and giant cells originate from the fusion of mononuclear phagocytes. Despite the pathophysiological importance of these cells, the mechanism by which their mononucleated precursors fuse remains poorly understood. Indeed, cell-cell fusion itself, whether it concerns that of sperm cells with oocytes in fertilization or myoblasts with myoblasts in muscle development, has not been investigated thoroughly. It is proposed that cell-cell fusion involves a set of proteins similar to those used by viruses to fuse with host cells be...

We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRP␣, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events.Membrane fusion is a ubiquitous event that occurs in a wide range of cell biological processes. While intracellular membrane fusion mediating interorganelle traffic is well studied, much less is known about cell-cell fusion mediating sperm cell-oocyte, myoblast-myoblast, and macrophage-macrophage fusion. These fusion events are required for fertilization, muscle, and osteoclast development, respectively. In the case of mononuclear phagocytes, fusion is associated not only with the differentiation of osteoclasts, cells which play a key role in the pathogenesis of osteoporosis, but also with the formation of giant cells that are present in chronic inflammatory reactions and in tumors. Despite the biological and pathophysiological importance of intercellular fusion events, the actual molecular mechanisms of cell-cell fusion are still unclear.

Cells of the mononuclear phagocyte lineage have the capability to adhere to and fuse with each other and to differentiate into osteoclasts and giant cells. To investigate the macrophage adhesion/fusion mechanism, we focused our attention on CD44, a surface glycoprotein known to play a role in hematopoietic cell–cell adhesion. We report that CD44 expression by macrophages is highly and transiently induced by fusogenic conditions both in vitro and in vivo. We show that CD44 ligands, hyaluronic acid, chondroitin sulfates, and osteopontin prevent macrophage multinucleation. In addition, we report that the recombinant extracellular domain of CD44 binds fusing macrophages and prevents multinucleation in vitro. These data suggest that CD44 may control the mononucleated status of macrophages in tissues by virtue of mediating cell–cell interaction.

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K ؉ current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K ؉ current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K ؉ current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 g/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels. ROMK, a cloned inward rectifying Kϩ channel from the renal outer medulla, is a key component of the small conductance K ϩ channel identified in the thick ascending limb and cortical collecting duct (CCD) 1 (1-3). This conclusion is based on observations that the conductance, open probability, opening and closing kinetics, and pH sensitivity of ROMK are similar to that of the native small conductance K ϩ channel (1, 3, 4). Moreover, both K ϩ channels are regulated by protein kinase A and protein kinase C (5-10). A difference between the native small conductance K ϩ channel and ROMK is that ROMK is insensitive to sulfonylurea agents, whereas the native small conductance K ϩ channel is inhibited by sulfonylurea agents (11-13). Three isoforms of ROMK, ROMK1, -2, and -3, have been found in the rat kidney (14). Based on in situ hybridization, ROMK1 is located in the apical membrane of principal cells in the CCD, whereas ROMK2 and -3 are expressed at the thick ascending limb (14). The principal cell in the CCD is responsible for Na ϩ reabsorption and K ϩ secretion, which takes place by K ϩ entering the cell across the basolateral membrane via Na,K-ATPase followed by diffusion into the lumen across the apical membrane through ROMK1-like channels (15).We have previously demonstrated that inhibition of PTP reduced the activity of the small conductance K ϩ channel in the apical membrane of the CCD of rat kidney (16). Moreover, we have reported that blocking ...

We have previously shown that inhibiting proteintyrosine kinase increased whereas inhibiting proteintyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylationinduced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamindependent internalization of the channel. ROMK11 is located in the apical membrane of the cortical collecting duct (CCD) and is generally believed to be a key component of the native small conductance K ϩ (SK) channel (2-6). The SK channels are the major contributors to the apical K ϩ conductance and are responsible for K ϩ secretion (4, 7). One important factor for regulating K ϩ secretion is the dietary K ϩ intake; a high K ϩ intake increases whereas a low K ϩ intake decreases K ϩ secretion (7). The low K ϩ intake-induced decrease in K ϩ secretion is at least partially achieved by reducing the number of SK channels in the apical membrane of the CCD (8).Our preceding experiments strongly indicated that the low K ϩ intake-induced decrease in SK channel number was mediated by protein-tyrosine kinase (PTK). This conclusion is supported by the observation that inhibition of PTK increased the number of the SK channels in the apical membrane of the CCD from rats on a K ϩ -deficient diet (8). In contrast, inhibition of PTP decreased the number of SK channels in the CCD from rats on a high K ϩ diet (9). Because the effect of inhibiting PTP on channel activity was blocked by 20% sucrose, we speculated that inhibiting PTP increases the endocytosis of the SK channels whereas inhibiting PTK augments the exocytosis of the SK channels into the cell membrane. This notion is supported by observations that inhibiting PTP with PAO reduced whereas inhibiting PTK with herbimycin A increased the membrane location of ROMK1 in oocytes injected with GF...

We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with (32)P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [(32)P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [(32)P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333-362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion.

. Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct. Am J Physiol Renal Physiol 286: F881-F892, 2004; 10.1152/ajprenal.00301.2003.-We previously demonstrated that dietary K intake regulates the expression of Src family PTK, which plays an important role in controlling the expression of ROMK1 in plasma membrane (Wei Y, Bloom P, Lin D-H, Gu RM, and Wang WH. Am J Physiol Renal Physiol 281: F206 -F212, 2001). In the present study, we used the immunofluorescence staining technique to demonstrate the presence of c-Src, a member of Src family PTK, in the thick ascending limb (TAL) and collecting duct. Confocal microscopy shows that c-Src is highly expressed in the renal cortex and outer medulla. Moreover, c-Src and ROMK are coexpressed in the same nephron segment. Also, the positive staining of c-Src is visible in tubules stained with Tamm-Horsfall glycoprotein or aquaporin-2. This suggests that c-Src is present in the TAL, cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). To study the role of PTK in the regulation of ROMK membrane expression in the TAL and CCD, we carried out immunocytochemical staining with ROMK antibody in the CCD or TAL from rats on either a high-K (HK) or K-deficient (KD) diet. A sharp membrane staining of ROMK can be observed in the TAL from rats on both HK and KD diets. However, a clear plasma membrane staining can be observed only in the CCD from rats on a HK diet but not from those on a KD diet. Treatment of the CCD from rats on a HK diet with phenylarsine oxide (PAO) decreases the positive staining in the plasma/subapical membrane and increases the ROMK staining in the intracellular compartment. However, PAO treatment did not significantly alter the staining pattern of ROMK in the TAL. Moreover, the biotinylation technique has also confirmed that neither herbimycin A nor PAO has significantly changed the biotin-labeled ROMK2 in HEK293 cells transfected with ROMK2 and c-Src. We conclude that c-Src is expressed in the TAL, CCD, and OMCD and that stimulation of PTK increases the ROMK channels in the intracellular compartment but decreases them in the apical/subapical membrane in the CCD. potassium secretion; endocytosis; exocytosis; dietary potassium intake; c-Src DIETARY K INTAKE is an important factor regulating renal K secretion: a high-K (HK) intake stimulates, whereas a low-K intake decreases, renal K secretion (4, 10, 11). However, the mechanism by which dietary K intake regulates K secretion is not completely understood. An increase in K intake has been shown to raise plasma aldosterone (4) and stimulate Na absorption in the cortical collecting duct (CCD). As a consequence, the driving force for K secretion increases sharply and K secretion is stimulated. Moreover, it has also been reported that a HK intake can increase K secretion by an aldosteroneindependent mechanism (16). We recently demonstrated that a HK diet decreases, whereas a low-K diet increases, the expression of c-Src, a nonreceptor type of PTK, in the...