We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

ABSTRACT. Thrombosis of hemodialysis vascular access grafts represents a major medical and economic burden. Experimental and clinical models suggest a role for antiplatelet agents in the prevention of thrombosis. The study was designed to determine the efficacy of the combination of aspirin and clopidogrel in the prevention of graft thrombosis. The study was a randomized, double-blind trial conducted at 30 hemodialysis units at Veterans Affairs medical centers. Participants undergoing hemodialysis with a polytetrafluoroethylene graft in the arm were randomized to receive either double placebos or aspirin (325 mg) and clopidogrel (75 mg) daily. Participants were to be monitored while receiving study medications for a minimum of 2 yr. The study was stopped after randomization of 200 participants, as recommended by the Data Safety and Monitoring Board because of a significantly increased risk of bleeding among the participants receiving aspirin and clopidogrel therapy. The cumulative incidence of bleeding events was significantly greater for those participants, compared with participants receiving placebos [hazard ratio, 1.98; 95% confidence interval (CI), 1.19 to 3.28; P = 0.007]. Twenty-three participants in the placebo group and 44 participants in the active treatment group experienced a bleeding event (P = 0.006). There was no significant benefit of active treatment in the prevention of thrombosis (hazard ratio, 0.81; 95% CI, 0.47 to 1.40; P = 0.45), although there was a trend toward a benefit among participants who had not experienced previous graft thrombosis (hazard ratio, 0.52; 95% CI, 0.22 to 1.26; P = 0.14). In the hemodialysis population, therapy with aspirin and clopidogrel was associated with a significantly increased risk of bleeding and probably would not result in a reduced frequency of graft thrombosis. E-mail: james.kaufman@med.va.gov

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.

Increased activation of specific protein kinase C (PKC) isoforms and increased nonenzymatic glycation of intracellular and extracellular proteins [the accumulation of advanced glycation end products (AGEs)] are major mechanistic pathways implicated in the pathogenesis of diabetic complications. Blocking PKC-beta(II) has been shown to decrease albuminuria in animal models of diabetes. To demonstrate a direct relationship between AGEs and the induction and translocation of PKC-beta(II), studies were carried out in rat neonatal mesangial cells, known to express PKC-beta(II) in association with rapid proliferation in post-natal development. Oxidative stress was studied by using the fluorescent probe dichlorfluorescein diacetate. Translocation of PKC-beta(II) was demonstrated by using immunofluorescence and Western blotting of fractionated mesangial cells. Induction of intracellular oxidative stress, increase in intracellular calcium, and cytosol to membrane PKC-beta(II) translocation (with no change in PKC-alpha) were demonstrated after exposure to AGE-rich proteins. These data support the hypothesis that AGEs cause mesangial oxidative stress and alterations in PKC-beta(II), changes that may ultimately contribute to phenotypic abnormalities associated with diabetic nephropathy.

Parathyroid hormone-related protein (PTHrP) is initially translated as a preprohormone which is posttranslationally processed to yield a family of mature secretory forms. Most attention has focused on the aminoterminal portion of the molecule which is homologous to parathyroid hormone. It is clear, however, that a midregion species of PTHrP is posttranslationally cleaved from the highly conserved mid-region of PTHrP, and that the amino terminus of this peptide is Ala 38 . The purposes of the current study were three: 1) to confirm that Arg 37 immediately preceding Ala 38 serves as a posttranslational processing site in the PTHrP precursor, 2) to determine the carboxyl terminus of the mid-region secretory species of PTHrP, and 3) to synthesize this authentic mid-region secretory form of PTHrP and determine whether it is biologically active. The results indicate that: 1) Arg 37 is indeed a processing site in the PTHrP precursor; 2) three distinct mid-region PTHrP species are generated by posttranslational processing, PTHrP(38 -94)amide, PTHrP(38 -95), and most likely, PTHrP(38 -101); and 3) synthetic mid-region PTHrP(38 -94)amide is active in four different biological systems. These studies confirm the finding that PTHrP is a prohormone. More importantly, they define a novel, biologically active highly conserved mid-region secretory form of PTHrP.Parathyroid hormone-related protein (PTHrP) 1 was initially discovered through its structural and functional homology with parathyroid hormone (for a review, see Refs.

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.

Parathyroid hormone-related protein (PTHrP) is widely expressed in normal adult and fetal tissues, where it acts in an autocrine/paracrine fashion, stimulates growth and differentiation, and shares early response gene characteristics. Since recovery from renal injury is associated with release of local growth factors, we examined the expression and localization of PTHrP in normal and ischemic adult rat kidney. Male SpragueDawley rats underwent complete bilateral renal artery occlusion for 45 min, followed by reperfusion for 15 min., and 2,6, 24, 48, and 72 h. Renal PTHrP mRNA levels, when compared with sham-operated animals, increased twofold after ischemia, and peaked within 6 h after reperfusion. PTH receptor, j-actin, and cyclophilin mRNA levels all decreased after ischemia. PTHrP immunohistochemical staining intensity increased in proximal tubular cells after ischemia, changing its location from diffusely cytoplasmic to subapical by 24 h after reperfusion. In addition, PTHrP localized to glomerular epithelial cells (visceral and parietal), but not to mesangial cells. PTHrP and PTH stimulated proliferation two-to threefold in cultured mesangial cells. We conclude that PTHrP mRNA and protein production are upregulated after acute renal ischemic injury, that PTHrP is present in glomerulus and in both proximal and distal tubular cells, and that PTHrP stimulates DNA synthesis in mesangial cells. The precise functions of PTHrP in normal and injured kidney remain to be defined. (J. Clin. Invest. 1993.

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