Renal stem cells in recovery from acute kidney injury.

The kidney has a dramatic capacity to regenerate after injury. Whether stem cells are the source of the epithelial progenitors replacing injured and dying tubular epithelium is currently an area of intense investigation. Studies from our laboratory and others have supported a model whereby many surviving renal epithelial cells after injury become dedifferentiated and take on mesenchymal characteristics. These cells proliferate to restore the integrity of the denuded basement membrane, and subsequently redifferentiate into a functional epithelium. An alternative possibility is that a minority of surviving intratubular cells possess stem cell properties and selectively proliferate after damage to neighboring cells. Some evidence exists to support this hypothesis but it has not yet been rigorously evaluated. A third hypothesis is that extratubular cells contribute to repair of damaged epithelium. Bone marrow-derived stem cells have been proposed to contribute to this process but our work and work of others indicates that the vast majority of tubular cells derive from an intrarenal source. Recent evidence suggests that interstitial cells may represent another extratubular stem cell niche. The fundamental unanswered questions in this field include whether renal stem cells exist in the adult, and if they do where are they located (interstitium, tubule, cortex, medulla) and what markers can be relied upon for the isolation and purification of these putative renal stem cells. In this review we focus on our current understanding of the potential role of renal and extrarenal stem cells in repair of the adult kidney and highlight some of the controversies in this field.

Renal stem cells in recovery from acute kidney injury.

The kidney has a dramatic capacity to regenerate after injury. Whether stem cells are the source of the epithelial progenitors replacing injured and dying tubular epithelium is currently an area of intense investigation. Studies from our laboratory and others have supported a model whereby many surviving renal epithelial cells after injury become dedifferentiated and take on mesenchymal characteristics. These cells proliferate to restore the integrity of the denuded basement membrane, and subsequently redifferentiate into a functional epithelium. An alternative possibility is that a minority of surviving intratubular cells possess stem cell properties and selectively proliferate after damage to neighboring cells. Some evidence exists to support this hypothesis but it has not yet been rigorously evaluated. A third hypothesis is that extratubular cells contribute to repair of damaged epithelium. Bone marrow-derived stem cells have been proposed to contribute to this process but our work and work of others indicates that the vast majority of tubular cells derive from an intrarenal source. Recent evidence suggests that interstitial cells may represent another extratubular stem cell niche. The fundamental unanswered questions in this field include whether renal stem cells exist in the adult, and if they do where are they located (interstitium, tubule, cortex, medulla) and what markers can be relied upon for the isolation and purification of these putative renal stem cells. In this review we focus on our current understanding of the potential role of renal and extrarenal stem cells in repair of the adult kidney and highlight some of the controversies in this field.

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density.

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.

Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor.

In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1beta processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10-30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3'-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, alpha-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and alpha-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1beta, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases.

Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore.

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

Modulation of P2X7 nucleotide receptor expression by pro- and anti-inflammatory stimuli in THP-1 monocytes.

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers maturation and release of the pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) in monocytes and macrophages. We report that interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synergistically induce P2X7R mRNA and functional responses in the human THP-1 monocytic cell line. Induction was dose dependent, with maximal functional activity requiring 1000 units/mL IFN-gamma and 10 ng/mL TNF-alpha and incubations of 36-72 h. The up-regulation of P2X7R function by lipopolysaccharide (LPS)/IFN-gamma and TNF-alpha/IFN-gamma was markedly attenuated by coincubation with prostaglandin E2 or the cell permeant cyclic AMP analog dibutryl cAMP (Bt2cAMP). Bt2cAMP did not significantly alter P2X7 function in HEK-293 cells stably transfected with the human P2X7 cDNA, indicating that Bt2cAMP does not exert a generalized effect on P2X7R synthesis or downstream signal transduction. These studies demonstrate that elevated cAMP negatively modulates P2X7R expression.

Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues.

1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and N-[1-[N-methyl-p-(5 isoquinolinesulfonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquinolinesulfonamide (KN-04) potently inhibit the human lymphocyte P2Z receptor, an ATP-gated cation channel [Br J Pharmacol 120:1483-1490 (1997)]. Although the molecular identity of the lymphocyte P2Z receptor has not been established, it shares many functional characteristics with the cloned P2X7 nucleotide receptor. We have tested whether these isoquinolines inhibit P2X receptor function in human embryonic kidney 293 cells that stably express the human or rat recombinant P2X7 receptors. ATP activation of cation currents and uptake of the organic dye ethidium were potently inhibited by KN-62 and KN-04 in human embryonic kidney cells expressing the human P2X7R but not the rat P2X7R, even though these species homologues share 80% amino acid identity. Introduction of the first 335 amino acids of the human P2X7R sequence conferred KN-62 sensitivity to the rat P2X7R; this suggests that isoquinolines interact with residues in the amino-terminal half (containing the large extracellular loop) of the human P2X7R. KN-62 and KN-04 also potently inhibited ATP-gated Ca2+ influx and ethidium uptake in several leukocyte cell lines (THP-1, BAC1.2f5, and BW5147) that natively express the human or murine P2X7R mRNA. The ability of isoquinoline sulfonamides to potently inhibit human and murine P2X7R signaling will be a useful tool for identifying P2Z/P2X7 functional responses in other cell types. The substantial differences in pharmacological sensitivity between rat and human P2X7R may also indicate structural domains important in channel/pore activation.

The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes.

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.

Functional consequences of mutations in the transmembrane domain and the carboxy-terminus of the murine AE1 anion exchanger.

We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.

NH4Cl activates AE2 anion exchanger in Xenopus oocytes at acidic pHi.

In the course of experiments to define regulation by intracellular pH (pHi) of the AE2 anion exchanger expressed in Xenopus oocytes, we discovered an unexpected regulation of AE2 by NH4+. Intracellular acidification produced by extracellular acidification or produced by equimolar substitution of NaCl with sodium acetate each inhibited AE2 activity. In contrast, intracellular acidification by equimolar substitution of NaCl with NH4Cl activated AE2-associated, trans-anion-dependent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- influx and efflux. Regulation by NH4+ was isoform specific, since neither erythroid nor kidney AE1 was activated. AE2 activation was maximal at <5 mM NH4Cl; was not mimicked by extracellular KCl, chloroquine, or polyamines; and was insensitive to amiloride, bumetanide, barium, and gadolinium. Whether NH4Cl acts directly on AE2 or on another target remains to be determined. Activation of AE2 by NH4+ may serve to sustain Cl-/HCO3- exchange activity in the presence of acidic pH in renal medulla, colon, abscesses, and other AE2-expressing acidic locales exposed to elevated NH4+ concentration.

Induction of the P2z/P2X7 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN-gamma in the human THP-1 monocytic cell line.

The activation of phospholipase D (PLD) was used as a marker for P2z nucleotide receptor (P2zR) activity in the human THP-1 monocytic cell line. While untreated THP-1 cells displayed little PLD response to the P2zR agonist 3'-O-(4-benzoyl)benzoyl (Bz)ATP, treatment with either IFN-gamma or bacterial LPS induced a BzATP-mediated stimulation of PLD activity. Treatment of cells with combinations of IFN-gamma and LPS resulted in synergistic induction. P2z receptors mediated these effects because: 1) reduction of extracellular divalent cation concentration increased agonist potency; 2) only BzATP or ATP acted as agonist nucleotides; 3) oxidized ATP, an inhibitor of the P2z receptor, abolished the response. The P2zR-stimulated PLD was rapidly activated (t(1/2) = 1.5 min), completely inhibited by KN-62, a calcium-calmodulin kinase II inhibitor, and only partially repressed by bisindolylmaleimide, a protein kinase C inhibitor. The P2zR-mediated PLD activity was distinguished from phorbol ester-stimulated PLD activity because the latter was slowly activated (t(1/2) > 15 min), unaffected by oxidized ATP or KN-62, and completely inhibited by bisindolylmaleimide. IFN-gamma and LPS treatment also synergistically induced P2zR-dependent changes in membrane permeability and cytolysis, as indicated by BzATP-mediated Ca2+ influx, ethidium bromide uptake, and lactate dehydrogenase release. Finally, IFN-gamma and LPS synergistically up-regulated mRNA encoding the P2X7 receptor, a recently cloned ATP-gated channel that exhibits a P2zR phenotype.

Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization.

Xenopus oocytes express endogenous Na+/H+ exchange activity but lack significant endogenous Cl-/HCO3- exchange activity. Coupled operation of Na+/H+ exchange and Cl-/HCO3- exchange contributes in many cell types to the cellular response to hypertonic stress. We therefore examined in Xenopus oocytes the osmotic regulation of chloride transport mediated by recombinant anion exchanger proteins AE2 and AE1. Hypotonicity was without effect on either anion transporter. Hypertonicity activated AE2-associated 36Cl- influx and efflux in a time- and osmolarity-dependent manner, whether incremental osmoles were charged or uncharged, but had no measurable effect on AE1 function. Hypertonic stimulation of AE2 was completely inhibited by Na+ removal or by addition of amiloride. In contrast, neither maneuver altered isotonic activity of AE2. Hypertonicity also induced amiloride-sensitive elevation of oocyte intracellular pH (pHi), and shifted the sigmoidal relationship of extracellular pH vs. AE2 activity > or = 0.5 units to the acid. Injection of pH 7.4 buffer into oocytes attenuated both hypertonic alkalinization and activation of AE2-associated 36Cl- influx, without inhibition of isotonic AE2 function. These data demonstrate that recombinant AE2 expressed in Xenopus oocytes is activated by increased pHi and that hypertonic activation of AE2 is secondary to hypertonic activation of Na+/H+ exchange.

Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes.

cRNA encoding the murine band 3-related protein AE2 was expressed in Xenopus oocytes. AE2-mediated transport function and regulation were analyzed by unidirectional 36Cl- influx and efflux studies. AE2 cRNA-injected oocytes took up 36Cl- as much as 40-fold faster than did water-injected oocytes. AE2-mediated 36Cl- uptake increased as a function of increasing uptake time, number of days after cRNA injection, and amount of injected cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent Km for external Cl- was 5.6 mM. AE2 was defined as a Cl-/anion exchanger by two criteria: 1) 36Cl- efflux from AE2-expressing oocytes was maximally stimulated by extracellular Cl- or nitrate; AE2-associated 36Cl- efflux was supported by substitution of extracellular Cl- with other anions in the rank order bromide > isethionate > or = gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl(-)-free media containing isethionate, gluconate, or glutamate decreased subsequent AE2-associated 36Cl- uptake from Cl- media in rough proportion to the degree of intracellular Cl- depletion, whereas preincubation in nitrate medium had no effect. AE2-associated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid at half-maximally inhibitory concentrations between 0.5 and 19 microM, depending on extracellular Cl- concentration, and progressed to irreversibility at 20 degrees C with a half-time of 20-30 min. Many additional inhibitors showed lower potency for AE2 than previously reported for AE1. Although AE2 expression did not change oocyte resting intracellular pH, AE2-associated 36Cl- influx and efflux were each decreased in acid incubation medium and increased in alkaline medium.