Renal Autocrine and Paracrine Signaling: A Story of Self-protection.

Autocrine and paracrine signaling in the kidney adds an extra level of diversity and complexity to renal physiology. The extensive scientific production on the topic precludes easy understanding of the fundamental purpose of the vast number of molecules and systems that influence the renal function. This systematic review provides the broader pen strokes for a collected image of renal paracrine signaling. First, we recapitulate the essence of each paracrine system one by one. Thereafter the single components are merged into an overarching physiological concept. The presented survey shows that despite the diversity in the web of paracrine factors, the collected effect on renal function may not be complicated after all. In essence, paracrine activation provides an intelligent system that perceives minor perturbations and reacts with a coordinated and integrated tissue response that relieves the work load from the renal epithelia and favors diuresis and natriuresis. We suggest that the overall function of paracrine signaling is reno-protection and argue that renal paracrine signaling and self-regulation are two sides of the same coin. Thus local paracrine signaling is an intrinsic function of the kidney, and the overall renal effect of changes in blood pressure, volume load, and systemic hormones will always be tinted by its paracrine status.

Regulation of renal aquaporin water channels in acute pyelonephritis.

Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.

The bacteria and the host: a story of purinergic signaling in urinary tract infections.

The local environment forces a selection of bacteria that might invade the urinary tract, allowing only the most virulent to access the kidney. Quite similar to the diet in setting the stage for the gut microbiome, renal function determines the conditions for bacteria-host interaction in the urinary tract. In the kidney, the term local environment or microenvironment is completely justified because the environment literally changes within a few micrometers. The precise composition of the urine is a function of the epithelium lining the microdomain, and the microenvironment in the kidney shows more variation in the content of nutrients, ion composition, osmolality, and pH than any other site of bacteria-host interaction. This review will cover some of the aspects of bacterial-host interaction in this unique setting and how uropathogenic bacteria can alter the condition for bacteria-host interaction. There will be a particular focus on the recent findings regarding how bacteria specifically trigger host paracrine signaling, via release of extracellular ATP and activation of P2 purinergic receptors. These finding will be discussed from the perspective of severe urinary tract infections, including pyelonephritis and urosepsis.

Lack of P2X7 Receptors Protects against Renal Fibrosis after Pyelonephritis with α-Hemolysin-Producing Escherichia coli.

Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7-/- mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7-/- mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7-/- mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7-/- mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections.

α-Haemolysin production, as a single factor, causes fulminant sepsis in a model of Escherichia coli-induced bacteraemia.

α-Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well-vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA-producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA-producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.

Prevention of P2 Receptor-Dependent Thrombocyte Activation by Pore-Forming Bacterial Toxins Improves Outcome in A Murine Model of Urosepsis.

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.

P2X1 receptor blockers reduce the number of circulating thrombocytes and the overall survival of urosepsis with haemolysin-producing Escherichia coli.

Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially bacteraemia. The number of sepsis cases has been steadily increasing over the last decades, and there are still no specific, molecular supportive therapies for sepsis to supplement antibiotic treatment. P2X1 receptors are expressed by a number of immune cells including thrombocytes, which presently have been established as an important player in the acute immune response to bacterial infections. P2X1 receptor-deficient mice have been shown to be relatively protected against urosepsis, with markedly reduced levels of circulating proinflammatory cytokines and intravascular coagulation. However, here we show that continuous intravenous infusion with P2X1 receptor antagonist markedly accelerates development of a septic response to induced bacteraemia with uropathogenic E. coli. Mice exposed to the P2X1 receptor antagonists die very early with haematuria, substantially elevated plasma levels of proinflammatory cytokines, massive intravascular coagulation and a concomitant reduction in circulating thrombocytes. Interestingly, infusion of P2X1 receptor antagonists causes a marked acute reduction in circulating thrombocytes and a higher number of bacteria in the blood. These data support the notion that the number of functional thrombocytes is important for the acute defence against bacteria in the circulation and that the P2X1 receptor potentially could be essential for this response.

Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response.

Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.

Erythrocyte P2X1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis.

BACKGROUND: Pore-forming proteins released from bacteria or formed as result of complement activation are known to produce severe cell damage. Inhibition of purinergic P2X receptors markedly reduces damage inflicted by cytolytic bacterial toxin and after complement activation in both erythrocytes and monocytes. P2X expression generally shows variation throughout the population. Here, we investigate correlation between P2X receptor abundance in blood cell plasma membranes and haematocrit during sepsis, in patients admitted to the emergency department (ED) or intensive care unit (ICU). METHOD: Patients admitted to the ED and successively transferred to ICU with the diagnosis sepsis (< 2 systemic inflammatory response syndrome (SIRS) criteria and suspected infection), were grouped as either blood pathogen-positive (14 patients) or blood pathogen-negative (20 patients). Blood samples drawn at ICU admission were analysed for P2X1 and P2X7 receptor abundance using indirect flow cytometry. RESULTS: Here, we find inverse correlation between P2X1 receptor expression and change in haematocrit (rs - 0.80) and haemoglobin (rs - 0.78) levels from admission to ED to arrival at ICU in patients with pathogen-positive sepsis. This correlation was not found in patients without confirmed bacteraemia. Patients with high P2X1 expression had a significantly greater change in both haematocrit (- 0.59 ± 0.36) and haemoglobin levels (- 0.182 ± 0.038 mg/dl) per hour, during the first hours after hospital admission compared to patients with low P2X1 expression (0.007 ± 0.182 and - 0.020 ± 0.058 mg/dl, respectively). CONCLUSION: High levels of P2X1 are correlated with more pronounced reduction in haematocrit and haemoglobin in patients with confirmed bacteraemia. This supports previous in vitro findings of P2X activation as a significant component in cell damage caused by pore-forming bacterial toxins and complement-dependent major attack complex. These data suggest a new potential target for future therapeutics in initial stages of sepsis.

Loop Diuretics Diminish Hemolysis Induced by α-Hemolysin from Escherichia coli.

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca2+-influx and activation of Ca2+-sensitive K+ and Cl- channels (KCa3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1-/-) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1-/- mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.

[Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia.

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca(2+)]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca(2+)]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca(2+)]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca(2+)]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca(2+)]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca(2+)]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli.

α-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular Ca2+ concentration ([Ca2+]i) in THP-1 monocytes. The HlyA- and LtxA-induced [Ca2+]i response is diminished by the P2 receptor antagonist in a pattern that fits the functional P2 receptor expression in these cells. Both toxins are capable of lysing THP-1 cells, with LtxA being more aggressive. Either desensitization or blockage of P2X1, P2X4, or P2X7 receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X7 receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to pore-forming cytolysins during infection or injury.

Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice.

The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the ß2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, ß2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.

Primary cilium-dependent sensing of urinary flow and paracrine purinergic signaling.

During the last 10 years or so, the renal research community has set the primary cilium into the lime light. From being viewed as a possible evolutionary rudiment, today the primary cilium has achieved the noble status of a physiologically relevant and necessary cellular structure. Its prime function in renal epithelium appears to be its ability to sense urinary flow. Much is still lacking to understand how the primary cilium senses flow. Transducer proteins, such as specific mechano-sensory ion channels, have been identified and are necessary for flow-dependent increases of epithelial [Ca(2+)](i). Other ciliary receptor proteins have been suggested, which may open the field of primary cilia sensing to become an even more dynamic topic of research. A flow-induced increase of [Ca(2+)](i) has been observed in all renal and other ciliated epithelial cells. Work over the last 5 years has addressed the mechanism underlying the flow-induced increase of [Ca(2+)](i). It has become apparent that an initial Ca(2+) influx triggers a global increase of epithelial [Ca(2+)](i). Eventually, it also became clear that mechanical stimulation of the epithelial cells triggers the release of ATP. Intriguingly, ATP is an auto- and paracrine signaling molecule that regulates electrolyte and water transport in the nephron by binding to apical and basolateral purinergic receptors. ATP inhibits transport at almost all sites from the proximal to the distal tubule and thus elicits a diuretic response. In the perspective of this review, the primary cilium is a sensory structure and the adequate stimulus is the mechanical deflection. The output signal is the released ATP, a paracrine factor that ultimately modulates the main function of the kidney, i.e. the enormous task of absorbing some 180 L of filtrate every day.

The primary cilium as sensor of fluid flow: new building blocks to the model. A review in the theme: cell signaling: proteins, pathways and mechanisms.

The primary cilium is an extraordinary organelle. For many years, it had the full attention of only a few dedicated scientists fascinated by its uniqueness. Unexpectedly, after decades of obscurity, it has moved very quickly into the limelight with the increasing evidence of its central role in the many genetic variations that lead to what are now known as ciliopathies. These studies implicated unique biological functions of the primary cilium, which are not completely straightforward. In parallel, and initially completely unrelated to the ciliopathies, the primary cilium was characterized functionally as an organelle that makes cells more susceptible to changes in fluid flow. Thus the primary cilium was suggested to function as a flow-sensing device. This characterization has been substantiated for many epithelial cell types over the years. Nevertheless, part of the central mechanism of signal transduction has not been explained, largely because of the substantial technical challenges of working with this delicate organelle. The current review considers the recent advances that allow us to fill some of the holes in the model of signal transduction in cilium-mediated responses to fluid flow and to pursue the physiological implications of this peculiar organelle.

Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

Furosemide-induced urinary acidification is caused by pronounced H+ secretion in the thick ascending limb.

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 µM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 µM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.

Sialic acid residues are essential for cell lysis mediated by leukotoxin from Aggregatibacter actinomycetemcomitans.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

Effects of high hydrostatic pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos.

Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.