Regulation of TNF-α by 1α,25-dihydroxyvitamin D3 in human macrophages from CAPD patients

Regulation of TNF-α by 1α,25-dihydroxyvitamin D3 in human macrophages from CAPD patients.BackgroundWe have previously reported that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] accumulates in the dialysis fluid of uremic patients treated by continuous ambulatory peritoneal dialysis (CAPD). It has been reported that this metabolite regulates the production of cytokines by monocytes/macrophages. Since tumor necrosis factor-α (TNF-α) initiates an inflammatory cascade during peritonitis, the aim of the present study was to investigate the effect of 1α,25(OH)2D3 on the production of TNF-α by human peritoneal macrophages (HPMs).MethodsHPMs were obtained from patients on CAPD. Cells were incubated with various concentrations of 1α,25(OH)2D3, 1α,24(S) dihydroxyvitamin D2 [1α,24(S)(OH)2D2] or 25-hydroxyvitamin D3 (25-OH-D3) for 16 hours. This was followed by lipopolysaccharide (LPS; 1 μg/mL) incubation for 2.5 to 6 hours. TNF-α protein production was determined by enzyme-linked immunosorbent assay. TNF-α mRNA was assayed by the reverse transcriptase-polymerase chain reaction procedure, using internal synthetic mRNA standards for quantitative results.ResultsIncubation of HPMs with 1α,25(OH)2D3 prior to stimulation with LPS dose dependently inhibited the expression of TNF-α on both mRNA and protein levels. Similar results were obtained with the less calcemic vitamin D2 analogue 1α,24(S)(OH)2D2. Incubation of HPMs with 25-OH-D3 also revealed a down-regulation of TNF-α expression. Since this down-regulatory effect was blocked by ketoconazole, it is likely that this effect was caused by the conversion of 25-OH-D3 into 1α,25(OH)2D3 by HPMs.Conclusions1α,25(OH)2D3 has a potent inhibitory effect on the production of TNF-α by LPS-activated HPMs. We hypothesize that 1α,25(OH)2D3 may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory response of the peritoneum, will moderate tissue damage during peritonitis

Renal cortical mitochondrial transport of calcium in chronic uremia

Renal cortical mitochondrial transport of calcium in chronic uremia. Calcium overload of tubular cells may occur in uremia, and may be the underlying functional abnormality in the continued deterioration of renal function in chronic renal failure. In order to study this question further, the effect of chronic uremia on the calcium transport properties and respiratory rates was examined in mitochondria (Mi) isolated from the cortex of the remnant kidneys of subtotally nephrectomized rats (SNX) and sham operated controls (C). Plasma calcium concentration was similar in both groups of rats, but a significant hyperphosphatemia was seen in SNX, 8.6 ± 0.6 mg%, as compared to 7.2 ± 0.2 mg% in C (P < 0.001). Mi calcium and phosphate concentrations (nmol/mg protein) were significantly elevated in SNX, 49.9 ± 7.9 and 35.1 ± 4.2, respectively, in SNX compared to C, 21.2 ± 4.2 and 21.4 ± 2.7, respectively (P < 0.01). Mi respiratory control ratio and ADP/O were similar in both experimental groups. Kinetic parameters for calcium uptake (Ca2+ concentrations in the medium of 1.25 to 16 µM) revealed initial velocities 1.5-fold higher in SNX Mi than in C. Mi retention of calcium in the presence of medium Ca2+ concentrations up to 500 µM was studied. Calcium retention was reduced in SNX: the Mi were unable to retain calcium at concentrations of 250 µM. The addition of ruthenium red to the medium substantially improved calcium retention by the uremic Mi. Chronic parathyroidectomy did not correct either the increased calcium uptake or the poor retention of uremic Mi. In conclusion: 1) Chronic uremia in rats is associated with a marked alteration in calcium transport of renal cortical Mi, which may result in Mi calcium overload. 2) The enhanced calcium uptake and poor calcium retention of uremic Mi is not PTH dependent. 3) Since Pi is known to cause a higher initial velocity of calcium uptake and to impair calcium retention of normal mitochondria, it seems that cellular accumulation of phosphate in chronic uremia may be involved in this abnormality

Acute phosphate depletion and in vitro rat proximal tubule injury: Protection by glycine and acidosis

Acute phosphate depletion and in vitro rat proximal tubule injury: Protection by glycine and acidosis.The effects of phosphate (PO4) removal from Krebs Henseleit buffer on freshly isolated rat proximal tubules (rPT) were assessed by measuring Ca2+ uptake (nmol/mg protein), cellular adenosine triphosphate (ATP) (nmol/mg), tissue K+ content (nmol/mg) and lactate dehydrogenase (LDH) as an index of cell integrity. Ca2+ uptake increased by 50% in rPT incubated in zero PO4 medium as compared to control (2.6 ±0.1 vs. 3.9 ±0.19, P < 0.001) and LDH release increased 2.5-fold from 14.2 ±0.6 to 31.6 ±1.6%, P < 0.001. Neither verapamil (200 µM) nor mepacrine (50 µM) reduced Ca2+ uptake or decreased LDH release suggesting that the increased Ca2+ uptake was not occurring through potential operated channels and that phospholipase-induced cell injury was not the cause of increased LDH release. Either glycine (2 mM) or extracellular fluid acidosis (pH 7.06), however, significantly diminished rPT injury and Ca2+ uptake. Specifically, as compared to the increased LDH released in untreated, PO4-depleted rPT, LDH release was diminished significantly by glycine treatment (31.0 ±0.9 vs. 15.5 ±1.6%, P < 0.001) or acidosis (30.3 ±0.04 vs. 19.2 ±0.9%, P < 0.01). Ca2+ uptake did not increase in glycine treated tubules (2.6 ±0.1 vs. 2.8 ±0.2 nmol/mg, NS) or in the presence of acidosis (2.6 ±0.1 vs. 2.97 ±0.17 nmol/mg, NS). ATP concentrations were markedly reduced by PO4 depletion (2.8±0.2 vs. 4.8±0.3 nmol/mg, P < 0.001) and remained at low levels during either acidosis or glycine-induced protection. ATP depletion was accompanied by loss of K+ from rPT and this was only modestly attenuated by either glycine or acidosis. Total cell PO4 was not significantly altered, however, perchloric acid (PCA) extractable free PO4 was reduced significantly (33.3 ±4.5 to 15.9 ±3.5 nmol/mg, P < 0.01). The rPT injury, associated with acute PO4 depletion, may be related to Ca2+ uptake since Ca2+ uptake and LDH release were both attenuated by glycine administration or acidosis

Effect of A₁R agonist on A₁R and A_2AR levels <i>in vivo.</i>

<p>Mice were administered i.p. with the A₁R agonist (CHA, 0.1 mg/kg) or with vehicle. PMC were scraped from the peritoneal surface and analyzed for (A) A_2AR and A₁R mRNA levels at 4 hours or (B+C) A_2AR and A₁R protein levels at 24 hours. (B) Densitometry of protein blot depicted in (C). A₁R and A_2AR mRNA levels were normalized to GAPDH and protein levels were normalized to β-actin. Results are presented as fold change from vehicle-treated animals. Data represent three experiments and are expressed as mean±SEM. * <i>p</i><0.05 between conditions per receptor, <i>n</i> = 4 for each experiment.</p

Effect of adenosine receptor subtype autoregulation on the inflammatory process.

<p>(A) Early expression of A₁R after bacterial inoculation decreases cAMP levels, enhances production of local pro-inflammatory cytokines and promotes leukocyte migration. (B) In a later phase of peritonitis A_2AR expression increase by A₁R which leads to increase in cAMP levels. High cAMP markedly decreases local pro-inflammatory cytokines and leukocyte recruitment, hence restraining inflammatory flames.</p

The anti-inflammatory effect of pretreatment with the A₁R agonist.

<p>Mice were treated with the A₁R agonist (CHA, i.p., 0.1 mg/kg) or vehicle 24 hours prior to bacterial inoculation. (A) Sera levels of IL-6 and TNFα at 12 hours. (B) Chemokine mRNA levels. 12 hours after inoculation PMC were scraped from the peritoneal membrane and total RNA was extracted, analyzed for MCP-1 and MIP-2 mRNA levels and normalized to β-actin. (C) Total cell count at 24 hours after inoculation. Cell exudates were collected from peritoneal lavage fluid. Data represent five experiments and are expressed as mean±SEM for serum cytokine levels and as mean±SEM fold of control for chemokine mRNA levels.* <i>p</i><0.05, ** <i>p</i><0.01, <i>n</i> = 5 for each experiment.</p

Effect of adenosine on A_2AR and A₁R levels <i>in vitro</i>.

<p>To simulate the gradual increase of adenosine that occurs during peritonitis, cultured primary PMC were treated with multiple and increasing concentrations of adenosine (0.1, 1 and 10 µM at 3 hour intervals). Total RNA was extracted after 9 hours and analyzed for A₁R and A_2AR mRNA levels. Results are normalized to β-actin. Data represent five experiments and are expressed as mean±SEM fold of control. * <i>p</i><0.05, ** <i>p</i><0.01 between expression levels of each receptor to expression at time 0, <i>n</i> = 3 for each experiment.</p