Cell-Free DNA in Plasma and Serum Indicates Disease Severity and Prognosis in Blunt Trauma Patients.

Background: Trauma is still a major cause of mortality in people < 50 years of age. Biomarkers are needed to estimate the severity of the condition and the patient outcome. Methods: Cell-free DNA (cfDNA) and further laboratory markers were determined in plasma and serum of 164 patients at time of admission to the emergency room. Among them were 64 patients with severe trauma (Injury Severity Score (ISS) ≥ 16), 51 patients with moderate trauma (ISS < 16) and 49 patients with single fractures (24 femur neck and 25 ankle fractures). Disease severity was objectified by ISS and Glasgow Coma Scale (GCS). Results: cfDNA levels in plasma and serum were significantly higher in patients with severe multiple trauma (SMT) than in those with moderate trauma (p = 0.002, p = 0.003, respectively) or with single fractures (each p < 0.001). CfDNA in plasma and serum correlated very strongly with each other (R = 0.91; p < 0.001). The AUC in ROC curves for identification of SMT patients was 0.76 and 0.74 for cfDNA in plasma and serum, respectively-this was further increased to 0.84 by the combination of cfDNA and hemoglobin. Within the group of multiple trauma patients, cfDNA levels were significantly higher in more severely injured patients and patients with severe traumatic brain injury (GCS ≤ 8 versus GCS > 8). Thirteen (20.3%) of the multiple trauma patients died during the first week after trauma. Levels of cfDNA were significantly higher in non-surviving patients than in survivors (p < 0.001), reaching an AUC of 0.81 for cfDNA in both, plasma and serum, which was further increased by the combination with hemoglobin and leukocytes. Conclusions: cfDNA is valuable for estimation of trauma severity and prognosis of trauma patients.

Effects of neuropeptides and vasoactive substances on microcirculation of the callus after tibial osteotomy in rabbits.

Previous studies have demonstrated a dynamic ingrowth of vessels into the developing callus. In this study, maturation and development of the regulation of microcirculation were followed in the callus of rabbits. In the first series, the effects of vasoactive substances on blood flow velocity, perfusion pressure, duration of effects and peripheral vascular resistance of the bone marrow in the femur and tibia were compared. In the second series, the same parameters were measured in the femur and in the developing callus 10 and 15 days following gap osteotomy of the tibia. There were no significant differences between the microcirculatory reactions of the intact femur and tibia. Basal blood flow could be verified in the callus on the 10th postoperative day. No vascular reactions could be elicited. Basal blood flow velocity was higher on the 15th day, when compared to the measurements on the 10th day. The substances elicited statistically significant differences in flow velocity, resistance and 50% recovery time in the callus on the 15th day. Blood flow reactions of the ipsilateral femoral and tibial bone marrow are identical, thus the femur can serve as a reference site for blood flow measurements in the callus. Regulation and maturation of callus microcirculation develop rapidly between the 10th and 15th days.

High osmolality induces the kidney-specific chloride channel CLC-K1 by a serum and glucocorticoid-inducible kinase 1 MAPK pathway.

The kidney-specific chloride channels CLC-K1/2 and their functionally important subunit barttin, by mediating solute transport in medulla, contribute to the osmotic gradient. We sought to determine whether they themselves are regulated by variations of osmolality. The expression of CLC-K1 and barttin mRNA and protein was significantly increased in a distal convoluted tubule cell line after a shift to high osmolar medium. This upregulation paralleled that of serum and glucocorticoid-inducible kinase 1 (SGK1), a gene known to be upregulated by cell shrinkage. Specific knockdown of SGK1 or addition of the p38 MAPK pathway inhibitor SB203580 abolished the induction of SGK1, CLC-K1 and barttin by high osmolarity suggesting that a functional MAPK pathway is required to mediate osmotic-driven induction of all three genes. The physiological relevance of our in vitro data was confirmed by water deprivation of male C57BL6 mice, which caused a significant increase in serum osmolality along with induction of CLC-K1, barttin and SGK1. Our study shows that change in intracellular volume, because of high osmolality, result in SGK1 upregulation and the subsequent increase of CLC-K1/barttin expression in distal renal tubular cells in vivo and in vitro.

Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension.

OBJECTIVE: The regulation of pulmonary prostacyclin synthesis is not completely understood. We tested the hypothesis that prostacyclin production is predominantly stimulated by hemodynamic factors, such as increased shear-stress, and is thus increased in rats with chronic hypoxic pulmonary hypertension. METHODS: To this end, we determined pulmonary prostacyclin synthase (PGIS) gene expression, circulating levels of the stable prostacyclin metabolite 6-keto prostaglandin F(1alpha) (6-keto-PGF(1alpha)), pulmonary endothelin (ET)-1 gene expression, and ET-1 plasma levels in rats exposed to 4 weeks of hypoxia (10% O(2)) in the presence or absence of either the nitric oxide (NO) donor molsidomine (MD, 15 mg/kg/day) or the ET-A receptor antagonist LU135252 (LU, 50 mg/kg/day). RESULTS: Right ventricular systolic pressure (RVSP), the cross-sectional medial vascular wall area of pulmonary arteries, and ET-1 production increased significantly during hypoxia. PGIS mRNA levels increased 1.7-fold, and 6-keto-PGF(1alpha) plasma levels rose from 8.2+/-0.8 to 12.2+/-2.2 ng/ml during hypoxia (each P<0.05 vs. normoxic controls). MD and LU reduced RVSP and pulmonary vascular remodeling similarly (each P<0.05 vs. hypoxia), but only MD inhibited pulmonary ET-1 formation (P<0.05 vs. hypoxia). Nevertheless, both drugs attenuated the increase in PGIS gene expression and plasma 6-keto-PGF(1alpha) levels (each P<0.05 vs. hypoxia). CONCLUSION: Our data suggest that prostacyclin production in hypertensive rat lungs is predominantly increased by hemodynamic factors while hypoxia, NO and ET-1 per are less important stimuli, and that this increase may serve as a compensatory mechanism to partially negate the hypoxia-induced elevation in pulmonary vascular tone.

Activation of bronchial epithelial cells in smokers without airway obstruction and patients with COPD.

STUDY OBJECTIVE: The aim of this study was to investigate the basal level as well as the tumor necrosis factor (TNF)-alpha- and interferon (IFN)-gamma-induced expression and release of the neutrophil chemoattractants interleukin (IL)-8 and growth-related oncogene (GRO)-alpha in primary bronchial epithelial cells (PBECs) from smokers without airflow obstruction and patients with COPD. In addition, the expression of both TNF-alpha-receptor subtypes--p55 TNF-receptor subtype (TNF-R55) and p75 TNF-receptor subtype (TNF-R75)--was quantified in PBECs. DESIGN: PBECs from eight smokers without airflow limitation and eight patients with COPD were stimulated with 50 ng/mL of TNF and 200 U/mL of IFN-gamma for 4 h along with unstimulated time controls. The transcriptional expression and protein release were quantitatively assessed by means of real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Basal level messenger RNA (mRNA) expression and protein release of IL-8 and GRO-alpha were not significantly different between both groups, although a trend toward higher IL-8 levels was seen in patients with COPD. TNF-alpha induced significantly higher mRNA amounts of IL-8 (p = 0.005) and GRO-alpha (p = 0.007) in patients with COPD. This was accompanied by higher protein release data for IL-8 (p = 0.005) and GRO-alpha (p = 0.007). IFN-gamma had no significant effect on the mRNA expression and protein release of IL-8 and GRO-alpha in either group. TNF-R55 and TNF-R75 were detectable in PBECs. However, no significant differences were found between both groups with respect to steady-state mRNA levels of TNF-alpha-receptor subtypes. CONCLUSION: PBECs from patients with COPD show significantly higher TNF-alpha-induced release of the neutrophil chemoattractant CXC-chemokines IL-8 and GRO-alpha compared to smokers without airflow limitation. This increased activation of PBECs may contribute to the predominance of neutrophils seen in the airway lumen of patients with COPD.

Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs.

We tested the hypothesis that pulmonary endothelial nitric oxide synthase (eNOS) gene expression is primarily regulated by hemodynamic factors and is thus increased in rats with chronic hypoxic pulmonary hypertension. Furthermore, we examined the role of endothelin (ET)-1 in this regulatory process, since ET-1 is able to induce eNOS via activation of the ET-B receptor. Therefore, chronic hypoxic rats (10% O(2)) were treated with the selective ET-A receptor antagonist LU-135252 (50 mg x kg(-1) x day(-1)). Right ventricular systolic pressure and cross-sectional medial vascular wall area of pulmonary arteries rose significantly, and eNOS mRNA levels increased 1.8- and 2.6-fold after 2 and 4 wk of hypoxia, respectively (each P < 0.05). Pulmonary ET-1 mRNA and ET-1 plasma levels increased significantly after 4 wk of hypoxia (each P < 0.05). LU-135252 reduced right ventricular systolic pressure, vascular remodeling, and eNOS gene expression in chronic hypoxic rats (each P < 0.05), whereas ET-1 production was not altered. We conclude that eNOS expression in chronic hypoxic rat lungs is modified predominantly by hemodynamic factors, whereas the ET-B receptor-mediated pathway and hypoxia seem to be less important.

Adrenal aldosterone biosynthesis is elevated in a model of chronic renal failure--role of local adrenal renin-angiotensin system.

BACKGROUND: Aldosterone seems to play a role in the development of chronic renal failure and proteinuria. We investigated the adrenal aldosterone production and the adrenal renin-angiotensin system (RAS) in rats with 5/6 nephrectomy with and without spironolactone treatment. METHODS: Sprague-Dawley rats underwent 5/6, 4/6 nephrectomy, heminephrectomy and sham operation. After 1 and 4 weeks creatinine clearance, urinary protein excretion, plasma aldosterone concentration, and plasma renin activity were measured. In adrenals mRNA expression of aldosterone synthase (CYP11B2) and genes of the RAS were measured. RESULTS: Creatinine clearance was significantly decreased and proteinuria significantly elevated in 5/6 nephrectomy. Treatment with spironolactone significantly reduced proteinuria after 8 but not after 30 days. With reduction of renal mass, renal renin mRNA and plasma renin activity were reduced significantly. In early 5/6 nephrectomy plasma aldosterone concentration was increased and in parallel adrenal CYP11B2 mRNA was increased significantly. Both were further augmented by spironolactone. Adrenal renin was up-regulated in 5/6 nephrectomy and further stimulated with spironolactone, possibly serving as a stimulus for the adrenal aldosterone synthesis. CONCLUSION: In early chronic renal failure after 5/6 nephrectomy adrenal aldosterone production is elevated despite a marked decrease of plasma renin activity. An up-regulated adrenal RAS may contribute to the observed increase in aldosterone.

Effects of vasoactive substances on the neurovascular structures and microcirculation in the developing callus 10 and 15 days after bone injury.

The developing callus requires sufficient oxygen and substrate supply. Despite the importance of these processes, we have limited understanding of regulation of the callus microcirculation. We aimed to assess the role of vasoactive substances in the microcirculation of the callus in a gap osteotomy model in the rabbit detected by laser-Doppler flowmetry. The reactions were elicited with locally applied vasoactive substances: epinephrine (E), calcitonine-gene related protein (CGRP), substance P (SP), sodium nitroprusside (SNP) and Ebrantil (Ebr) on the 10th and 15th postoperative days. Changes of the circulatory parameters were compared to changes in the ipsilateral femoral bone marrow. Perfusion pressure, maximal change of the blood flow and 50% recovery time (50RT) of the flow reactions and peripheral micro vascular resistance (MVR) was calculated. Systemic blood pressure (BP) was measured directly with an arterial catheter. Reactive neurovascular structures, sensitive to neuropeptides and vasoactive substances, appear at a very early stage of callus formation. On the 10th postoperative day, 2/3 of the blood flow velocity of the intact tibia has already returned, and the values are higher on the 15th postoperative day than those of the intact tibia. The basal blood flow velocities (prior to administration of any substance) are significantly higher on the 15th postoperative day than on the 10th.

Calcitonin gene-related peptide, substance P, nitric oxide and epinephrine modulate bone marrow micro circulation of the rabbit tibia and femur.

Different bones have different blood supplies, which may influence bone healing. Therefore, elucidation of the mechanisms involved in the regulation of bone marrow blood flow in different bones is of high clinical importance. To assess the micro circulation of bone marrow of the femur and tibia simultaneously, flow velocities were continuously measured by a two-channel laser-Doppler flowmeter. The probes were introduced into the femoral and tibial diaphysis, respectively, in the anesthetized rabbit. Changes in micro circulation of the bone marrow were elicited by intra-arterial bolus injections of vasoactive substances: epinephrine (E), calcitonine-gene related peptide (CGRP), substance P (SP), sodium nitroprusside (SNP), E and Ebrantil. Systemic arterial blood pressure was recorded with an electro-manometer. Micro vascular resistance (MVR) and 50% recovery time (50RT) to baseline flow level were calculated from the measured data. Flow velocity in the femur was significantly higher. Epinephrine considerably reduced micro vascular blood flow, which could be significantly warded off by Ebrantil. CGRP and SP did not change MVR. Application of SNP resulted in reduction of flow velocity, but it also decreased MVR. No statistically significant differences were found between reactions of the micro circulation in the two marrows. These results suggest that there are no significant differences between the blood flow response patterns of these two bone marrow sites, thus the regulation patterns of the micro circulation of the two bones are also similar.

Gene expression of 5-, 12-, and 15-lipoxygenases and leukotriene receptors along the rat nephron.

The arachidonate signaling pathways comprise prostanoids formed by cyclooxygenases, EETs, and HETEs formed by cytochrome P-450 (CYP) enzymes and HETEs and leukotrienes generated by lipoxygenases. Whereas the intrarenal localization of cyclooxygenases and of some CYP enzymes along the nephron has already been determined, the localization of lipoxygenases and leukotriene-forming enzymes together with leukotriene receptors in the kidney is less clear. This study therefore aimed to determine the expression of 5-, 12-, and 15-lipoxygenases as well as the leukotriene receptors along the rat nephron. The kidneys were dissected into cortex and outer and inner medulla, and the microdissected nephron segments were collected after a collagenase digestion. mRNA abundance was determined by RT-PCR and real-time PCR. 15-LOX mRNA showed a characteristic expression pattern along the distal nephron. 12-LOX mRNA was only found in the glomerulus. Similarly, 5-LOX mRNAs together with 5-LOX-activating protein mRNAs were expressed in the glomerulus and also in the vasa recta. The leukotriene A4 hydrolase was found in all nephron segments, whereas leukotriene C4 synthase mRNA could not be found in any nephron segment. The leukotriene receptor B4 and the cysteinyl leukotriene receptor type 1 were selectively expressed in the glomerulus, whereas cysteinyl receptor type 2 was not found in any nephron segment. Our data suggest that the glomerulus is a major source and target for 5- and 12-HETE and for leukotrienes. The collecting duct system, on the other hand, appears to be a major source of 15-HETE.

Role of renocortical cyclooxygenase-2 for renal vascular resistance and macula densa control of renin secretion.

This study aimed to assess the role of cyclooxygenase-2 (COX-2)-derived prostanoids for the macula densa control of renal afferent arteriolar resistance and for renin secretion. For this purpose, studied were the effects of blocking macula densa salt transport by the loop diuretic bumetanide (100 microM) on renal perfusate flow and on renin secretion in isolated perfused rats, in which renocortical COX-2 expression was prestimulated in vivo by treatment with the angiotensin-converting enzyme inhibitor ramipril, with low-salt diet, or with a combination of both. These maneuvers stimulated COX-2 expression in an order of ramipril + low salt>> low salt > ramipril > controls. Flow rates through isolated kidneys at a constant pressure of 100 mmHg were dependent on the pretreatment regimen, in the way that they went in parallel with COX-2 expression. The COX-2 inhibitor NS-398 (10 microM) lowered flow rates depending on the COX-2 expression level and was most pronounced therefore after pretreatment with low salt + ramipril. NS-398 did not change the increase of flow in response to bumetanide but attenuated the stimulation of renin secretion in response to bumetanide in a manner depending on the expression level of COX-2. These findings suggest that in states of increased renocortical expression of COX-2, overall renal vascular resistance and the macula densa control of renin secretion become dependent on COX-2-derived prostanoids.

Expression and release of interleukin-8 by human bronchial epithelial cells from patients with chronic obstructive pulmonary disease, smokers, and never-smokers.

BACKGROUND: One of the consistently observed features in chronic obstructive pulmonary disease (COPD) are markedly increased neutrophils in the airways which are accompanied by increased levels of interleukin-8 (IL-8) in induced sputum and bronchoalveolar lavage fluid. To some extent, IL-8 may derive from bronchial epithelial cells since airway epithelium plays a crucial role in initiating and augmenting host defense mechanisms. OBJECTIVES: We hypothesized that a marked increase in bronchoepithelial IL-8 expression and release may be found in the airway epithelium of COPD patients compared to 'healthy' smokers and never-smokers. METHODS: Primary bronchoepithelial cell cultures from COPD patients, smokers, and never-smokers were established. The unstimulated and TNFalpha-induced IL-8 release was measured by enzyme-linked immunosorbent assay. In addition, mRNA expression levels were quantified by means of reverse transcriptase polymerase chain reaction and light cycler measurements. RESULTS: In subjects with COPD the constitutive and stimulated IL-8 release was significantly higher compared to 'healthy' smokers and control subjects, whereas no differences were seen between smokers and the control group. Quantitative assessment of transcript levels confirmed these data, displaying significantly higher mRNA levels in primary bronchial epithelial cells from COPD patients compared to controls (p < 0.05) in uninduced and stimulated conditions (p < 0.05). CONCLUSIONS: These results suggest that patients with chronic obstructive airflow limitation are characterized by a significant upregulation of bronchial epithelial IL-8 expression levels and secretion, indicating specific differences in epithelial cell activation in COPD patients compared to smokers and control subjects.

Nephron specific regulation of chloride channel CLC-K2 mRNA in the rat.

BACKGROUND: This study investigated the influence of salt intake on the nephron specific gene expression of the kidney chloride channel CLC-K2. To this end, male Sprague-Dawley rats were fed a low (0.02% wt/wt), normal (0.6% wt/wt), or high salt (8% wt/wt) diet for ten days, or they received the loop diuretic furosemide (12 mg/kg/day) for six days. METHODS: Expression and regulation of messenger RNA for CLC-K2 was demonstrated by RNase protection assay and in situ hybridization in kidney cortex, outer medulla and inner medulla. Tubular localization and regulation were determined precisely by reverse transcription-polymerase chain reaction (RT-PCR) and real time PCR of microdissected nephron segments. RESULTS: In situ hybridization analysis and RNase protection assay of the total kidney revealed a down-regulation of CLC-K2 mRNA in the high salt diet rats and an up-regulation of CLC-K2 mRNA in furosemide treated rats, which were restricted to the outer medulla. Microdissection of collagenase treated kidney revealed CLC-K2 mRNA expression in the outer medullary thick ascending limb (mTAL), cortical thick ascending limb (cTAL), distal convoluted tubule (DCT), connecting tubule and cortical collecting duct (CNT/CCD), and outer medullary collecting duct (OMCD), whereas no signals were detected in proximal convoluted and straight tubules (PCT and PST), descending thin limb from the outer medulla (dTL), descending and ascending thin limb from the inner medulla (TL), inner medullary collecting duct (IMCD) and glomeruli (glom). Using RT-PCR and real time PCR, the changing levels of CLC-K2 mRNA after furosemide treatment or high salt diet were restricted to the mTAL, whereas CLC-K2 mRNA levels in cTAL and OMCD were not changed in furosemide or high salt rats compared to time paired controls. CONCLUSIONS: Given that CLC-K2 expressed in the thick ascending limb of Henle's loop is responsible for net chloride reabsorption in this part of the nephron, our findings suggest that in states of surplus salt and in states of severe salt deprivation, selective regulation of CLC-K2 mRNA plays a role in the adaptation of the kidney to different salt loads.

Parallel down-regulation of chloride channel CLC-K1 and barttin mRNA in the thin ascending limb of the rat nephron by furosemide.

In the past few years the pivotal role of kidney Cl(-)channels (ClC-K) channels in maintaining salt and water homeostasis in the kidney has been established. The aim of the present study was to investigate the influence of the loop diuretic furosemide on the gene expression of the kidney chloride channel ClC-K1 and its recently described functional subunit barttin. Male Sprague Dawley rats received the loop diuretic furosemide (12 mg/kg/day) for 6 days. Rats had free access to 0.9% NaCl, 0.1%KCl solution to prevent volume depletion. Localisation and regulation of ClC-K1 and barttin mRNA was analysed by RNase protection and in situ hybridisation. Nephron-specific regulation was investigated by microdissection and real-time PCR quantification. In furosemide-treated rats ClC-K1 mRNA decreased to half in the inner medulla. In the renal cortex and outer medulla ClC-K1 mRNA levels were weak and did not change. Under furosemide treatment barttin mRNA was regulated in parallel with ClC-K1 mRNA. A significant mRNA decrease occurred after furosemide treatment in inner medulla (0.50 fold), whereas cortical and outer medulla levels remained unaffected. (35)S in situ hybridisation confirmed the regulation and distribution seen in the RNase protection assay experiments. Microdissection of the inner medullary collecting duct and thin limb of Henle's loop followed by real-time PCR revealed that CLC-K1 and barttin mRNA regulation in inner medulla was limited to the thin limb; mRNA levels in collecting ducts were not affected by furosemide treatment. Our findings imply that during furosemide treatment selective down-regulation of ClC-K1 and barttin mRNAs in thin limb plays a role in maintaining salt and water homeostasis.

Barttin increases surface expression and changes current properties of ClC-K channels.

The term Bartter syndrome encompasses a heterogeneous group of autosomal recessive salt-losing nephropathies that are caused by disturbed transepithelial sodium chloride reabsorption in the distal nephron. Mutations have been identified in the NKCC2 (Na(+)-K(+)-2Cl(-)) cotransporter and ROMK potassium channel, which cooperate in the process of apical sodium chloride uptake, and ClC-Kb chloride channels, which mediate basolateral chloride release. Recently, mutations in barttin, a protein not related to any known ion transporter or channel, were described in BSND, a variant of Bartter syndrome associated with sensorineural deafness. Here we show that barttin functions as an activator of ClC-K chloride channels. Expression of barttin together with ClC-K in Xenopus oocytes increased ClC-K current amplitude, changed ClC-K biophysical properties, and enhanced ClC-K abundance in the cell membrane. Co-immunoprecipitation revealed a direct interaction of barttin with ClC-K. We performed in situ hybridization on rat kidney slices and RT-PCR analysis on microdissected nephron segments to prove co-expression of barttin, ClC-K1 and ClC-K2 along the distal nephron. Functional analysis of BSND-associated point mutations revealed impaired ClC-K activation by barttin. The results demonstrate regulation of a CLC chloride channel by an accessory protein and indicate that ClC-K activation by barttin is required for adequate tubular salt reabsorption.