On-Site Laser Photobiomodulation Treatment of Crushed Muscle Due to Prolonged Pressure in Rats.

BACKGROUND AND OBJECTIVES: Crush injuries and prolonged pressure on muscles lead to bruises and sprains and, in most of the cases, cause distraction of the muscle and release of particles into the blood stream, causing renal and systemic complications in severe cases. Laser photobiomodulation treatment (i.e., laser phototherapy) is a method suggested to decrease the pressure damage in the first 24-48 hours after muscle injury, allowing a faster and more complete physical rehabilitation. We studied the efficacy of non-invasive laser photobiomodulation treatment as an on-site treatment for crush-injured gastrocnemius muscles, developing a moderate muscle crush injury model and aiming at decreasing damage extent while regaining physical competence faster. STUDY DESIGN/MATERIALS AND METHODS: Muscle crush injury was performed on 30 female Wistar rats using direct pressure for 10 minutes on the gastrocnemius muscle in both left and right hindlimbs. Immediately after the injury, only the left hindlimb were irradiated for 16 minutes (with 780 nm laser with a power of 250 mW, the energy at the target was 240 J, and the fluence was 1019 J/cm2 ) for 1, 3, or 7 consecutive days, and sacrificed accordingly. During the follow-up period, 1, 3, or 7 days, both gastrocnemius muscles (of the treated and untreated hindlimbs) were evaluated for electrophysiology and functionality. RESULTS: The laser photobiomodulation treatment showed a significant electrophysiological and functional recovery of the gastrocnemius muscle during the first 3 days after injury, in comparison with the untreated hindlimb. CONCLUSIONS: These preliminary results are promising, showing a significant effect of the laser photobiomodulation treatment during the first 3 days after the induction of the muscle crush injury, which is the most critical period in the clinical aspect. These findings suggest a therapeutic approach, which may help restore the muscle after crush injury.

Fucoidin, an inhibitor of leukocyte adhesion, exacerbates acute ischemic renal failure and stimulates nitric oxide synthesis.

OBJECTIVES: To lessen renal ischemic injury caused by fucoidin, a substance capable of reducing tissue infiltration by neutrophils, and to seek a possible interrelationship with the nitric oxide system which may also modulate leukocyte infiltration. MATERIAL AND METHODS: Acute ischemic renal failure was induced in rats by uninephrectomy followed by 60 min of clamping of the renal artery. The rats were injected with fucoidin (25 mg/kg) or fucoidin+nitroprusside (2.5 mg/kg) before reperfusion, and urine was collected for 24 h afterwards. Serum and urine were examined for creatinine sodium and protein; creatinine clearance and fractional excretion of sodium (FENa) were calculated. The renal tissue of the sacrificed animals was examined histologically for tissue damage and histochemically for myeloperoxidase, a marker of neutrophil infiltration. The nitric oxide system was evaluated by measuring urinary nitrates and inducible nitric oxide synthase messenger RNA (iNOs mRNA). RESULTS: Renal failure was more severe in the fucoidin group than the nitroprusside group (creatinine clearance 0.11+/-0.08 ml/min for ischemia+fucoidin versus 0.26+/-0.11 ml/min for ischemia only; p<0.002). Adding nitroprusside to fucoidin lessened the decline in creatinine clearance (0.13+/-0.13 ml/min; p=NS). Fucoidin was associated with greater tubular damage, as evidenced by increased FENa (7.2%+/-2.8% vs 1.51%+/-1.96% for ischemia only; p<0.001). Nitroprusside weakened this trend. Fucoidin caused an increase in the fractional excretion of nitrates, a response accompanied by increased iNOS mRNA. CONCLUSIONS: Fucoidin failed to protect the kidney from ischemic damage and was even nephrotoxic. It also stimulated the formation of iNOS RNA.

Anaemia as a contributor to morbidity and mortality in congestive heart failure.

Anaemia is present in approximately 40% of cases of congestive heart failure (CHF) and is associated with a higher mortality, a lower left ventricular ejection fraction, a lower cardiac functional status, a higher rate of hospitalization, signs of malnutrition, a lower exercise capacity, a progressive fall in renal function, an increased need for high dose diuretics, hyponatraemia, an increased plasma volume, a reduced red cell volume and a lower quality of life. In both uncontrolled and controlled studies, correction of the anaemia with subcutaneous erythropoietin and, in some cases, with the addition of intravenous iron, has been shown to improve these parameters. A vicious circle is present between CHF, chronic kidney insufficiency (CKI) and anaemia, each capable of causing or being caused by the other, the so-called cardio renal syndrome. If larger randomized, controlled, double-blind studies confirm these observations, correction of the anaemia may prove to be a useful addition to the prevention and progression of both CHF and CKI. Cooperation between nephrologists, cardiologists and other internists to identify and treat these anaemic CHF patients early will help prevent progression of both the cardiac and renal disease.

The interaction between heart failure, renal failure and anemia - the cardio-renal anemia syndrome.

BACKGROUND: Many patients with congestive heart failure (CHF) have chronic kidney insufficiency (CKI) and anemia. AIMS: The purpose of this review is to clarify the relationship between these three factors and to study the effect of correction of anemia in CHF and CKI. FINDINGS: Anemia, CHF and CKI are each capable of causing or worsening each other. Thus they form a vicious circle which can result in progressive CHF, CKI and anemia. Aggressive therapy of CHF, CKI and control of the associated anemia with erythropoietin and i.v. iron can prevent the progression of CHF and CKI, reduce hospitalization, and improve quality of life. CONCLUSION: CHF patients are a major source of end-stage renal failure patients and deserve special attention. If treated well and early, progressive heart failure and renal failure can be prevented. Cooperation between nephrologists, cardiologists, and other internists will improve the care of all three conditions and prevent their progression.

Augmented arginine uptake, through modulation of cationic amino acid transporter-1, increases GFR in diabetic rats.

BACKGROUND: It is suggested that either arginine or its metabolites, nitric oxide and polyamines play a role in the renal hemodynamic alterations observed in the early stages of diabetes. Yet, the regulation of arginine transport in diabetic kidneys has never been studied. METHODS: Arginine uptake was determined in glomeruli harvested from control rats; diabetic rats (2 weeks following an intraperitoneal injection of streptozotocin, 60 mg/kg body weight); rats, 4 days following left nephrectomy (a nondiabetic model of hyperfiltration); diabetes + lysine (0.5% in the drinking water to attenuate arginine uptake); and control + lysine. RESULTS: Glomerular arginine transport was significantly increased in diabetic rats, but remained unchanged following uninephrectomy. Lysine abolished the increase in arginine uptake in diabetic rats but had no effect in controls. The increase in creatinine clearance observed in diabetes was completely abolished by lysine. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and immunohistochemistry, we found a significant increase in glomerular cationic amino acid transporter-1 (CAT-1) expression in diabetic animals, which was unaffected by lysine. When human endothelial cells were incubated with arginine end products no effect on arginine transport was observed. However, only in the presence of 0.5 mM/L sodium nitroprusside (SNP) an augmented steady-state CAT-1 mRNA was demonstrated by RT-PCR. CONCLUSION: In a rat model of early diabetes, glomerular arginine uptake is elevated through modulation of CAT-1 expression, thus, contributing to the pathogenesis of hyperfiltration. Increased nitric oxide formation may play a role in this process.

The cardio-renal anaemia syndrome: does it exist?

Many patients in our nephrology department who have anaemia and chronic kidney insufficiency (CKI) show evidence of congestive heart failure (CHF). This triad of anaemia, CKI and CHF is known as the cardio-renal anaemia syndrome. The three conditions form a vicious circle, in which each condition is capable of causing or being caused by another. Anaemia can increase the severity of CHF and is associated with a rise in mortality, hospitalization and malnutrition. Anaemia can also further worsen renal function and cause a more rapid progression to dialysis than is found in patients without anaemia. Uncontrolled CHF can cause rapid deterioration of renal function and anaemia. CKI can also cause anaemia, as well as worsen the severity of CHF, and is associated with increased mortality and hospitalization in patients with CHF. Aggressive therapy against CHF with all the conventional medications at the accepted doses often fails to improve the CHF if anaemia is also present but is not treated. In studies in which the anaemia was corrected with s.c. erythropoietin and, in some cases, with i.v. iron, however, the cardiac function improved, as assessed by measurement of the left ventricular ejection fraction and oxygen utilization during maximal exercise. Symptomatic patient functioning improved, as monitored by shortness of breath and fatigue on exertion, and the need for hospitalization and oral and i.v. diuretics markedly decreased. The quality of life, as judged by different criteria, also improved. The glomerular filtration rate, which fell rapidly when the anaemia was untreated, stabilized in patients when their anaemia was treated. Nephrologists need to assess the cardiac status of all patients with CKI carefully, and this includes an echocardiogram along with possibly measuring the levels of B-type natriuretic peptide. Nephrologists also need to use the indicated agents for CHF at the recommended doses, while cardiologists and internists need to be more aware of the importance and lethal effects of even mild anaemia and the benefits of its treatment in CHF and CKI. Cooperation between these specialists will allow better and much earlier treatment of the anaemia, CHF and CKI, and prevent the deterioration of all three conditions.

Erythropoietin should be part of congestive heart failure management.

BACKGROUND: Up to 64% of patients referred to nephrologists with chronic kidney insufficiency (CKI) have evidence of congestive heart failure (CHF), and most of these patients are also anemic. We have called this triad of anemia, CKI, and CHF the cardio renal anemia (CRA) syndrome. The 3 components of this syndrome form a vicious circle, with each one capable of causing or worsening the other 2. Anemia is found in one-third to one-half of CHF patients and can either cause or worsen the CHF, and can increase the mortality, hospitalization, and malnutrition in this condition. Anemia is also associated with a worsening of renal function in CHF and CKI, causing a more rapid progression to dialysis than is found in those without anemia. Uncontrolled CHF can cause rapid deterioration of renal function and may also cause anemia. Chronic kidney insufficiency can cause anemia and worsen the CHF. METHODS: Aggressive therapy of CHF with all the accepted CHF medications in the accepted doses will often fail to improve the CHF if anemia is also present but is not corrected. However, when the anemia was corrected with subcutaneous erythropoietin and, in some cases, with intravenous iron, the cardiac and patient function and quality of life improved, the need for hospitalization and for high-dose oral and intravenous diuretics was strikingly reduced, and renal function, which had previously been deteriorating, stabilized. RESULTS: Nephrologists should carefully assess the cardiac status of all CKI patients, including routinely getting an echocardiogram and possibly measuring B-type natriuretic peptide. Where CHF is present, the indicated CHF agents in the indicated doses should be used. CONCLUSION: Studies show that most cardiologists and internists do not recognize, investigate, or treat the anemia frequently seen in their CHF patients. In our experience cooperation between nephrologists and these specialists has increased their awareness about anemia, resulting in its earlier correction, and thus preventing the deterioration of the CHF, the CKI, and the anemia itself.

Effect of tumor necrosis factor-alpha on endothelial and inducible nitric oxide synthase messenger ribonucleic acid expression and nitric oxide synthesis in ischemic and nonischemic isolated rat heart.

OBJECTIVES: The present study aimed to investigate the influence of endogenous tumor necrosis factor-alpha (TNF-alpha) that was synthesized during ischemia and exogenous TNF-alpha on endothelial and inducible nitric oxide synthase (eNOS and iNOS) messenger ribonucleic acid (mRNA) expression and nitric oxide (NO) production in the isolated rat heart. BACKGROUND: Tumor necrosis factor-alpha is recognized as being a proinflammatory cytokine with a significant cardiodepressant effect. One of the proposed mechanisms for TNF-alpha-induced cardiac contractile dysfunction is increased NO production via iNOS mRNA upregulation, but the role of NO in TNF-alpha-induced myocardial dysfunction is highly controversial. METHODS: Isolated rat hearts studied by a modified Langendorff model were randomly divided into subgroups to investigate the effect of 1-h global cardioplegic ischemia or the effect of 1-h perfusion with exogenous TNF-alpha on the expression of eNOS mRNA and iNOS mRNA and on NO production. RESULTS: After 1 h of ischemia, there were significant increases in TNF levels in the effluent (from hearts), and eNOS mRNA expression had declined (from 0.91 +/- 0.08 to 0.68 +/- 0.19, p < 0.001); but there were no changes in iNOS mRNA expression, and NO was below detectable levels. Perfusion of isolated hearts with TNF-alpha had a cardiodepressant effect and decreased eNOS mRNA expression to 0.67 +/- 0.04 (p < 0.002). Inducible nitric oxide synthase mRNA was unchanged, and NO was below detectable levels. CONCLUSIONS: We believe this is the first study to directly show that TNF-alpha does not increase NO synthesis and release but does downregulate eNOS mRNA in the ischemic and nonischemic isolated rat heart.

Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats.

The decrease in glomerular filtration rate (GFR) that is characteristic of sepsis has been shown to result from inhibition of glomerular endothelial nitric oxide synthase (eNOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). Although l-arginine is the sole precursor for NO biosynthesis, its intracellular availability in glomeruli from septic animals has never been investigated. Arginine uptake was measured in freshly harvested glomeruli from the following experimental groups: 1) untreated rats; 2) rats pretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3) rats treated with LPS as above with either l-N(6)-(1-iminoethyl)lysine hydrochloride (l-NIL), a selective iNOS antagonist, or 7-nitroindazole, a selective neuronal NOS antagonist; and 4) rats treated with l-NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with a y(+) system. Arginine uptake was augmented in glomeruli from LPS-treated rats. Treatment with l-NIL completely abolished this effect whereas l-NIL alone had no effect. Similar results were obtained when primary cultures of rat mesangial cells were preincubated with LPS (10 microg/ml for 24 h) with or without l-NIL. Using RT-PCR, we found that in vivo administration of LPS resulted in a significant increase in glomerular cationic amino acid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA was undetected. Northern blotting further confirmed a significant increase in glomerular CAT-2 by LPS. In mesangial cells, the expression of both CAT-1 and CAT-2 mRNA was augmented after incubation with LPS. In conclusion, in vivo administration of LPS augments glomerular arginine transport through upregulation of steady-state CAT-2 mRNA while downregulating CAT-1 mRNA. These results may correspond to the changes in glomerular iNOS and eNOS activity in sepsis.

Glomerular arginine transport is attenuated in chronic renal failure in rats.

UNLABELLED: L-Arginine supplementation was found to have a reno-protective effect in different models of chronic renal failure (CRF) in spite of normal plasma levels. Experiments were designed to determine if changes in glomerular uptake of arginine occur in 5/6 nephrectomized rats as a model of CRF. Renal function and glomerular uptake of radiolabeled arginine {[3H] L-arginine} was measured in sham operated, 5/6 nephrectomy, and right nephrectomy rats. Renal failure and proteinuria was found in the CRF animals only. Arginine uptake by glomeruli harvested from rats with CRF was significantly lower than that by glomeruli from sham operated and unilateral nephrectomy rats. IN CONCLUSION: glomerular arginine uptake is decreased in CRF. This phenomenon can explain the beneficial effect of L-arginine supplementation in CRF.

L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats.

BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF.

The increased proliferation of cultured neuroblastoma cells treated with vasoactive intestinal peptide is enhanced by simultaneous inhibition of neutral endopeptidase.

Vasoactive intestinal peptide (VIP) stimulates the neuroblastoma cell line (NMB) to proliferate. Neuropeptide activity can be inhibited by neutral endopeptidases that function intracellularly and in the extracellular milieu. NMB cells express neutral endopeptidase (NEP) activity that can be specifically inhibited by phosphoramidon (PA). Our data now show that phosphoramidon treatment increases the efficacy of VIP-stimulated neuroblastoma proliferation. These results suggest that membrane endopeptidases modulate VIP-associated cell proliferation and enhancement of endopeptidase activity may serve as a target for cancer therapy.

A possible role of prolactin on growth and maturation of the gut during development in the rat.

The growth and maturation of the gastrointestinal tract during development is influenced by diverse genetic and growth factors. Since prolactin is abundant in amniotic fluid and breast milk, we hypothesized that it may also affect gut development. The effect of prolactin on thymidine incorporation and tissue alkaline phosphatase, maltase and lactase activity was studied on jejunal explants from fetal, newborn and 2 week-old rats. The results were compared with the effects of epidermal growth factor (EGF) under identical experimental conditions. Prolactin induced a significant increase in proliferation and a two- to threefold increase in maltase and alkaline phosphatase activity of the newborn explants. The effect of prolactin in this group compared to that of EGF was significantly greater with respect to proliferation, and almost identical with respect to the hydrolases studied. These results suggest that prolactin might have a role in the process of growth and maturation of the gut mucosa during ontogeny.

Erythropoietin and G-CSF receptors in human tumor cells: expression and aspects regarding functionality.

AIMS AND BACKGROUND: Recombinant human erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF) are used to stimulate hematopoiesis in patients with malignant diseases. These cytokines transduce their biological signal via the Epo receptor (EpoR) and G-CSF receptor (G-CSF-R) into the cell. We therefore investigated in human tumor cell lines the expression of these receptors in tumor cells as well as their response to Epo and G-CSF. METHODS AND STUDY DESIGN: The expression of EpoR and G-CSF-R mRNA was analyzed with reverse transcription-polymerase chain reaction (RT-PCR). EpoR protein expression was further monitored with Western blot and immunocytochemistry analysis. The cellular response to various concentrations of Epo was evaluated using 3[H]-thymidine uptake, Northern blot of c-fos expression and tyrosine kinase activity assay. The proliferation after G-CSF incubation was analyzed with the MTS assay. RESULTS: In this study EpoR mRNA and protein were detected in various human tumor cell lines. Treatment with Epo did not influence the proliferation rate of examined EpoR-positive tumor cell lines. Epo did not stimulate the tyrosine kinase activity nor did it affect the c-fos mRNA in these cell lines. G-CSF-R mRNA was only detected in two myeloid cell lines. Treatment with G-CSF did not increase the proliferation of these cells. CONCLUSIONS: These results demonstrate that Epo and G-CSF did not modulate the growth rate of examined receptor-positive tumor cell lines; the presence of the Epo receptor seems not essential for cell growth of these tumor cells in cell culture.

Garlic attenuates nitric oxide production in rat cardiac myocytes through inhibition of inducible nitric oxide synthase and the arginine transporter CAT-2 (cationic amino acid transporter-2).

It is now accepted that allicin, the main biologically active compound in garlic, exhibits antioxidant activity. The present study was designed to test the hypothesis that the antioxidant activity of garlic can be partially attributed to the inhibition of nitric oxide (NO) production by cytokine-induced NO synthase (iNOS). Cardiac myocytes cultured from neonatal Wistar rats were stimulated by lipopolysaccharide (LPS) and incubated for 24 h with various concentrations of allicin. This resulted in marked inhibition of nitrite production. Interestingly, a low concentration of allicin (10 microM) was significantly more potent in abrogating the effect of LPS on nitrite production than a higher concentration (40 microM). Allicin decreased steady-state iNOS mRNA levels, and this effect was maximal when a lower concentration was used (10 microM compared with 40 microM). In order to explore additional effects of allicin on NO generation that might counteract the effect on iNOS, we assessed the effects of higher allicin concentrations on arginine transport. Allicin inhibited the uptake of 1 mM extracellular arginine in a concentration-dependent manner. The expression of the two arginine transporters that are expressed in cardiac myocytes [CAT-1 (cationic amino acid transporter-1) and CAT-2] was studied using reverse transcription-PCR. A concentration of 200 microM allicin abolished the expression of CAT-2 mRNA, 100 microM significantly attenuated it, whereas 50 microM had no effect. Allicin had no effect on steady-state CAT-1 mRNA levels. Our results suggest that allicin inhibits iNOS activity through two different mechanisms: at lower concentrations it decreases iNOS mRNA levels, whereas at higher concentrations it inhibits arginine transport through down-regulation of CAT-2 mRNA.