Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target.

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.

Renal transplantation in the elderly: a long-term, single-centre experience.

BACKGROUND: End-stage renal failure increases with advancing age and renal transplantation should be considered in end-stage renal failure patients older than 60 years. However, there is a paucity of data on long-term patient and graft survival in this population. METHODS: From October 1983 to March 1999, 310 renal transplantations were performed at Geneva University Hospital in 283 patients, of which 49 were done in 48 patients older than 60 years (mean age 65.6+/-4.1 years). The following data were analysed at 1, 5, and 10 years, and compared between the patients >60 years and <60 years old: actuarial patient and graft survival, serum creatinine, causes of graft loss, and patient death. RESULTS: Patient survival at 10 years was 81% for patients <60 years and 44% for patients >60 years. Graft survival at 10 years was 59% for patients <60 years and 32% for patients >60 years. Graft survival at 10 years censored for death with functioning graft was 65% for patients <60 years and 81% for patients >60 years. Main causes of mortality in the older patients were related to cardiovascular events (47%), neoplasia (41%), and sepsis (18%). Overall, recipient and donor age were not predictive factors for graft survival, as shown by multiple logistic regression. CONCLUSIONS: Renal transplantation should be considered in patients older than 60 years, since graft survival is excellent in this population. Although these patients have a shorter life expectancy, they benefit from renal transplantation similarly to younger kidney transplant recipients.

Cyclosporine toxicity in renal transplant recipients detected by nailfold capillaroscopy with Na-fluorescein.

BACKGROUND: Cyclosporine represented a major advance in the medical management of patients with organ transplantation, but its use is limited by the frequent occurrence of hypertension and renal toxicity diagnosed by invasive renal biopsy. Renal histology shows a specific arteriolopathy. It was hypothesized that cyclosporine may also induce subclinical microvascular changes in the skin that might be detected noninvasively by a combination of dynamic capillaroscopy [capillary blood cell velocity (CBV)] with and without intravenous Na-fluorescein (NaF) injection and laser Doppler fluxmetry (LDF). METHODS: The nailfold skin microcirculation was evaluated in 112 consecutive renal transplant recipients (54 +/- 11 years old; 70 males and 42 females) receiving cyclosporine. The investigation was made the same day as a routine renal biopsy performed in all patients more than two years after transplantation. Renal biopsies were blindly classified as positive (N = 33) when significant specific signs of cyclosporine toxicity were clearly observed (AH2-AH3) and were otherwise negative (AH0-AH1, N = 79) according to the Banff classification. RESULTS: Time to fluorescence peak after NaF injection (tpNaF) was significantly longer in patients with positive biopsies than in patients with negative biopsies (13.9 +/- 8.1 vs. 17.5 +/- 9.4 sec, P = 0.009). All patients but three with negative biopsies (93%) had a tpNaF less than 10 seconds (sensitivity 91%, negative predictive value 93%). On the other hand, CBV, LDF, plasma levels of cyclosporine, and endothelin were similar in the two groups. CONCLUSION: Nailfold fluorescence capillaroscopy is an accurate and simple mean to rule out cyclosporine toxicity in renal transplant recipients. A normal test could avoid invasive renal biopsy in about 40% of the patients. Renal biopsy would, however, still be indicated when the test is abnormal.

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

L-type voltage-gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells.

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.

Kinetics of cellular viability in warm versus cold ischemia conditions of kidney preservation. A biometric study.

We have determined the kinetics of the cellular viability ratio (CVR), defined as the number of living cells over the total cell count, in pig kidneys using propidium iodide and fluorescein diacetate staining, as a function of time and preservation conditions. The kidneys were preserved in warm or cold ischemia in order to mimic the conditions of transplantation from non-heart-beating donors or multiple removal with optimal preservation of the graft, respectively. To determine the CVR, the cells were obtained by a fine-needle aspiration biopsy, which minimizes the damage to the graft. A biometric analysis by regression enabled the determination of the time dependence for warm ischemia (CVR(t) = 80.0 x e(-0.733-t)(+2.7/-0.36)) and for cold ischemia (CVR(t) = 80.0 x e(-0.022-t)(+1.57/-0.64)) with a confidence interval of 95%. These master curves allow us to predict, under the described conditions, the CVR after a given ischemia time. The half-life of the cells can be deduced from the time-dependent CVR(t), and is 0.64 hr (38 min) for warm ischemia and 21.4 hr for cold ischemia. Further, the CVR for a given kidney can be used to assess its condition at removal: if the CVR is below 48% at 2 hr after removal, one can conclude that the organ has suffered a period of warm ischemia.

Protein synthesis within dendrites: ionic and neurotransmitter modulation of synthesis of particular polypeptides characterized by gel electrophoresis.

This study evaluates whether physiological variables differentially affect the local synthesis of protein constituents of synapses in subcellular fractions containing pinched-off dendrites (synaptodendrosomes). Synaptodendrosomes were pulse-labeled in a medium containing 35S-methionine with 3 or 25 mM KCl and in the presence or absence of 0.5 mM EGTA or 10 microM glutamate. Synaptodendrosomes were then subfractionated to prepare synaptic plasma membranes and synaptic junctional complexes. The protein constituents of the synaptic plasma membrane and synaptic junctional complex fractions that were locally synthesized were identified using SDS-PAGE and two-dimensional gel electrophoresis and the extent of labeling of individual bands was analyzed using a Phosphorimager. Analysis of incorporation into individual bands resolved by SDS-PAGE revealed that depolarizing conditions (25 mM KCl) increased the extent of labeling of different bands to a different extent (ranging from 10-70% increases in labeling). Addition of 0.5 mM EGTA decreased the extent of labeling of the same group of bands in both 3 mM KCl and 25 mM KCl conditions. Addition of 10 microM glutamate reduced incorporation especially in the synaptodendrosomes incubated in 25 mM KCl. Two-dimensional gel electrophoresis analyses revealed that the labeled spots that showed differential labeling under the different conditions did not correspond to the most prominent Coomassie-stained spots. These results indicate that the proteins that are synthesized in synaptodendrosomes and regulated by physiological variables are not amongst the more abundant protein constituents of the fractions. Taken together, these results are consistent with the idea that protein synthesis within dendrites may be regulated by synaptic activity.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Purification and characterization of p68/70, regeneration-associated proteins from goldfish brain.

Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu(2+)-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5.2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a heterodimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydrophobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton X-114. Analysis of hydrolytic products indicated p68/70 to be a glycoprotein, containing 11% carbohydrate.

Effects of oral calcitriol on bone mineral density in patients with end-stage renal failure.

Evolution of bone mineral density (BMD) at various skeletal sites and the influence of calcitriol on BMD are still poorly documented in patients with terminal renal failure. Using dual photon absorptiometry, we investigated the changes in BMD at the levels of lumbar spine, femoral neck and midfemoral shaft in 21 patients with end-stage renal failure (ESRF) treated with calcitriol (mean dosage +/- SEM: 0.21 +/- 0.02 microgram/day) and compared them to 25 patients with ESRF but not treated with calcitriol (control group) over a period of 20.3 +/- 1.5 and 17.2 +/- 1.2 months, respectively. Lumbar spine BMD increased by 7.7 +/- 3.2%/year in the treated group and decreased by 2.5 +/- 1.3%/year in the control group (P < 0.005). Femoral shaft BMD increased more in treated than in control group (+ 6.7 +/- 2.3 vs. + 1.4 +/- 2.0%/year; P < 0.05) and femoral neck BMD remained stable. PTH levels increased by 92 +/- 121 and 1033 +/- 254 pmol/year (P < 0.01) in the treated group and the controls, respectively. Osteocalcin changes were -2.7 +/- 3.7 and +20.1 +/- 11.7 micrograms/liter (P < 0.05) per year in the same groups. These results indicate that low doses of oral calcitriol in patients with end-stage renal failure were associated with an increase in BMD at the levels of lumbar spine and femoral shaft, and with a stabilization of serum PTH and osteocalcin concentrations.

Effects of dialysate composition during hemodialysis on left ventricular function.

To determine the effects of dialysate composition during hemodialysis on left ventricular systolic and diastolic function, 12 patients treated by chronic hemodialysis (mean age 40.8 +/- 2.7 years), without overt heart disease, were studied by Doppler-echocardiography successively before and after acetate hemodialysis (AHD), bicarbonate hemodialysis (BHD), and acetate-free biofiltration (AFB). The three types of hemodialysis resulted in a comparable decrease of the body weight. Mean arterial blood pressure decreased by 5 mm Hg (NS), 8 mm Hg (NS) and 10 mm Hg (P < 0.05) during AHD, BHD and AFB, respectively. There was a significant increase of the heart rate and the shortening fraction of the left ventricular diameter after AHD, but not after BHD and AFB. Mean total systemic resistance increased by 20% after AHD, 18% after BHD and by 7% after AFB (all changes NS). During each type of hemodialysis there was a significant reduction of the peak velocity of the early diastolic rapid filling wave (peak E) without change of the peak filling velocity during atrial contraction (peak A). During AHD and BHD the pressure half-time of the early filling phase (TP/2) increased, and the velocity-integral of the early diastolic filling phase (E-area) decreased. However, TP/2 and E-area did not change significantly after AFB.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of altered loading conditions during haemodialysis on left ventricular filling pattern.

Changes in the circulating volume associated with haemodialysis result in modification of left ventricular loading conditions. To determine the influence of haemodialysis on Doppler indices of left ventricular filling, 12 patients (mean age 40.8 +/- 2.7 (SEM) years) with renal insufficiency but without overt heart disease were studied by Doppler-echocardiography immediately before and after haemodialysis. Haemodialysis resulted in a decrease in body weight from 68.0 +/- 3.8 kg to 65.0 +/- 3.7 kg (P < 0.01). Heart rate and blood pressure did not change significantly during haemodialysis. Left ventricular diastolic dimension (M-mode) decreased from 53.5 +/- 1.1 mm to 49.5 +/- 1.9 mm (P < 0.05), whereas the shortening fraction did not change. Haemodialysis elicited marked changes in the early diastolic rapid filling wave (E wave) recorded by pulsed Doppler at the level of the mitral annulus. Peak velocity of the early rapid filling phase (peak E) decreased significantly from 95.3 +/- 8.2 cm.s-1 to 63.0 +/- 5.7 cm.s-1 (P < 0.001) and mid-diastolic deceleration of transmitral velocity decreased from 437.3 +/- 54.2 cm.s-2 to 239.7 +/- 54.4 cm.s-2 (P < 0.01). The peak filling velocity during atrial contraction (peak A) did not change (79.7 +/- 6.3 cm.s-1 vs 74.1 +/- 4.7 cm.s-1; P = NS). The ratio peak E/peak A decreased from 1.19 +/- 0.06 to 0.85 +/- 0.04 (P < 0.01) during haemodialysis. The results provide further evidence for the pronounced preload-dependence of Doppler indices of left ventricular diastolic function.

Bone mineral density in patients with end-stage renal failure.

Renal osteodystrophy is a well-recognized complication of chronic renal failure (CRF) and is associated with a marked morbidity. Bone mineral density (BMD) has been shown to be the best predictor of fracture risk in different circumstances. In this cross-sectional study, we measured BMD using dual photon absorptiometry at three skeletal sites of functional importance such as the lumbar spine (LS), the femoral shaft (FS) and the femoral neck (FN) in 106 patients with end-stage renal failure (11 predialysis patients and 95 patients on maintenance dialysis). These skeletal sites are characterized by various relative amounts of trabecular and cortical bone. The results indicate that decreased bone mass was detectable in all skeletal sites before the beginning of dialysis and that BMD was negatively related to dialysis duration in LS and FS. Nevertheless, the deleterious effects of renal osteodystrophy were more pronounced at the FS level, where cortical bone is predominant. A separate analysis of BMD in both sexes revealed that females presented a more important bone loss in both cortical and trabecular bone than males. We did not find any significant difference in BMD at the three measured sites between patients on continuous ambulatory peritoneal dialysis and on regular hemodialysis. This study emphasizes the need to pay more attention to the prevention of bone loss in patients on CRF before the start of dialysis therapy, and to the fact that the female population might display a more pronounced susceptibility to bone loss.

Safety and efficacy of recombinant human erythropoietin treatment of anaemia associated with multiple myeloma in haemodialysed patients.

Recombinant human erythropoietin (rHuEpo) was used to treat the anaemia of four haemodialysed patients (3 males, 1 female) with advanced multiple myeloma; the type of serum M component was IgG kappa in all cases. During the 6-month period preceding rHuEpo therapy the patients received multiple blood transfusions (range 4-22 units of packed red cells per patient). After the first month of treatment haematocrit increased from 23 +/- 3 (SD) to 32 +/- 4% and during the last 3 months the maintenance dose of rHuEpo was 143 +/- 37 U/kg per week to achieve a mean haematocrit of 35 +/- 1%. After introduction of rHuEpo, blood transfusions were no longer required and the patients reported an improvement in wellbeing. No apparent worsening of multiple myeloma has been observed over the treatment period ranging from 5 to 34 months (cumulative duration of treatment 55 months). Anti-hypertensive therapy was started in one case and increased in two patients. We conclude that rHuEpo appears to be effective and safe in treating anaemia associated with multiple myeloma in patients requiring haemodialysis.

Traumatic rupture of the urinary tract in a patient presenting nephrogenic diabetes insipidus associated with hydronephrosis and chronic renal failure: case report and review of the literature.

We report the case of a 34-year-old Japanese man suffering from a nephrogenic diabetes insipidus (NDI) associated with bilateral hydronephrosis, hydroureters and enlarged trabeculated bladder without obstruction. He also presented with chronic renal failure which has rarely occurred in similar cases. The patient was admitted after a traumatic rupture of the left urinary tract which had never been described until now in NDI. He was treated successfully by transient peritoneal and vesical drainages. This paper focuses on the very rare complication of chronic renal failure secondary to hydronephrosis in cases of NDI. The literature of this association is reviewed.

Hemostatic disturbances induced by two hollow-fiber hemodialysis membranes.

The effects on hemostasis of two high-flux membranes in hollow-fiber configuration, polyamide (PAM) and polyacrylonitrile (AN69), were analyzed in a cross-over study involving ten chronic hemodialyzed patients. Blood samples were obtained at arterial and venous sites of the extracorporeal circuit before dialysis and at 15, 30 and 180 min. Primary hemostasis: PAM induced an early significant drop in platelet counts, but at 180 min there was no longer any difference between membranes. Beta-thromboglobulin release by PAM was significantly higher at all time points. Coagulation: thrombin-antithrombin III complexes (TAT) and fibrinopeptide A increased significantly, the highest values being found with AN69. With both membranes the arterio-venous differences in TAT levels were negative throughout the sessions. Fibrinolysis: no significant differences were observed. In conclusion, both membranes induced hemostatic changes. Although these two hollow-fiber dialyzers look relatively similar, the changes observed were different, polyamide acting mainly on primary hemostasis and polyacrylonitrile on coagulation.

[Kidney retransplantation: results and prognostic factors]. / Retransplantation rénale: résultats et facteurs de pronostic.

48 non primary renal transplants were performed in 40 recipients during the 1973-1990 period in our institution (40 second grafts, 6 third grafts and 1 four and fifth grafts). Despite poor HLA matching our second graft survival rates compare favorably with others (80% and 70%, 1 and 5 year graft survival rates). The type of immunosuppression (including ciclosporine A or not) and the duration of the first graft had an influence on the outcome of second grafts. Our experience with repeated retransplantation is limited, but graft survival appears to be poor: most of the grafts were rejected within 2 years (or less). However patient survival was not affected by overimmunosuppression following multiple grafts.