Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury.

The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal-truncated EGF-R under the control of the kidney-specific type 1 gamma-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.

Aggressive acute CD3+, CD56- T cell large granular lymphocyte leukemia with two stages of maturation arrest.

In the majority of clonal expansions of CD3+ large granular lymphocytes (LGL), referred to as T-LGL leukemia, patients have a chronic disease, often manifested by severe neutropenia, rheumatoid arthritis, and mild to moderate splenomegaly. The characteristic leukemic phenotype is CD3+, CD8+, CD16+, CD57+ and CD56-. Here we report an unusual case of T-LGL (CD3cyt+, CD3surface-, CD16+, CD56-) with clinicopathological features (acute presentation, large tumor mass, and systemic illness with highLGL counts at diagnosis) similar to those described for patients with CD3-natural killer (NK)-LGL leukemia. Two distinct stages of maturation arrest were observed: in the lymph node abnormal cells were CD4+, CD8+ whereas the majority of circulating leukemic cells expressed only CD8. TCR gamma (TCR gamma) gene configuration demonstrated that these originated from the same T cell clone, suggesting a maturation process between the two populations, or preferential passage of CD8 single positive cells into the blood.

Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations.

Detection of clonal T cell receptor gamma (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10(-3) at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent Vgamma and Jgamma PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vgamma and Jgamma segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10(-2)-10(-3) level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TAL1-negative and -positive cases. A combination of fluorescent multiplex and Vgamma-Jgamma-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

Prostaglandin E2-like activity of 20:3n-9 platelet lipoxygenase end-product.

5,8,11-Icosatrienoic acid (20:3n-9), a fatty acid associated with platelet hyperactivity, was oxygenated by platelet lipoxygenase. The end-product of this pathway was purified by high-performance liquid chromatography (HPLC) and characterized as 12-hydroxy-5,8,10-icosatrienoic acid [12-OH-20:3(5,8,10)] by capillary gas-liquid mass spectrometry. When tested upon platelet aggregation, 12-OH-20:3(5,8,10) exhibited a biphasic effect. At low concentrations (below 5 X 10(-7) M) it potentiated aggregation but inhibited it at higher levels, a pattern similar to that obtained with prostaglandin E2. However, since the amounts of 12-OH-20:3(5,8,10) generated under thrombin stimulation are in the range of concentrations with potentiating effects, it seems that the 12-OH derivative is responsible for the hyperaggrebility of 20:3n-9-rich platelets.

Nutritional effects of feeding a ketoanalogue mixture in growing and adult uremic rats.

Insufficient protein diets supplemented with ketoanalogue/essential amino acid (KA/EAA) mixtures are proposed to maintain nutrition and to retard renal deterioration. We compared in growing and in adult uremic rats diets containing limited or usual amounts of protein (12%, 20% for growing rats, and 10% and 16% for adult rats) with diets containing 50% or 60% less casein plus a KA/EAA mixture providing KA at an equimolar amount of removed EAA or at higher amounts. The latter supplement caused stunting, the former caused no anorexia, a slight growth deficit when added to the lowest basal casein diets, and almost normal growth when added to higher casein diets. Growth was normal with EAA supplements. The plasma EAA changes were unrelated to intake and to growth. Thus, KA utilization is maximal, provided that basal protein is sufficient and KA are not in excess.

Optimal dietary substitution of racemic ketoanalogues for isoleucine in growing normal and uremic rats.

Dietary ketoanalogues (KAs) were shown to replace their essential amino acids with a 50% efficiency for valine and leucine. We determined the optimal concentration of the racemic KA of isoleucine (KMVA) in uremic and control rats: nutrition responses were compared between a diet containing optimal isoleucine concentration and diets containing various KMVA concentrations. Isomolar replacement of isoleucine produced anorexia, stunting, and poor nitrogen balance. Doubling KMVA partially improved these indices. Tripling KMVA lessened urea production and improved growth up to that obtained with the isoleucine diet in uremic but not in control rats (20% lower). A further KMVA increase produced no further benefit. Among plasma branched-chain amino acids, only alloisoleucine was affected; it increased with increasing KMVA concentration, being maximum after tripling KMVA. Racemic KMVA could replace isoleucine with a 35% efficiency but supported no growth acceleration in uremic rats and no maximal growth in control rats. Plasma alloisoleucine rose without adverse nutrition effects.

Uremia-induced disturbances in hepatic carbohydrate metabolism: enhancement by sucrose feeding.

A high-sucrose (S) diet accentuates anorexia and stunts growth in uremic (U) rats, and an oral S load induces a greater hyperfructosemia in U rats than in control (C) rats. Four studies were performed to determine the roles of S feeding and an acute S load on liver carbohydrate (CHO) metabolism in U and C rats (eight to 10 rats per group). We also examined the plasma responses to either water or a S load. Levels of the main metabolites of glycolysis, gluconeogenesis, and glycogenesis were measured under basal conditions (7 hours' postmeal) in U and C rats fed either a cornstarch diet (study I) or S diet (study II) and at 30 and 60 minutes after an intragastric S load (studies III and IV) in s-fed U and C rats. The weight gain, food intake, and plasma creatinine and urea levels of the rats in the four studies were comparable. Weight gain and liver weight (g/100 g body weight) were lower in U than in C rats. In the plasma, baseline levels of lactate were decreased by uremia and S feeding and those of glucose (G) were increased by S feeding. The increases in plasma G and fructose (F) levels after a S load were greater in U rats than in C rats, whereas those of plasma lactate were comparable. In the liver under basal conditions, uremia markedly decreased levels of glycogen, F-1,6-diphosphate (F-1,6-diP), F-2,6-diP, 3-glycero-phosphate (3-glycero-P), dihydroxyacetone phosphate (DHAP), pyruvate, lactate, and adenosine triphosphate (ATP), and the phosphorylation state (ATP/adenosine diphosphate [ADP] x inorganic phosphorus [PI]), increased phosphoenolpyruvate (PEP), ADP, and Pi levels, but did not affect the cytosolic redox state (pyruvate/lactate). In addition to uremia, S feeding further decreased levels of glycogen, F-2,6-diP, 3-glycero-P, and ATP. After S loading, liver F levels increased more in U than in C rats, but glycogen and 3-glycero-P levels increased less in U than in C rats. Liver lactate and pyruvate levels increased more in U than in C rats, and the pyruvate/lactate and DHAP/3-glycero-P ratios were higher in U than in C rats after a S load. The ATP level and the phosphorylation state in U rats increased 30 minutes later in U than in C rats. Our findings indicate that uremia causes a depletion in liver glycogen, which is enhanced by S feeding and could be partially attributed to decreased glycogen synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Potentiating effect of 5,8,11-eicosatrienoic acid on human platelet aggregation.

5,8,11-Eicosatrienoic acid (20:3 omega 9), a fatty acid increased in the platelet phospholipids of man and animals fed saturated fats, was either added to human platelets simultaneously with the aggregating agents, or incorporated into the platelet phospholipids by preincubation. 20:3 omega 9 markedly increased the response of platelets to all aggregating agents tested when added simultaneously with the agent, but solely to thrombin and ionophore, after incorporation into the platelet phospholipids. The potentiating effects of 20:3 omega 9 on thrombin aggregation do not appear to be related to prostaglandin formation, but rather to the production of a monohydroxy derivative through the lipoxygenase pathway.

[Growth factors. Role in the progression of renal lesions]. / Facteurs de croissance. Rôle dans la progression des lésions rénales.

FROM PATHOPHYSIOLOGY TO THERAPEUTICS: Nephrologists are faced with the continuing problem of helping patients avoid the onset or retard the development of end-stage renal failure. Despite the treatments available, the risk is still high for patients and the cost a heavy burden for the public health budget. These facts underline the importance of a detailed understanding of the mechanisms leading to the destruction of renal parenchyma in order to develop therapeutic strategies capable of slowing the inevitable progression of kidney lesions. GROWTH FACTORS: It is currently recognized that a major reduction in the number of functional nephrons, whatever the initial cause, leads in itself to a progressive deterioration of healthy nephrons and finally to complete destruction of the kidney. The underlying mechanisms remain largely unknown. One possible mechanism would involve an overexpression of several growth factors in the damaged renal parenchyma. We present in this review experimental data obtained with various approaches, including pharmacological and/or dietetic modulations and the establishment of transgenic mouse lines, to demonstrate the key role played by growth factors in the progression of renal lesions. The pathways followed by these growth factors in the process of renal destruction as well as certain elements leading to their overexpression are also discussed.

Using transgenic mice to analyze the mechanisms of progression of chronic renal failure.

An understanding of the mechanisms underlying the formation of renal lesions is necessary for the development of strategies aiming to delay the progression of chronic renal failure. The generation of transgenic mice in the past 20 years has contributed significantly to the study of this phenomenon. Overexpression and/or inactivation of single factors in renal tissue demonstrated that molecules such as growth factors, proto-oncogenes, and renin-angiotensin system elements play major roles in renal deterioration. Several mouse models of renal injury have been developed in the past 10 yr. Transgenic mice that exhibit a normal phenotype under physiologic conditions allow analysis of the roles of single factors in the progression of chronic renal failure when renal injury models are used. Using this strategy, it was demonstrated that vascular adaptation, which is a process that involves the endothelin/nitric oxide balance, is essential for the survival of mice after nephron reduction and that the epidermal growth factor/activator protein-1/Bcl-2 pathway is involved in the development of renal lesions after renal injury, possibly via adjustment of the proliferation/apoptosis balance. Moreover, it was demonstrated that selective inhibition of epidermal growth factor signaling in the kidney successfully prevents the progression of chronic renal failure. These results indicate the power of transgenesis for elucidation of the pathogenesis of renal disease.

[Experimental approach to nutritional problems in chronic renal insufficiency]. / Abord expérimental des problèmes nutritionnels dans l'insuffisance rénale chronique.

The many published studies of experimental chronic renal failure (CRF) include a few findings which are similar to those reported in children with the naturally occurring disease. Experimental CRF has proved a useful model for investigating changes in eating behaviors: lack of appetite for sweet foods and selection of foods with high protein contents was comparable to behaviors exhibited by children. Optimal protein intake was found to be close to the minimum recommended intake for "optimal" growth (different from maximum growth in rats). Excessive protein intake had detrimental effects on renal function and growth with conventional dehydrated feeds, but water intake may have a greater impact than blood urea nitrogen and acidosis. A 50% reduction in protein intake with adequate amounts of essential amino acids ensured normal growth and slowed progression of renal lesions. Replacement of protein by mixtures of ketoanalogs was more likely to be responsible for growth failure; where similar growth rates were achieved, there was no evidence of a beneficial effect on renal lesions. Diets with high sucrose contents were poorly tolerated by CRF rats and were associated with fructose "intolerance" and reduced liver energy stores.

Species differences in mammalian renal function: 5-HIAA, a reported marker of tubular secretion in humans, is handled exclusively by filtration in the rat.

Renal clearance of endogenous 5-HIAA has been shown to be similar to that of PAH in humans. The present study compared 5-HIAA and PAH clearances in rats. It showed that the clearance of 5-HIAA in rats was identical to that of inulin and creatinine. This suggests that in this species, 5-HIAA is only filtered and not secreted by the tubules in contrast to what is observed in humans.

Refractoriness of diabetic platelets to inhibitory prostaglandins.

Inhibition of collagen-induced platelet aggregation by either endothelial extracts, prostacyclin, prostaglandin E1 or prostaglandin D2 was investigated. The inhibition was less efficient with diabetic platelets than with platelets from normal donors. The refractoriness of diabetic platelets to inhibitory prostaglandins was observed both with platelet-rich plasma and platelets isolated from their plasma. Moreover levels of cyclic AMP in resting platelets and after stimulation by either PGE1 or PGD2 were lower in diabetic platelets than in normal platelets. It is concluded that the weaker response of diabetic platelets to inhibitory prostaglandins could be related to their content in cyclic AMP.

[Kidney functional reserve. An experimental study]. / Réserve fonctionnelle rénale. Etude expérimentale.

The renal functional reserve (RFR), the increase in glomerular filtration rate (GFR) induced by a protein load, seems to be diminished or even lost in renal failure. Our experimental study was undertaken to determine whether the RFR is lost beyond a given level of nephron reduction, using different protein loads. In the first two studies, RFRs were evaluated during an oral protein load consisting in a high-protein diet (30% casein) compared to a low-protein diet (7% casein). Each diet was given to SD rats (200 g) either for three weeks immediately after nephrectomy (Nx) or for four days one month after Nx. Nx was subtotal and consisted in removal of 65 to 85% of the mass of the renal parenchyma. The GFR evaluated by inulin clearance measurements increased considerably after a prolonged (+188%) or short-lived (+35%) oral protein load if less than 70% of the renal mass had been removed. Beyond this threshold of nephron amputation, the high-protein diet had no effect on the GFR, despite an increase in the residual renal mass (+200% in moderate and severe Nxs). In a third study, the GFR was measured one month after Nx and the effects of an infusion of amino acids (vamine) or of a placebo were compared, each rat serving as his own control. Extent of Nx was 0%, 50%, 65-70%, and 80%. Regardless of the extent of nephron reduction, the GFR increased under vamine, but interindividual variations in each group were marked (+5 to +70%).(ABSTRACT TRUNCATED AT 250 WORDS)