Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells.

The effects of flow-induced mechanical strains on the phenotype of proximal tubular cells were addressed in vivo and in vitro by subjecting LLC-PK(1) and mouse proximal tubular cells to different levels of flow. Laminar flow (1 ml/min) induced a reorganization of the actin cytoskeleton and significantly inhibited the expression of plasminogen activators [tissue-type (tPA) activity: 25% of control cells; tPA mRNA: 70% of control cells; urokinase (uPA) mRNA: 56% of control LLC-PK(1) cells]. In vivo, subtotal nephrectomy (Nx) decreased renal fibrinolytic activity and uPA mRNA content detectable in proximal tubules. Nx also induced a reinforcement of the apical domain of the actin cytoskeleton analyzed by immunofluorescence. These effects of flow on tPA and uPA mRNA were prevented in vitro when reorganization of the actin cytoskeleton was blocked by cytochalasin D and were associated, in vitro and in vivo, with an increase in shear stress-responsive element binding activity detected by an electrophoretic mobility shift assay in proximal cell nuclear extracts. These results demonstrate that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

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.

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.

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.

[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.

Sodium restriction decreases AP-1 activation after nephron reduction in the rat: role in the progression of renal lesions.

Renal hyperplasia and hypertrophy are early events after nephron reduction which precede progressive destruction of the remnant kidney. Restriction of dietary sodium content was shown to reduce renal lesions following nephron reduction. AP-1 is a transcription factor, resulting from heterodimerization of fos and jun proteins, which mediates the effects of mitogenic growth factors. To elucidate the role of AP-1 in growth processes involved in renal deterioration, we evaluated whether restriction of dietary sodium content (0.25 vs. 0.50% sodium w/w) affected AP-1-DNA binding and hyperplasia in the remnant kidney after nephron reduction (70% nephrectomy). Cell proliferation, evaluated by PCNA immunostaining, increased progressively from day 7 to day 60 in glomeruli, proximal and distal tubules and loops of Henle of nephrectomized (Nx) rats compared to control sham-operated (C) animals. AP-1-DNA binding activity increased 7 and 14 days after surgery, but it was reduced below C values at day 60. c-fos and c-jun expression were also reduced in Nx rats at day 60. Sodium restriction significantly reduced the number of PCNA-stained cells in glomeruli and tubules at days 14 and 60, but not at day 7, whereas it decreased AP-1 activation at all times of the study. This effect was associated to a marked reduction of renal lesions in Nx rats. In conclusion, we showed that, after nephron reduction, the beneficial effect of sodium restriction was associated with a reduction of hyperplasia and AP-1 activation, but that the latter did not parallel delayed cell proliferation rate in remaining nephrons. Thus, we propose that different transduction pathways are involved in cell proliferation after nephron reduction, according to the time of evolution of renal lesions.

Abnormal intestinal intraepithelial lymphocytes in refractory sprue.

BACKGROUND & AIMS: The etiology of refractory sprue is unclear. To gain insight into its pathogenesis, the phenotype and T-cell receptor (TCR) gene rearrangement status of intestinal lymphocytes were analyzed in a group of patients with clinical or biological features of celiac disease but either initially or subsequently refractory to a gluten-free diet. METHODS: Intestinal biopsy specimens were obtained from 26 adults: 6 patients with refractory sprue, 7 patients with active celiac disease, and 13 normal controls. The phenotype of intestinal lymphocytes was studied by immunohistochemistry and, in 3 patients with refractory sprue, by cytometry of lymphocytes purified from intestinal biopsy specimens. TCR rearrangements were assessed by studying TCRgammaV-J junctional regions from DNA extracted from intestinal biopsy specimens and purified intestinal lymphocytes. RESULTS: In the 6 patients with refractory sprue, but not in normal controls or patients with active celiac disease, the intestinal epithelium was massively infiltrated by small lymphocytes that lacked CD8, CD4, and TCR, contained intracytoplasmic but not surface CD3epsilon chains, and exhibited restricted TCRgamma gene rearrangements. CONCLUSIONS: Refractory sprue is associated with an abnormal subset of intraepithelial lymphocytes containing CD3epsilon and restricted rearrangements of the TCRgamma chain but lacking surface expression of T-cell receptors.

Subtotal nephrectomy alters tubular function: effect of phosphorus restriction.

Few studies have examined tubular function after subtotal nephrectomy (Nx) and conservative treatments. The effects of 70% and 80% Nx (associated with dietary phosphate restriction in the latter case) on the apical brush border membrane (BBM) enzymes 5'-nucleotidase, gamma glutamyl-transferase and alkaline-phosphatase, and one BBM Na-phosphate cotransporter (NaPi-2) were studied in rats after a six week period. Changes in activity and mRNA abundance of the BBM enzymes and in NaPi-2 protein and mRNA abundance were compared with changes in the distal markers of Na,K-ATPase activity and epidermal growth factor (EGF) production. The activity, but not the mRNA of BBM enzymes, was moderately reduced by the 70% Nx. Both the mRNA and activity of gamma glutamyl-transferase and alkaline-phosphatase were decreased in the 80% Nx, and the NaPi-2 mRNA, protein and Na,K-ATPase activities were also reduced. These effects (except for 5'nucleotidase and Na,K-ATPase) were partly reversed by phosphate restriction. Overproduction of EGF occurred after the 70% Nx, was blunted in the 80% Nx, and then partially restored by phosphate restriction. Aggravation of tubular alteration was associated with enhanced renal hyperplasia (increased DNA mass), reduced GFR and hyperphosphatemia, and high PTH levels, but reduced cAMP excretion. Improvement following phosphate restriction was associated with reduced hyperplasia and lowering of phosphatemia and PTH levels. These data demonstrate that Nx selectively affected BBM function through transcriptional changes that were partially reversed by phosphate restriction. Regulatory factors involved in these changes may include intracellular phosphate content and growth factors, but not the PTH effects that are impaired in 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.

Na-K-ATPase along rat nephron after subtotal nephrectomy: effect of enalapril.

Tubular overwork is thought to be a promoter of the tubular hypertrophy and renal failure that occur in response to renal mass reduction. Because Na-K-adenosinetriphosphatase (Na-K-ATPase) is an index of tubular work, we evaluated the effects of subtotal nephrectomy and of enalapril therapy, which delays the evolution of renal lesions, on tubular hypertrophy and Na-K-ATPase activity along the rat nephron. Within 6 wk, 70% reduction of renal mass engendered hypertrophy of the proximal convoluted tubule (PCT), thick ascending limb (TAL), and collecting duct (CD), as well as parallel increments in Na-K-ATPase activity per millimeter tubule length (Na-K-ATPase activity per unit surface area was not modified by subtotal nephrectomy). Chronic enalapril therapy prevented part of the hypertrophy (but not Na-K-ATPase stimulation) of the PCT and the whole stimulation of Na-K-ATPase (but not hypertrophy) in the CD, whereas it had no effect on the TAL. Enalapril effect on Na-K-ATPase in CD might result from reduced bradykinin metabolism, as the reduction in urinary excretion of bradykinin observed in subtotally nephrectomized rats was prevented by enalapril therapy.

Subtotal but not unilateral nephrectomy induces hyperplasia and protooncogene expression.

It is generally accepted that renal compensatory growth after unilateral nephrectomy (Uni) is due to prominent hypertrophy with no involvement of protooncogenes. Neither the balance between hypertrophy and hyperplasia nor the expression of the early-growth-related genes has been studied after subtotal nephrectomy (Nx). The occurrence of cystic tubular dilatations after Nx may suggest an excessive cell proliferation in this model. We measured DNA, RNA, and protein content, number of nuclei per tubular section, as well as c-fos, c-jun, c-myc, c-H-ras, c-sis, and c-erb-B2 protooncogene expression in kidneys taken at time of surgery and 2, 7, and 14 days after sham operation (control rats), Uni, or Nx. After Uni, hyperplasia was greater than expected (+79% for DNA at day 14) and was associated with moderate hypertrophy (+11% for protein/DNA ratio). After Nx, compensatory growth was due only to hyperplasia (+117% for DNA at day 14), with unchanged protein/DNA ratio (vs. Uni, P < 0.02). The greater hyperplasia after Nx was confirmed by nuclei counting. The protooncogene mRNA expression was constantly absent in control and Uni rats, whereas that of c-fos and c-jun genes was detected in Nx rats at day 14 with a 2- to 12-fold increment. The c-fos and c-jun protein levels were also increased at that time in Nx rats. This suggests the following: 1) the cellular events following Uni and Nx are not the same, and 2) the late protooncogene expression in Nx exclusively could favor a particular type of cell proliferation possibly more related with cystic formation than with actual compensatory growth.

Glomerular response to acute protein load is not blunted by high-protein diet or nephron reduction.

Inulin clearance (CIn) was measured in the presence of varying degrees of renal excision (NX, 0-85% of renal mass by weight), in anesthetized rats fed on high-protein (HP, 30%), median-protein (MP, 10%), or low-protein (LP, 7%) diets, before and during amino acid (AA) infusion or before and after an intragastric protein load. CIn was higher in rats fed HP than in rats fed LP in controls (3.4 vs. 2.1 ml/min) and in rats with NX up to 70% after feeding for 3 wk (1.4 vs. 0.7 ml/min) or 4 days (1.5 vs. 1.1 ml/min). The difference decreased from 0% to 70% NX, and disappeared when NX exceeded 70%. Acute AA infusion and intragastric loads always increased CIn with wide individual variations. The increase was greater in rats fed HP than in rats fed MP and LP (+1.4 vs. 0.8 and 1.1 ml/min for 0% NX), diminished with greater NX (0.7 vs. 0.2 and 0.4 ml/min for 70% NX), and was very small for NX above 70%. However, when expressed as the percent of baseline values, the mean CIn increment after acute stimulation remained constant (30-45%), regardless of renal ablation and of diet. Thus preexisting hyperfiltration resulting from diet or from renal ablation does not suppress the glomerular response to an acute protein load, and acute loads afford no advantages over baseline glomerular filtration rate (GFR) measurements. By contrast, chronic protein feeding increases GFR only when nephron loss is not too severe.

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)

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.

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.

Renal effect of anti-hypertensive drugs depends on sodium diet in the excision remnant kidney model.

Angiotensin converting enzyme inhibitors (ACEI) are believed to protect remnant kidney, but all previous studies used the ligation model which causes severe hypertension, and very few have compared drugs in rats having similar control of blood pressure (BP). We compared rats with uremia obtained by 70% excision of total renal mass, a model which causes mild, late hypertension. Study I compared the effects of enalapril (E), cicletanine (C) and placebo (P) in uremic (U) rats fed a 0.50% (normal-high) Na diet. Study II compared the effects of E, C, P, and guanfacine (G) in U rats fed a diet restricted to 0.25% Na (normal-low). In study I, UP rats developed progressive hypertension (140, 146, 160 and 166 mm Hg at 3, 6, 9 and 12 weeks), proteinuria (240 mg/day at 9 and 12 weeks) which were not affected by E or C. The occurrence of end-stage renal disease (ESRD) led to the sacrifice of all rats after three months. All three groups had similar severe renal lesions (over 25% sclerosed glomeruli in 5 of 10 UP, 9 of 14 UE, 7 of 14 UC rats, with huge cystic tubular dilatations). In study II, rats could be sacrificed later (6 months) and had evidence of less severe renal disease. All the drugs tested prevented hypertension throughout the study (P less than 0.001), with lowest values in UE rats. E and G, but not C, reduced proteinuria. Renal damage was reduced with E and G, but not with C, despite similar BP in C and G rats. Thus, in contrast with what was obtained in the ligation model, ACEI affected neither the BP nor the renal lesions of rats made uremic by renal excision and fed a 0.50% Na diet. Moderate Na restriction improved the consequences of nephron loss and restored the anti-hypertensive effect of drugs. However, these drugs had a different effect on renal preservation: it was dramatic with E, good with G, and undetectable with C.