Laurent Baud

Targeting the Calpain/Calpastatin System as a New Strategy to Prevent Cardiovascular Remodeling in Angiotensin II–Induced Hypertension

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II–induced hypertrophy of left ventricle. Ang II–dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II–induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Peroxisome Proliferator-Activated Receptor β/δ Exerts a Strong Protection from Ischemic Acute Renal Failure

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells.

Human recombinant tumor necrosis factor‐α (TNF) was found to stimulate the production of prostaglandins (PG) by cultured rat mesangial cells. This effect was demonstrable from 6 h, was dose dependent and affected the synthesis of PGE2, PGF2α, and 6‐keto‐PGF1α. It required both RNA and protein synthesis but was not associated with a modification of cell proliferation. TNF also stimulated adenosine 3′–5′ cyclic monophosphate (cAMP) levels in the mesangial cell culture medium. Indomethacin suppressed the effect of TNF on PGs but only reduced that on cAMP, indicating that PG production partly mediates the increase in cAMP. These findings demonstrate that mesangial cells can be a target for TNF and that the mechanism of TNF action includes stimulation of both PG production and cAMP levels.

Glomerular hyperfiltration in adult sickle cell anemia: a frequent hemolysis associated feature.

BACKGROUND AND OBJECTIVES Sickle cell anemia-associated nephropathy is a growing matter of concern because renal failure affects most aging sickle cell anemia patients. Glomerular damage is a common feature revealed by a microalbuminuria or a macroalbuminuria. Although glomerular hyperfiltration has been described for decades in this population, its prevalence in young adults is unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS To address this issue, as well as the clinical and biologic correlates of hyperfiltration, a single-center, cross-sectional study of 280 homozygous SS disease patients was performed. RESULTS The prevalence of hyperfiltration assessed by Modification of Diet in Renal Disease estimated GFR was 51%. Among patients with hyperfiltration, 49% had hyperfiltration alone, whereas 36% and 15% had an associated microalbuminuria or macroalbuminuria, respectively. Estimated GFR sensitivity and specificity for hyperfiltration were 94% and 63%, respectively, in a selected subgroup of 48 patients (measured GFR was assessed by urinary (51)Cr EDTA clearance). In patients with no albuminuria, hyperfiltration status was significantly associated with a young age (years), the absence of alpha thalassemia, a lower hemoglobin level (g/dl), and a lower fetal hemoglobin. The role of chronic hemolysis was further strengthened by multivariate analysis showing a correlation between estimated GFR and a low plasma fetal hemoglobin level, a young age, and a high reticulocyte count (r(2) = 0.54). CONCLUSIONS Together, the data suggest that the pathophysiology of hyperfiltration would rather be attributable to the hemolysis-associated vasculopathy rather than a viscosity-vaso-occlusive process.

INSULIN ACTIVATES NUCLEAR FACTOR KAPPA B IN MAMMALIAN CELLS THROUGH A RAF-1-MEDIATED PATHWAY

We examined the effect of insulin on nuclear factor κB (NF-κB) activity in Chinese hamster ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr1162 and Tyr1163 autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-κB. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-κB: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with IκB degradation and/or NF-κB•IκB complex dissociation; 3) was independent of both PMA-sensitive and atypical (ζ) protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-κB DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-κB-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-κB in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.

Calpain Activation and Secretion Promote Glomerular Injury in Experimental Glomerulonephritis: Evidence from Calpastatin-Transgenic Mice

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.

In vitro production of tumour necrosis factor and prostaglandin E2 by peripheral blood mononuclear cells from tuberculosis patients

We investigated the production of tumour necrosis factor‐alpha (TNF‐α) and prostaglandin E2 (PGE2) by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy controls. PBMC from tuberculosis patients generated constitutively more TNF‐α than did control PBMC. This production was significantly higher for patients with high‐grade fever and cachexia. The increase of TNF‐α production by PBMC from tuberculosis patients was associated with a comparatively weaker elevation of PGE2 synthesis which did not parallel fever or weight loss. In vitro treatment of control PBMC with the tuberculin purified protein derivative (PPD) promoted an increased TNF‐α production which was similar to that of untreated PBMC from tuberculosis patients. Thus, the increased TNF‐α production in tuberculosis could be explained by the in vivo exposure of PBMC to mycobacterial antigens. In contrast, the concentration of PGE; was weaker in the medium of untreated PBMC from tuberculosis patients than in the medium of PPD‐treated control PBMC, suggesting that PGE, synthesis by PBMC was limited in tuberculosis by unidentified factors.

Leukotrienes and other lipoxygenase products of arachidonic acid synthesized in the kidney

Lipoxygenase products are synthesized in the kidney. Rabbit medulla and murine and human glomeruli produce 12- and 15-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Minor amounts of leukotrienes are formed under normal conditions, but it is likely that the resident renal cells are capable of synthesizing these metabolites. Rat glomeruli and papillae possess the enzymes necessary to process leukotriene C4 into leukotrienes D4 and E4. However, the enzyme activity of the papillae is masked due to the presence of an inhibitor detected in the 10,000 g supernate of the papillary homogenate. 12-HETE synthesis is markedly increased in glomeruli from rats with nephrotoxic serum nephritis and leukotriene B4 synthesis in glomeruli from rats with cationic bovine gamma-globulin-induced glomerulonephritis. In vivo consequences of the association between the resident glomerular cells and the bone marrow-derived cells have been studied in vitro in co-incubation experiments. Glomeruli release factors that stimulate the cyclo-oxygenase and lipoxygenase pathways in macrophages. Co-incubation of glomeruli, platelets, and polymorphonuclear leukocytes results in the formation of 12,20-diHETE and an excess of 12-HETE. Lipoxygenase products, regardless of their origin, modify the renal functions. Leukotriene C4 binds specifically to rat glomeruli and human cultured glomerular epithelial cells. Leukotrienes C4 or D4 administered in vivo cause renal vasoconstriction and a decline in the glomerular filtration rate. In vitro, these two sulfidopeptide leukotrienes promote epithelial cell proliferation and produce mesangial cell contraction. The lipoxygenase pathway is also implicated in the attachment of macrophages to glomeruli and in the oxidative burst of glomerular mesangial cells during phagocytosis. The future use of specific inhibitors of the synthesis or antagonists of the lipoxygenase products, particularly the leukotrienes, should provide a tool for evaluating the role of these metabolites in renal diseases.

Calpastatin Controls Polymicrobial Sepsis by Limiting Procoagulant Microparticle Release

RATIONALE Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release. OBJECTIVES We investigated the contribution of calpain in eliciting tissue damage during sepsis. METHODS To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. MEASUREMENTS AND MAIN RESULTS In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy. CONCLUSIONS These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.

Determinants of Osteopenia in Male Renal-Stone–Disease Patients with Idiopathic Hypercalciuria

BACKGROUND AND OBJECTIVES Bone demineralization is frequent in renal-stone formers with hypercalciuria. Although this pathologic link has been recognized for decades, the underlying mechanisms and risk factors associated with osteopenia/osteoporosis in this population remain partially understood. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This study retrospectively analyzed determinants of low bone mineral density (BMD) in 65 idiopathic hypercalciuric male renal-stone formers. Clinical and biologic evaluation included BMD measurement, bone-remodeling markers, analysis of calcium metabolism with oral calcium load test, and dietary inquiry. RESULTS Patients with osteopenia (n=23, 35% of the population) presented significantly higher fasting calciuria as compared with normal bone density patients (n=42) (calcium/creatinine ratio was 0.32 versus 0.24 mmol/mmol; P=0.006). Analysis of the whole population revealed a negative association between fasting hypercalciuria and BMD (P = 0.003), independent of confounding variables including body-mass index and tobacco consumption. The fasting calcium/creatinine ratio above 0.25 mmol/mmol was associated with a 3.8-fold increase in the risk of low BMD. CONCLUSION In our study, fasting hypercalciuria after a 2-day calcium-restricted diet appears as the only biologic factor associated with low BMD, suggesting a bone-calcium efflux. Our results support the view of a parathyroid-independent pathologic process that remains to be identified. Hypercalciuric patients with low BMD do not excrete more calcium in 24-hour urine samples than patients without low BMD.