Myocardial fibrosis and apoptosis, but not inflammation, are present in long-term experimental diabetes.

The aim of this paper is to study the myocardial damage secondary to long-term streptozotocin-induced type 1 diabetes mellitus (DM1). Normotensive and spontaneously hypertensive rats (SHR) received either streptozotocin injections or vehicle. After 22 or 6 wk, DM1, SHR, DM1/SHR, and control rats were killed, and the left ventricles studied by histology, quantitative PCR, Western blot, ELISA, and electromobility shift assay. Cardiomyocyte cultures were also performed. The expression of profibrotic factors, transforming growth factor-beta (TGF-beta1), connective tissue growth factor, and matrix proteins was increased, and the TGF-beta1-linked transcription factors phospho-Smad3/4 and activator protein-1 were activated in the DM1 myocardium. Proapoptotic molecules FasL, Fas, Bax, and cleaved caspase-3 were also augmented. Myocardial injury in long-term hypertension shared these features. In addition, hypertension was associated with activation of NF-kappaB, increased inflammatory cell infiltrate, and expression of the mediators [interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, monocyte chemoattractant protein 1, vascular cell adhesion molecule 1, angiotensinogen, and oxidants], which were absent in long-term DM1. At this stage, the combination of DM1 and hypertension resulted in nonsignificant additive effects. Moreover, the coexistence of DM1 blunted the inflammatory response to hypertension. Anti-inflammatory IL-10 and antioxidants were induced in long-term DM1 and DM1/SHR hearts. Myocardial inflammation was, however, observed in the short-term model. In cultured cardiomyocytes, IL-10, TGF-beta1, and catalase blocked the glucose-stimulated expression of proinflammatory genes. Fibrosis and apoptosis are features of long-term myocardial damage in experimental DM1. Associated hypertension does not induce additional changes. Myocardial inflammation is present in hypertension and short-term DM1, but is not a key feature in long-term DM1. Local reduction of proinflammatory factors and expression of anti-inflammatory and antioxidant molecules may underlie this effect.

Apoptosis: from advances in PD to therapeutic targets in DM.

A high glucose concentration is shared by peritoneal dialysis (PD) and diabetes mellitus (DM). High glucose leads to tissue injury in diabetes. Peritoneal dialysis research has emphasized the role of glucose degradation products in tissue injury. Apoptosis induction is one of the mechanisms of tissue injury induced both by glucose and glucose degradation products. We now review the role of apoptosis and its regulation by glucose degradation products in antibacterial defense and loss of renal function in diabetes mellitus and peritoneal dialysis. The pathogenic role of the recently identified glucose degradation product 3,4-di-deoxyglucosone- 3-ene (3,4-DGE) is detailed. Available therapeutic strategies include the use of peritoneal dialysis solutions containing a low concentration of glucose degradation products. Based on preclinical results, specific targeting of apoptosis regulatory factor should be explored in the clinical setting.

Modulation of renal tubular cell survival: where is the evidence?

Tubular cell loss is prominent both in acute and chronic renal failure. Apoptosis and its regulatory mechanisms contribute to cell number regulation in the kidney. The potential role of apoptosis ranges from induction and progression to repair of renal injury. However, therapeutic interest has focused in preventing the apoptotic loss of tubular cells that leads to acute and chronic renal failure. Death ligands and receptors, such as tumor necrosis factor and Fas ligand, proapoptotic and antiapoptotic Bcl2 family members and caspases have all been shown to participate in apoptosis regulation in the course of renal cell injury. Nevertheless, the precise role of these proteins is unclear, and the participation of most known apoptosis regulatory proteins has not been studied. We now review the role of apoptosis in renal injury, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis, with emphasis in acute renal failure.

TWEAK favors phosphate-induced calcification of vascular smooth muscle cells through canonical and non-canonical activation of NFκB.

Vascular calcification (VC) is associated with increased cardiovascular mortality in aging, chronic kidney disease (CKD), type 2 diabetes mellitus (T2DM) and atherosclerosis. TNF-like weak inducer of apoptosis (TWEAK) recently emerged as a new biomarker for the diagnosis and prognosis of cardiovascular diseases. TWEAK binding to its functional receptor Fn14 was reported to promote several steps of atherosclerotic plaque progression. However, no information is currently available on the role of TWEAK/Fn14 on the development of medial calcification, which is highly prevalent in aging, CKD and T2DM. This study explored the involvement of TWEAK in human vascular smooth muscle cells (h-VSMCs) calcification in vitro. We report that TWEAK binding to Fn14 promotes inorganic phosphate-induced h-VSMCs calcification, favors h-VSMCs osteogenic transition, decreasing acta2 and myh11 and increasing bmp2 mRNA and tissue non-specific alkaline phosphatase (TNAP), and increases MMP9 activity. Blockade of the canonical NFκB pathway reduced by 80% TWEAK pro-calcific properties and decreased osteogenic transition, TNAP and MMP9 activity. Blockade of non-canonical NFκB signaling by a siRNA targeting RelB reduced by 20% TWEAK pro-calcific effects and decreased TWEAK-induced loss of h-VSMCs contractile phenotype and MMP9 activity, without modulating bmp2 mRNA or TNAP activity. Inhibition of ERK1/2 activation by a MAPK kinase inhibitor did not influence TWEAK pro-calcific properties. Our results suggest that TWEAK/Fn14 directly favors inorganic phosphate-induced h-VSMCs calcification by activation of both canonical and non-canonical NFκB pathways. Given the availability of neutralizing anti-TWEAK strategies, our study sheds light on the TWEAK/Fn14 axis as a novel therapeutic target in the prevention of VC.

Albumin-induced apoptosis of tubular cells is modulated by BASP1.

Albuminuria promotes tubular injury and cell death, and is associated with faster progression of chronic kidney disease (CKD) to end-stage renal disease. However, the molecular mechanisms regulating tubular cell death in response to albuminuria are not fully understood. Brain abundant signal protein 1 (BASP1) was recently shown to mediate glucose-induced apoptosis in tubular cells. We have studied the role of BASP1 in albumin-induced tubular cell death. BASP1 expression was studied in experimental puromycin aminonucleoside-induced nephrotic syndrome in rats and in human nephrotic syndrome. The role of BASP1 in albumin-induced apoptosis was studied in cultured human HK2 proximal tubular epithelial cells. Puromycin aminonucleoside induced proteinuria and increased total kidney BASP1 mRNA and protein expression. Immunohistochemistry localized the increased BASP1 to tubular cells. BASP1 expression colocalized with deoxynucleotidyl-transferase-mediated dUTP nick-end labeling staining for apoptotic cells. Increased tubular BASP1 expression was observed in human proteinuric nephropathy by immunohistochemistry, providing evidence for potential clinical relevance. In cultured tubular cells, albumin induced apoptosis and increased BASP1 mRNA and protein expression at 6-48 h. Confocal microscopy localized the increased BASP1 expression in albumin-treated cells mainly to the perinuclear area. A peripheral location near the cell membrane was more conspicuous in albumin-treated apoptotic cells, where it colocalized with actin. Inhibition of BASP1 expression by a BASP1 siRNA protected from albumin-induced apoptosis. In conclusion, albumin-induced apoptosis in tubular cells is BASP1-dependent. This information may be used to design novel therapeutic approaches to slow CKD progression based on protection of tubular cells from the adverse consequences of albuminuria.

Apoptotic cell death in renal injury: the rationale for intervention.

Cell number abnormalities are frequent in renal diseases, and range from the hypercellularity of postinfectious glomerulonephritis to the cell depletion of chronic renal atrophy. Recent research has shown that apoptosis and its regulatory mechanisms contribute to cell number regulation in the kidney. The potential role of apoptosis ranges from induction and progression to repair of renal injury. Death ligands and receptors, such as tumor necrosis factor and Fas ligand, proapoptotic and antiapoptotic Bcl2 family members and caspases have all been shown to participate in apoptosis regulation in the course of renal cell injury. However, the precise role of these proteins is unclear, and the participation of most known apoptosis regulatory proteins has not been studied. We now review the role of apoptosis in renal injury, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis, with emphasis on the acute tubular necrosis.

MIF, CD74 and other partners in kidney disease: tales of a promiscuous couple.

Macrophage migration inhibitory factor (MIF) is increased in kidney and urine during kidney disease. MIF binds to and activates CD74 and chemokine receptors CXCR2 and CXCR4. CD74 is a protein trafficking regulator and a cell membrane receptor for MIF, D-dopachrome tautomerase (D-DT/MIF-2) and bacterial proteins. MIF signaling through CD74 requires CD44. CD74, CD44 and CXCR4 are upregulated in renal cells in diseased kidneys and MIF activation of CD74 in kidney cells promotes an inflammatory response. MIF or CXCR2 targeting protects from experimental kidney injury, CD44 deficiency modulates kidney injury and CXCR4 activation promotes glomerular injury. However, the contribution of MIF or MIF-2 to these actions of MIF receptors has not been explored. The safety and efficacy of strategies targeting MIF, CD74, CD44 and CXCR4 are under study in humans.

Lethal activity of FADD death domain in renal tubular epithelial cells.

Fas-associated death domain (FADD) is an adaptor protein that is required for the transmission of the death signal from lethal receptors of the tumor necrosis factor superfamily. FADD contains a death domain (DD) and a death effector domain (DED). As death receptors contribute to renal tubular injury and tubular cell FADD increases in acute renal failure, we have studied the function of FADD in tubular epithelium. FADD expression was studied in kidney samples from mice. In order to study the contribution of FADD to renal tubular cell survival, FADD or FADD-DD were overexpressed in murine tubular epithelium. FADD is expressed in renal tubules of the healthy kidney. Both FADD and FADD-DD induce apoptosis in primary cultures of murine tubular epithelium and in the murine cortical tubular cell line. Death induced by FADD-DD has apoptotic morphology, but differs from death receptor-induced apoptosis in that it is not blocked by inhibitors of caspases. Neither an inhibitor of serine proteases nor overexpression of antiapoptotic BclxL prevented cell death. However, the combination of caspase and serine protease inhibition was protective. FADD and FADD-DD overexpression decreased nuclear factor kappa B activity. These data suggest that FADD has a death regulatory function in renal tubular cells that is independent of death receptors. FADD-DD is sufficient to induce apoptosis in these cells. This information is relevant to understanding the role of FADD in tubular injury.

Cytokine cooperation in renal tubular cell injury: the role of TWEAK.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFalpha/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury.