Protein load impairs factor H binding promoting complement-dependent dysfunction of proximal tubular cells

Intrarenal complement activation plays an important role in the progression of chronic kidney disease. A key target of the activated complement cascade is the proximal tubule, a site where abnormally filtered plasma proteins and complement factors combine to promote injury. This study determined whether protein overloading of human proximal tubular cells (HK-2) in culture enhances complement activation by impairing complement regulation. Addition of albumin or transferrin to the cells incubated with diluted human serum as a source of complement caused increased apical C3 deposition. Soluble complement receptor-1 (an inhibitor of all 3 activation pathways) blocked complement deposition while the classical and lectin pathway inhibitor, magnesium chloride–EGTA, was, ineffective. Media containing albumin as well as complement had additive proinflammatory effects as shown by increased fractalkine and transforming growth factor-β mRNA expression. This paralleled active C3 and C5b-9 generations, effects not shared by transferrin. Factor H, one of the main natural inhibitors of the alternative pathway, binds to heparan sulfate proteoglycans. Both the density of heparan sulfate and factor H binding were reduced with protein loading, thereby enhancing the albumin- and serum-dependent complement activation potential. Thus, protein overload reduces the ability of the tubule cell to bind factor H and counteract complement activation, effects instrumental to renal disease progression

Sirtuin3 Dysfunction Is the Key Determinant of Skeletal Muscle Insulin Resistance by Angiotensin II

<div><p>Background</p><p>Angiotensin II promotes insulin resistance. The mechanism underlying this abnormality, however, is still poorly defined. In a different setting, skeletal muscle metabolism and insulin signaling are regulated by Sirtuin3.</p><p>Objective</p><p>Here, we investigate whether angiotensin II-induced insulin resistance in skeletal muscle is associated with Sirtuin3 dysregulation and whether pharmacological manipulation of Sirtuin3 confers protection.</p><p>Study Design</p><p>Parental and GLUT4-myc L6 rat skeletal muscle cells exposed to angiotensin II are used as <i>in vitro</i> models of insulin resistance. GLUT4 translocation, glucose uptake, intracellular molecular signals such as mitochondrial reactive oxygen species, Sirtuin3 protein expression and activity, along with its downstream targets and upstream regulators, are analyzed both in the absence and presence of acetyl-L-carnitine. The role of Sirtuin3 in GLUT4 translocation and intracellular molecular signaling is also studied in Sirtuin3-silenced as well as over-expressing cells.</p><p>Results</p><p>Angiotensin II promotes insulin resistance in skeletal muscle cells via mitochondrial oxidative stress, resulting in a two-fold increase in superoxide generation. In this context, reactive oxygen species open the mitochondrial permeability transition pore and significantly lower Sirtuin3 levels and activity impairing the cell antioxidant defense. Angiotensin II-induced Sirtuin3 dysfunction leads to the impairment of AMP-activated protein kinase/nicotinamide phosphoribosyltransferase signaling. Acetyl-L-carnitine, by lowering angiotensin II-induced mitochondrial superoxide formation, prevents Sirtuin3 dysfunction. This phenomenon implies the restoration of manganese superoxide dismutase antioxidant activity and AMP-activated protein kinase activation. Acetyl-L-carnitine protection is abrogated by specific Sirtuin3 siRNA.</p><p>Conclusions</p><p>Our data demonstrate that angiotensin II-induced insulin resistance fosters mitochondrial superoxide generation, in turn leading to Sirtuin3 dysfunction. The present results also highlight Sirtuin3 as a therapeutic target for the insulin-sensitizing effects of acetyl-L-carnitine.</p></div

Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function

Ang II inhibits Sirt3 deacetylase activity and impairs mitochondrial antioxidant defense.

<p>(A) Western blot of acetylated proteins in mitochondria from control and Ang II-treated L6 myotubes in the absence and presence of ALCAR or MnTBAP. The arrow marks the band corresponding to MnSOD that was revealed on the same membrane after stripping and incubation with an anti-MnSOD specific antibody. Expression of the loading protein VDAC was analyzed by western blot in the same samples run in parallel. (B) MnSOD activity in isolated mitochondria. Results are mean ± SE (n = 5). °°P < 0.01,°°°P < 0.001 <i>vs</i>. Control; ***P < 0.001 <i>vs</i>. Ang II.</p

Ang II promotes insulin resistance via mitochondrial ROS.

<p>Effect of Ang II on (A) mitochondrial O₂^• generation, (B) insulin (Ins)-stimulated GLUT4 transport (densitometric analysis, top, and representative western blot, bottom) and (C) Ins-stimulated 2-deoxyglucose (2-DG) uptake in L6 myotubes in the absence and presence of ALCAR (0.6 mM). MFI, mean fluorescence intensity; CPM, crude plasma membrane. (D) Effect of Ang II on surface GLUT4-myc density in Ins-stimulated cells in the absence and presence of ALCAR or MnTBAP (0.1 mM). Results are mean ± SE (n = 3, A-C; n = 6, D). °P < 0.05, °°P < 0.01, °°°P < 0.001 <i>vs</i>. Control; •P < 0.05, ••P < 0.01, •••P < 0.001 <i>vs</i>. Ins; ***P < 0.001 <i>vs</i>. Ang II; #P < 0.05, ##P < 0.01, ###P < 0.001 <i>vs</i>. Ang II + Ins.</p