Endothelin-targeted new treatments for proteinuric and inflammatory glomerular diseases: focus on the added value to anti-renin-angiotensin system inhibition.

Chronic kidney disease (CKD) is the main cause of end-stage renal disease worldwide arising as a frequent complication of diabetes, obesity, and hypertension. Current therapeutic options, mainly based of inhibition of the renin-angiotensin system (RAS), provide imperfect renoprotection if started at an advanced phase of the disease, and treatments that show or even reverse the progression of CKD are needed. The endothelin (ET) system contributes to the normal renal physiology; however, robust evidence suggests a key role of ET-1 and its cognate receptors, in the progression of CKD. The effectiveness of ET receptor antagonists in ameliorating renal hemodynamics and fibrosis has been largely demonstrated in different experimental models. A significant antiproteinuric effect of ET receptor antagonists has been found in diabetic and non-diabetic CKD patients even on top of RAS blockade, and emerging evidence from ongoing clinical trials highlights their beneficial effects on a wide range of kidney disorders.

Shiga toxin triggers endothelial and podocyte injury: the role of complement activation.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the offending agent in post-diarrhea-associated hemolytic uremic syndrome (HUS), a disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure, with thrombi occluding the renal microvasculature. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Glomerular endothelial cells are susceptible to the toxic effects of Stxs that, via nuclear factor kappa B (NF-κB) activation, induce the expression of genes encoding for adhesion molecules and chemokines, culminating in leukocyte adhesion and platelet thrombus formation on the activated endothelium. Complement activation via the alternative pathway has been seen in patients during the acute phase of STEC-associated HUS. Experimental evidence has highlighted the role of complement proteins in driving glomerular endothelium toward a thrombogenic phenotype. At the glomerular level, podocytes are also an important target of Stx-induced complement activation. Glomerular injury as a consequence of podocyte dysfunction and loss is thus a mechanism that might affect long-term renal outcomes in the disease. New approaches to targeting the complement system may be useful therapeutic options for patients with STEC-HUS.

Shiga toxin promotes podocyte injury in experimental hemolytic uremic syndrome via activation of the alternative pathway of complement.

Shiga toxin (Stx)-producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS.

ß-arrestin-1 drives endothelin-1-mediated podocyte activation and sustains renal injury.

Activation of endothelin-A receptor (ET(A)R) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through ß-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ET(A)R activation and increased ß-arrestin-1 expression. Activated ET(A)R recruited ß-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and ß-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1-induced migration as confirmed by Snail knockdown experiments. Silencing of ß-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ET(A)R-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; ß-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ET(A)R antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular ß-arrestin-1 and Snail. Increased ß-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ET(A)R antagonism for a group of diseases still needing a specific treatment.

MYO1E mutations and childhood familial focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS: We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS: We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS: MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Angiotensin II contributes to diabetic renal dysfunction in rodents and humans via Notch1/Snail pathway.

In nondiabetic rat models of renal disease, angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. Herein, we wanted to explore the role of Ang II in diabetic nephropathy by a translational approach spanning from in vitro to in vivo rat and human studies, and to dissect the intracellular pathways involved. In isolated perfused rat kidneys and in cultured human podocytes, Ang II down-regulated nephrin expression via Notch1 activation and nuclear translocation of Snail. Hairy enhancer of split-1 was a Notch1-downstream gene effector that activated Snail in cultured podocytes. In vitro changes of the Snail/nephrin axis were similar to those in renal biopsy specimens of Zucker diabetic fatty rats and patients with advanced diabetic nephropathy, and were normalized by pharmacological inhibition of the renin-angiotensin system. Collectively, the present studies provide evidence that Ang II plays a relevant role in perpetuating glomerular injury in experimental and human diabetic nephropathy via persistent activation of Notch1 and Snail signaling in podocytes, eventually resulting in down-regulation of nephrin expression, the integrity of which is crucial for the glomerular filtration barrier.

The complement C3a and C5a signaling in renal diseases: a bridge between acute and chronic inflammation.

The complement system, a cornerstone of the innate immune defense, typically confers protection against pathogens. However, in various clinical scenarios the complement's defensive actions can harm host cells, exacerbating immune and inflammatory responses. The central components C3 and C5 undergo proteolytic cleavage during complement activation, yielding small active fragments C3a and C5a anaphylatoxins. Traditionally these fragments were associated with inflammation via the specific receptors C3a receptor (R), C5aR1 and C5aR2. Recent insights, however, spotlight the excessive C3a/C3aR and C5a/C5aR1 signaling as culprits in diverse disorders of inflammatory and autoimmune etiology. This is particularly true for several kidney diseases, where the potential involvement of anaphylatoxins in renal damage is supported by the enhanced renal expression of their receptors and the high levels of C3a and C5a in both plasma and urine. Furthermore, the production of complement proteins in the kidney, with different renal cells synthesizing C3 and C5, significantly contributes to local tissue injury. In the present review, we discuss the different aspects of C3a/C3aR and C5a/C5aR signaling in acute and chronic kidney diseases, and explore the therapeutic potential of emerging targeted drugs for future clinical applications.

Alternative pathway activation of complement by Shiga toxin promotes exuberant C3a formation that triggers microvascular thrombosis.

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti-P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

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.

Membranous nephropathy associated with IgG4-related disease.

Immunoglobulin G4 (IgG4)-related systemic disease is a rare condition characterized by high levels of circulating IgG4 and IgG4-positive plasma cell infiltrates in various organs, including the pancreas, salivary glands, biliary tract, liver, lung, and kidney. We describe a case of a 54-year-old man with IgG4-related systemic disease presenting with autoimmune pancreatitis and Mikulicz disease. Steroid therapy decreased circulating IgG4 levels and promoted regression of clinical signs. Thereafter, an increase in serum IgG4 values was followed by the occurrence of nephrotic-range proteinuria. Kidney biopsy showed membranous nephropathy with no IgG4-positive cell infiltrates. A search for circulating immune complexes was negative, and antibodies against M-type phospholipase A(2) receptor could not be detected. Western blot analyses identified circulating IgG3 reacting with superoxide dismutase 2. This case suggests that membranous nephropathy represents an additional renal manifestation of IgG4-related systemic disease, with a pathogenesis possibly associated with neoproduction of autoantibodies targeting podocyte antigen(s).

Fractalkine and CX3CR1 mediate leukocyte capture by endothelium in response to Shiga toxin.

Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-kappaB and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IkappaBalpha inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.

Shiga toxin-associated hemolytic uremic syndrome: pathophysiology of endothelial dysfunction.

Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli O157:H7 has become a global threat to public health, as a primary cause of a worldwide spread of hemorrhagic colitis complicated by diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure that mainly affects early childhood. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Endothelial cells, mainly those located in the renal microvasculature, are primary targets of the toxic effects of Stx1 and 2. Stxs bound to their specific globotriaosylceramide (Gb3Cer) receptor on the cell surface trigger a cascade of signaling events, involving NF-κB activation, that induce expression of genes encoding for adhesion molecules and chemokines, and culminate in the adhesion of leukocytes to endothelial cells, thereby increasing the endothelial susceptibility to leukocyte-mediated injury. Activated endothelial cells in response to Stxs lose the normal thromboresistance phenotype and become thrombogenic, initiating microvascular thrombus formation. Evidence is emerging that complement activation in response to Stxs favors platelet thrombus formation on endothelial cells, which may play a role in amplifying the inflammation-thrombosis circuit in Stx-associated HUS.

Proteasomal processing of albumin by renal dendritic cells generates antigenic peptides.

The role of dendritic cells (DC) that accumulate in the renal parenchyma of non-immune-mediated proteinuric nephropathies is not well understood. Under certain circumstances, DC capture immunologically ignored antigens, including self-antigens, and present them within MHC class I, initiating an autoimmune response. We studied whether DC could generate antigenic peptides from the self-protein albumin. Exposure of rat proximal tubular cells to autologous albumin resulted in its proteolytic cleavage to form an N-terminal 24-amino acid peptide (ALB1-24). This peptide was further processed by the DC proteasome into antigenic peptides that had binding motifs for MHC class I and were capable of activating syngeneic CD8+ T cells. In vivo, the rat five-sixths nephrectomy model allowed the localization and activation of renal DC. Accumulation of DC in the renal parenchyma peaked 1 wk after surgery and decreased at 4 wk, concomitant with their appearance in the renal draining lymph nodes. DC from renal lymph nodes, loaded with ALB1-24, activated syngeneic CD8+ T cells in primary culture. The response of CD8+ T cells of five-sixths nephrectomized rats was amplified with secondary stimulation. In contrast, DC from renal lymph nodes of five-sixths nephrectomized rats treated with the proteasomal inhibitor bortezomib lost their capacity to stimulate CD8+ T cells in primary and secondary cultures. These data suggest that albumin can be a source of potentially antigenic peptides upon renal injury and that renal DC play a role in processing self-proteins through a proteasome-dependent pathway.

C3a receptor blockade protects podocytes from injury in diabetic nephropathy.

Renal activation of the complement system has been described in patients with diabetic nephropathy (DN), although its pathological relevance is still ill-defined. Here, we studied whether glomerular C3a, generated by uncontrolled complement activation, promotes podocyte damage, leading to proteinuria and renal injury in mice with type 2 diabetes. BTBR ob/ob mice exhibited podocyte loss, albuminuria, and glomerular injury accompanied by C3 deposits and increased C3a and C3a receptor (C3aR) levels. Decreased glomerular nephrin and α-actinin4 expression, coupled with integrin-linked kinase induction, were also observed. Treatment of DN mice with a C3aR antagonist enhanced podocyte density and preserved their phenotype, limiting proteinuria and glomerular injury. Mechanistically, ultrastructural and functional mitochondrial alterations, accompanied by downregulation of antioxidant superoxide dismutase 2 (SOD2) and increased protein oxidation, occurred in podocytes and were normalized by C3aR blockade. In cultured podocytes, C3a induced cAMP-dependent mitochondrial fragmentation. Alterations of mitochondrial membrane potential, SOD2 expression, and energetic metabolism were also found in response to C3a. Notably, C3a-induced podocyte motility was inhibited by SS-31, a peptide with mitochondrial protective effects. These data indicate that C3a blockade represents a potentially novel therapeutic strategy in DN for preserving podocyte integrity through the maintenance of mitochondrial functions.

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-beta 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.

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections have become a threat to public health globally because of the severe illnesses that they can trigger, such as hemorrhagic colitis and the post-diarrheal hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Glomerular endothelial cells are primary targets of Stx which, after binding to its specific receptor globotriaosylceramide, upregulates proinflammatory proteins involved both in the recruitment and adhesion of leukocytes and thrombus formation at the site of endothelial injury. In this review, we discuss the role of complement activation in promoting glomerular microvascular dysfunction, providing evidence from experimental models and patients with STEC-HUS. Within the glomerulus, an important target for Stx-induced complement activation is the podocyte, a cell type that is in close contact with endothelial cells and participates in maintaining the filtration barrier. Recently, podocyte injury and loss have been indicated as potential risk factors for long-term renal sequelae in patients with STEC-HUS. Therapeutic approaches targeting the complement system, that may be useful options for patients with STEC-HUS, will also be discussed.

SGLT2 inhibitor dapagliflozin limits podocyte damage in proteinuric nondiabetic nephropathy.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have pleiotropic properties beyond blood glucose-lowering effects and modify important nonglycemic pathways, leading to end-organ protection. SGLT2 inhibitors display renoprotective effects in diabetic kidney disease, which creates a rationale for testing the therapeutic potential of this drug class in nondiabetic chronic kidney disease. Here, we have shown that dapagliflozin provided glomerular protection in mice with protein-overload proteinuria induced by bovine serum albumin (BSA), to a similar extent as an ACE inhibitor used as standard therapy for comparison. Dapagliflozin limited proteinuria, glomerular lesions, and podocyte dysfunction and loss. We provide the observation that SGLT2 was expressed in podocytes and upregulated after BSA injections. Through in vitro studies with cultured podocytes loaded with albumin we have identified what we believe to be a novel mechanism of action for SGLT2 inhibitor that directly targets podocytes and relies on the maintenance of actin cytoskeleton architecture. Whether SGLT2 inhibitors represent a possible future therapeutic option for some patients with proteinuric glomerular disease who do not have as yet an effective treatment will require ad hoc clinical studies.

A previously unrecognized role of C3a in proteinuric progressive nephropathy.

Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh(-/-)) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh(-/-) +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis.

Effects of MCP-1 inhibition by bindarit therapy in a rat model of polycystic kidney disease.

BACKGROUND/AIMS: Experimental and clinical evidence suggested that monocyte chemoattractant protein-1 (MCP-1/CCL2) has a role in the development of interstitial inflammation and renal failure in polycystic kidney disease (PKD). We investigated whether bindarit, an inhibitor of MCP-1/CCL2 synthesis, could influence the evolution of PKD in PCK rats. METHODS: PCK rats were treated from 5 to 15 weeks of age with vehicle or bindarit. Sprague-Dawley rats served as control. For in vitro studies, murine podocytes were exposed to albumin with or without bindarit. RESULTS: MCP-1 mRNA was upregulated in the kidney of PCK rats and reduced by bindarit. Treatment limited overexpression of MCP-1 protein by epithelial cells of dilated tubules and cysts, and interstitial inflammatory cells. Excessive renal accumulation of monocytes/macrophages was lowered by bindarit by 41%. Serum creatinine slightly increased in PCK rats on vehicle and was similar to controls after bindarit. Kidney and liver cysts were not affected by treatment. Bindarit significantly reduced progressive proteinuria of PCK rats. The antiproteinuric effect was associated with the restoration of the defective nephrin expression in podocytes of PCK rats. Bindarit limited podocyte foot process effacement and ameliorated slit diaphragm frequency. In cultured podocytes, bindarit reduced MCP-1 production in response to albumin and inhibited albumin-induced cytoskeletal remodeling and cell migration. CONCLUSION: This study showed that although bindarit did not prevent renal cyst growth, it limited interstitial inflammation and renal dysfunction and reduced proteinuria in PKD. Thus, bindarit could be considered a therapeutic intervention complementary to therapies specifically acting to block renal cyst growth.

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

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: Parental and GLUT4-myc L6 rat skeletal muscle cells exposed to angiotensin II are used as in vitro 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. RESULTS: 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. CONCLUSIONS: 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.