Therapeutic Small Interfering RNA Targeting Complement C3 in a Mouse Model of C3 Glomerulopathy.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.

Empagliflozin protects glomerular endothelial cell architecture in experimental diabetes through the VEGF-A/caveolin-1/PV-1 signaling pathway.

In addition to having blood glucose-lowering effects, inhibitors of sodium glucose cotransporter 2 (SGLT2) afford renoprotection in diabetes. We sought to investigate which components of the glomerular filtration barrier could be involved in the antiproteinuric and renoprotective effects of SGLT2 inhibition in diabetes. BTBR (black and tan, brachyuric) ob/ob mice that develop a type 2 diabetic nephropathy received a standard diet with or without empagliflozin for 10 weeks, starting at 8 weeks of age, when animals had developed albuminuria. Empagliflozin caused marked decreases in blood glucose levels and albuminuria but did not correct glomerular hyperfiltration. The protective effect of empagliflozin against albuminuria was not due to a reduction in podocyte damage as empagliflozin did not affect the larger podocyte filtration slit pore size nor the defective expression of nephrin and nestin. Empagliflozin did not reduce the thickening of the glomerular basement membrane. In BTBR ob/ob mice, the most profound abnormality seen using electron microscopy was in the endothelial aspect of the glomerular capillary, with significant loss of endothelial fenestrations. Remarkably, empagliflozin ameliorated the subverted microvascular endothelial ultrastructure. Caveolae and bridging diaphragms between adjacent endothelial fenestrae were seen in diabetic mice and associated with increased expression of caveolin-1 and the appearance of PV-1. These endothelial abnormalities were limited by the SGLT2 inhibitor. Although no expression of SGLT2 was found in glomerular endothelial cells, SGLT2 was expressed in the podocytes of diabetic mice. VEGF-A, which is a known stimulus for endothelial caveolin-1 and PV-1, was increased in podocytes of BTBR ob/ob mice and normalized by SGLT2 inhibitor treatment. Thus, empagliflozin's protective effect on the glomerular endothelium of diabetic mice could be due to a limitation of the paracrine signaling of podocyte-derived VEGF-A that resulted in a reduction of the abnormal endothelial caveolin-1 and PV-1, with the consequent preservation of glomerular endothelial function and permeability. © 2022 The Pathological Society of Great Britain and Ireland.

Endothelial Glycocalyx of Peritubular Capillaries in Experimental Diabetic Nephropathy: A Target of ACE Inhibitor-Induced Kidney Microvascular Protection.

Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss.

Sirtuin 3 Deficiency Aggravates Kidney Disease in Response to High-Fat Diet through Lipotoxicity-Induced Mitochondrial Damage.

Sirtuin 3 (SIRT3) is the primary mitochondrial deacetylase that controls the antioxidant pathway and energy metabolism. We previously found that renal Sirt3 expression and activity were reduced in mice with type 2 diabetic nephropathy associated with oxidative stress and mitochondrial abnormalities and that a specific SIRT3 activator improved renal damage. SIRT3 is modulated by diet, and to assess whether Sirt3 deficiency aggravates mitochondrial damage and accelerates kidney disease in response to nutrient overloads, wild-type (WT) and Sirt3-/- mice were fed a high-fat-diet (HFD) or standard diet for 8 months. Sirt3-/- mice on HFD exhibited earlier and more severe albuminuria compared to WT mice, accompanied by podocyte dysfunction and glomerular capillary rarefaction. Mesangial matrix expansion, tubular vacuolization and inflammation, associated with enhanced lipid accumulation, were more evident in Sirt3-/- mice. After HFD, kidneys from Sirt3-/- mice showed more oxidative stress than WT mice, mitochondria ultrastructural damage in tubular cells, and a reduction in mitochondrial mass and energy production. Our data demonstrate that Sirt3 deficiency renders mice more prone to developing oxidative stress and mitochondrial abnormalities in response to HFD, resulting in more severe kidney diseases, and this suggests that mitochondria protection may be a method to prevent HFD-induced renal injury.

Addition of cyclic angiotensin-(1-7) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy.

The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.

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.

MicroRNA-184 is a downstream effector of albuminuria driving renal fibrosis in rats with diabetic nephropathy.

AIMS/HYPOTHESIS: Renal fibrosis is a common complication of diabetic nephropathy and is a major cause of end-stage renal disease. Despite the suggested link between renal fibrosis and microRNA (miRNA) dysregulation in diabetic nephropathy, the identification of the specific miRNAs involved is still incomplete. The aim of this study was to investigate miRNA profiles in the diabetic kidney and to identify potential downstream targets implicated in renal fibrosis. METHODS: miRNA expression profiling was investigated in the kidneys of 8-month-old Zucker diabetic fatty (ZDF) rats during overt nephropathy. Localisation of the most upregulated miRNA was established by in situ hybridisation. The candidate miRNA target was identified by in silico analysis and its expression documented in the diabetic kidney associated with fibrotic markers. Cultured tubule cells served to assess which of the profibrogenic stimuli acted as a trigger for the overexpressed miRNA, and to investigate underlying epigenetic mechanisms. RESULTS: In ZDF rats, miR-184 showed the strongest differential upregulation compared with lean rats (18-fold). Tubular localisation of miR-184 was associated with reduced expression of lipid phosphate phosphatase 3 (LPP3) and collagen accumulation. Transfection of NRK-52E cells with miR-184 mimic reduced LPP3, promoting a profibrotic phenotype. Albumin was a major trigger of miR-184 expression. Anti-miR-184 counteracted albumin-induced LPP3 downregulation and overexpression of plasminogen activator inhibitor-1. In ZDF rats, ACE-inhibitor treatment limited albuminuria and reduced miR-184, with tubular LPP3 preservation and tubulointerstitial fibrosis amelioration. Albumin-induced miR-184 expression in tubule cells was epigenetically regulated through DNA demethylation and histone lysine acetylation and was accompanied by binding of NF-κB p65 subunit to miR-184 promoter. CONCLUSIONS/INTERPRETATION: These results suggest that miR-184 may act as a downstream effector of albuminuria through LPP3 to promote tubulointerstitial fibrosis, and offer the rationale to investigate whether targeting miR-184 in association with albuminuria-lowering drugs may be a new strategy to achieve fully anti-fibrotic effects in diabetic nephropathy.

B7-1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy.

The incidence of progressive kidney disease associated with diabetes continues to rise worldwide. Current standard therapy with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers achieves only partial renoprotection, increasing the need for novel therapeutic approaches. Previous studies described B7-1 induction in podocytes of patients with proteinuria, including those with FSGS and type 2 diabetic nephropathy (DN). These findings sparked great excitement in the renal community, implying that abatacept, a costimulatory inhibitor that targets B7-1, could be a novel therapy for diabetic renal disease. Given previous concerns over the value of B7-1 immunostaining and the efficacy of abatacept in patients with recurrent FSGS after renal transplantation, we investigated B7-1 expression in human and experimental DN before embarking on clinical studies of the use of B7-1 targeting strategies to treat proteinuria in DN. Immunohistochemical analysis of kidney specimens using different antibodies revealed that B7-1 is not induced in podocytes of patients with DN, independent of disease stage, or BTBR ob/obmice, a model of type 2 diabetes. These results do not support the use of abatacept as a therapeutic strategy for targeting podocyte B7-1 for the prevention or treatment of DN.

Key pathways in renal disease progression of experimental diabetes.

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus and the leading cause of end-stage kidney disease. Both diabetes and chronic kidney disease are risk factors for cardiovascular disease, and diabetic patients with renal involvement are three times more likely to eventually die of cardiovascular disease than diabetic patients without signs of renal failure. In type 2 diabetes, microalbuminuria is a marker of renal dysfunction and a crucial predictor of cardiovascular disease. Inhibitors of angiotensin II synthesis/activity, while preventing micro- or macroalbuminuria, also reduced cardiovascular events in diabetic patients. However, the effectiveness of renin angiotensin system blocking agents depends on the time when treatment is started, and imperfect renoprotection may occur if therapy begins at an advanced disease phase. This raises the need to identify novel multidrug approaches that simultaneously inhibit additional pathways other than angiotensin II for those diabetic patients who remain at high risk of both poor renal and cardiovascular outcomes. Studies in animal models of diabetes have contributed to defining relevant cellular mechanisms underlying the pathogenesis of DN that could represent possible targets for therapies. The pathogenesis of DN is multifactorial, involving a complex series of molecular processes. In this review, we report evidence obtained in experimental models of DN on some specific processes and pathways implicated in DN that may be crucial for managing this disease.

Progression of renal injury toward interstitial inflammation and glomerular sclerosis is dependent on abnormal protein filtration.

Chronic proteinuric renal diseases, independent from the type of the initial insult, have in common a loss of selectivity of the glomerular barrier to protein filtration. Glomerular sclerosis is the progressive lesion affecting the glomerular capillary wall, the primary site at which the protein filtration is abnormally enhanced by disease. Dysfunction of podocytes, that serve to maintain the intact barrier, is a central event in lesion development. However, glomerular injury is signalled to tubular and interstitial structures largely in advance of nephron destruction. Glomerular ultrafiltration of excessive amounts of plasma-derived proteins and associated factors incites tubulointerstitial damage and might amplify an inherent susceptibility of the kidney to become dysfunctional in several disease conditions. Thus, noxious substances in the proteinuric ultrafiltrate promote apoptotic responses and multiple changes in the phenotype of tubule cells with generation of inflammatory and fibrogenic mediators. The severity of tubular interstitial damage has long been recognized to be highly correlated to the degree of deterioration of renal failure even better than glomerular lesions. This review focuses on pathways of tubular injury and apoptosis that in turn promote nephron-by-nephron degeneration and interstitial fibrosis during proteinuria contributing to multifaceted processes of kidney scarring and function loss.

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.

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.

The Nrf2 pathway in the progression of renal disease.

The Nrf2/Keap1 system regulates the transcription of antioxidant and cytoprotective genes through direct Nrf2 binding to responsive elements in the promoter region of target genes or via Keap1-induced NF-kB inhibition. The association between oxidative stress and inflammation with progression of chronic kidney diseases (CKDs) directed attention towards bardoxolone methyl and its analogues, potent Nrf2/Keap1 inducers, as a potential modality of renoprotective intervention. In a phase II clinical trial (BEAM), bardoxolone methyl was shown to increase the estimated glomerular filtration rate (eGFR) in patients with CKD associated with type 2 diabetes. The study generated great interest but raised concerns as well, on the adverse event profile of the drug. Experiments in rats with type 2 diabetic nephropathy treated with bardoxolone methyl analogues reproduced some drawbacks of bardoxolone methyl therapy in humans. Despite these warnings, a long-term phase III trial (BEACON) was started that was prematurely terminated because of an excess serious adverse events and mortality. Lessons from the above studies suggest that before jumping into use in clinical practice, adequately designed experiments in animal models are needed to provide insights into pathogenetic mechanisms as well as unexpected side effects.

Lack of the lectin-like domain of thrombomodulin worsens Shiga toxin-associated hemolytic uremic syndrome in mice.

Shiga toxin (Stx)-producing Escherichia coli is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The pathophysiology of renal microvascular thrombosis in Stx-HUS is still ill-defined. Based on evidence that abnormalities in thrombomodulin (TM), an anticoagulant endothelial glycoprotein that modulates complement and inflammation, predispose to atypical HUS, we assessed whether impaired TM function may adversely affect evolution of Stx-HUS. Disease was induced by coinjection of Stx2/LPS in wild-type mice (TM(wt/wt)) and mice that lack the lectin-like domain of TM (TM(LeD/LeD)), which is critical for its anti-inflammatory and cytoprotective properties. After Stx2/LPS, TM(LeD/LeD) mice exhibited more severe thrombocytopenia and renal dysfunction than TM(wt/wt) mice. Lack of lectin-like domain of TM resulted in a stronger inflammatory reaction after Stx2/LPS with more neutrophils and monocytes/macrophages infiltrating the kidney, associated with PECAM-1 and chemokine upregulation. After Stx2/LPS, intraglomerular fibrin(ogen) deposits were detected earlier in TM(LeD/LeD) than in TM(wt/wt) mice. More abundant fibrin(ogen) deposits were also found in brain and lungs. Under basal conditions, TM(LeD/LeD) mice exhibited excess glomerular C3 deposits, indicating impaired complement regulation in the kidney that could lead to local accumulation of proinflammatory products. TM(LeD/LeD) mice with HUS had a higher mortality rate than TM(wt/wt) mice. If applicable to humans, these findings raise the possibility that genetic or acquired TM defects might have an impact on the severity of microangiopathic lesions after exposure to Stx-producing E. coli infections and raise the potential for using soluble TM in the treatment of Stx-HUS.

Efficacy of GalNAc C3 siRNAs in factor H-deficient mice with C3 glomerulopathy.

Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh+/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh-/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function.

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.

Liver factor B silencing to cure C3 glomerulopathy: Evidence from a mouse model of complement dysregulation.

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.

Thyroid hormone treatment counteracts cellular phenotypical remodeling in diabetic organs.

Diabetes mellitus and alterations in thyroid hormone (TH) signaling are closely linked. Though the role of TH signaling in cell differentiation and growth is well known, it remains unclear whether its alterations contribute to the pathobiology of diabetic cells. Here, we aim to investigate whether the administration of exogenous T3 can counteract the cellular remodeling that occurs in diabetic cardiomyocytes, podocytes, and pancreatic beta cells. Treating diabetic rats with T3 prevents dedifferentiation, pathological growth, and ultrastructural alterations in podocytes and cardiomyocytes. In vitro, T3 reverses glucose-induced growth in human podocytes and cardiomyocytes, restores cardiomyocyte cytoarchitecture, and reverses pathological alterations in kidney and cardiac organoids. Finally, T3 treatment counteracts glucose-induced transdifferentiation, cell growth, and loss in pancreatic beta cells through TH receptor alpha1 activation. Our studies indicate that TH signaling activation substantially counteracts diabetes-induced pathological remodeling, and provide a potential therapeutic approach for the treatment of diabetes and its complications.

Add-On Cyclic Angiotensin-(1-7) with Cyclophosphamide Arrests Progressive Kidney Disease in Rats with ANCA Associated Glomerulonephritis.

Rapidly progressive crescentic glomerulonephritis associated with anti-neutrophil cytoplasmic antibodies (ANCA-GN) is a major cause of renal failure. Current immunosuppressive therapies are associated with severe side effects, intensifying the need for new therapeutic strategies. The activation of Mas receptor/Angiotensin-(1-7) axis exerted renoprotection in chronic kidney disease. Here, we investigated the effect of adding the lanthionine-stabilized cyclic form of angiotensin-1-7 [cAng-(1-7)] to cyclophosphamide in a rat model of ANCA-GN. At the onset of proteinuria, Wistar Kyoto rats with ANCA-GN received vehicle or a single bolus of cyclophosphamide, with or without daily cAng-(1-7). Treatment with cAng-(1-7) plus cyclophosphamide reduced proteinuria by 85% vs. vehicle, and by 60% vs. cyclophosphamide, and dramatically limited glomerular crescents to less than 10%. The addition of cAng-(1-7) to cyclophosphamide protected against glomerular inflammation and endothelial rarefaction and restored the normal distribution of parietal epithelial cells. Ultrastructural analysis revealed a preserved GBM, glomerular endothelium and podocyte structure, demonstrating that combination therapy provided an additional layer of renoprotection. This study demonstrates that adding cAng-(1-7) to a partially effective dose of cyclophosphamide arrests the progression of renal disease in rats with ANCA-GN, suggesting that cAng-(1-7) could be a novel clinical approach for sparing immunosuppressants.

Low Nephron Number Induced by Maternal Protein Restriction Is Prevented by Nicotinamide Riboside Supplementation Depending on Sirtuin 3 Activation.

A reduced nephron number at birth, due to critical gestational conditions, including maternal malnutrition, is associated with the risk of developing hypertension and chronic kidney disease in adulthood. No interventions are currently available to augment nephron number. We have recently shown that sirtuin 3 (SIRT3) has an important role in dictating proper nephron endowment. The present study explored whether SIRT3 stimulation, by means of supplementation with nicotinamide riboside (NR), a precursor of the SIRT3 co-substrate nicotinamide adenine dinucleotide (NAD+), was able to improve nephron number in a murine model of a low protein (LP) diet. Our findings show that reduced nephron number in newborn mice (day 1) born to mothers fed a LP diet was associated with impaired renal SIRT3 expression, which was restored through supplementation with NR. Glomerular podocyte density, as well as the rarefaction of renal capillaries, also improved through NR administration. In mechanistic terms, the restoration of SIRT3 expression through NR was mediated by the induction of proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α). Moreover, NR restored SIRT3 activity, as shown by the reduction of the acetylation of optic atrophy 1 (OPA1) and superoxide dismutase 2 (SOD2), which resulted in improved mitochondrial morphology and protection against oxidative damage in mice born to mothers fed the LP diet. Our results provide evidence that it is feasible to prevent nephron mass shortage at birth through SIRT3 boosting during nephrogenesis, thus providing a therapeutic option to possibly limit the long-term sequelae of reduced nephron number in adulthood.