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.

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.

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.

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.

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.

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.

Amnion epithelial cells are an effective source of factor H and prevent kidney complement deposition in factor H-deficient mice.

Complement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh-/- mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh-/- mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh-/- mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.

Characterization of a Rat Model of Myeloperoxidase-Anti-Neutrophil Cytoplasmic Antibody-Associated Crescentic Glomerulonephritis.

BACKGROUND/AIM: Necrotizing crescentic glomerulonephritis (GN) associated with anti-neutrophil cytoplasmic antibodies (ANCA) against myeloperoxidase (MPO) is a devastating disease that quickly progresses to kidney failure. Current therapies are broadly immunosuppressive and associated with adverse effects. We wanted to set up a model that could be suitable for testing narrowly targeted therapies. METHODS: The model was constructed in male Wistar Kyoto rats through injections of human MPO (hMPO) and pertussis toxin, followed by a sub-nephritogenic dose of sheep anti-rat glomerular basement membrane (GBM) serum to boost the disease. Rats were monitored for 35 days. Rats given hMPO alone, saline, or human serum albumin with or without anti-GBM serum were also studied. RESULTS: Rats receiving hMPO developed circulating anti-hMPO and anti-rat MPO antibodies. Challenging hMPO-immunized rats with the anti-GBM serum led to more glomerular neutrophil infiltration and MPO release, and severe haematuria, heavy proteinuria, and higher blood urea nitrogen than hMPO alone. Pauci-immune GN developed with crescents, affecting 25% of glomeruli. The majority of crescents were fibrocellular. Necrotizing lesions and Bowman capsule ruptures were detected. Cells double positive for claudin-1 (a marker of parietal epithelial cells [PECs]) and neural cell adhesion molecule (NCAM; progenitor PECs) were present in crescents. Double staining for NCAM and Ki-67 established proliferative status of progenitor PECs. Podocyte damage was associated with endothelial and GBM changes by electron microscopy. Monocyte/macrophages and CD4+ and CD8+ T cells accumulated in glomeruli and the surrounding area and in the tubulointerstitium. Lung haemorrhage also manifested. CONCLUSION: This model reflects histological lesions of human ANCA-associated rapidly progressive GN and may be useful for investigating new therapies.

CER-001 ameliorates lipid profile and kidney disease in a mouse model of familial LCAT deficiency.

OBJECTIVE: CER-001 is an HDL mimetic that has been tested in different pathological conditions, but never with LCAT deficiency. This study was designed to investigate whether the absence of LCAT affects the catabolic fate of CER-001, and to evaluate the effects of CER-001 on kidney disease associated with LCAT deficiency. METHODS: Lcat-/- and wild-type mice received CER-001 (2.5, 5, 10 mg/kg) intravenously for 2 weeks. The plasma lipid/ lipoprotein profile and HDL subclasses were analyzed. In a second set of experiments, Lcat-/- mice were injected with LpX to induce renal disease and treated with CER-001 and then the plasma lipid profile, lipid accumulation in the kidney, albuminuria and glomerular podocyte markers were evaluated. RESULTS: In Lcat-/- mice a decrease in total cholesterol and triglycerides, and an increase in HDL-c was observed after CER-001 treatment. While in wild-type mice CER-001 entered the classical HDL remodeling pathway, in the absence of LCAT it disappeared from the plasma shortly after injection and ended up in the kidney. In a mouse model of renal disease in LCAT deficiency, treatment with CER-001 at 10 mg/kg for one month had beneficial effects not only on the lipid profile, but also on renal disease, by limiting albuminuria and podocyte dysfunction. CONCLUSIONS: Treatment with CER-001 ameliorates the dyslipidemia typically associated with LCAT deficiency and more importantly limits renal damage in a mouse model of renal disease in LCAT deficiency. The present results provide a rationale for using CER-001 in FLD patients.

Histological Examination of the Diabetic Kidney.

The increasing prevalence of diabetes worldwide has led to a concomitant rise in diabetic kidney disease (DKD) as a major cause of end-stage renal disease. Glomerular lesions constitute the most striking and consistent features identified in biopsies from patients with DKD, although tubulointerstitial injury has an important and often under-recognized role in the progression to overt nephropathy. In advanced stages of the disease, podocyte detachment is a pivotal event in the loss of glomerular filtration barrier integrity and may explain, at least in part, the inability of current therapies to halt renal function decline. This chapter details the systematic method that can be used to study renal tissue samples from diabetic patients, and the specific role of different imaging techniques, such as light microscopy, immunofluorescence microscopy, and transmission and scanning electron microscopy in detecting histologic lesions specific to DKD.

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.

Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties. METHODS: The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 106 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigenpos cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h. RESULTS: Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system. CONCLUSIONS: Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients.

Fenofibrate attenuates cardiac and renal alterations in young salt-loaded spontaneously hypertensive stroke-prone rats through mitochondrial protection.

OBJECTIVES: The simultaneous presence of cardiac and renal diseases is a pathological condition that leads to increased morbidity and mortality. Several lines of evidence have suggested that lipid dysmetabolism and mitochondrial dysfunction are pathways involved in the pathological processes affecting the heart and kidney. In the salt-loaded spontaneously hypertensive stroke-prone rat (SHRSP), a model of cardiac hypertrophy and nephropathy that shows mitochondrial alterations in the myocardium, we evaluated the cardiorenal effects of fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist that acts by modulating mitochondrial and peroxisomal fatty acid oxidation. METHODS: Male SHRSPs aged 6-7 weeks were divided in three groups: standard diet (n = 6), Japanese diet with vehicle (n = 6), and Japanese diet with fenofibrate 150 mg/kg/day (n = 6) for 5 weeks. Cardiac and renal functions were assessed in vivo by MRI, ultrasonography, and biochemical assays. Mitochondria were investigated by transmission electron microscopy, succinate dehydrogenase (SDH) activity, and gene expression analysis. RESULTS: Fenofibrate attenuated cardiac hypertrophy, as evidenced by histological and MRI analyses, and protected the kidneys, preventing morphological alterations, changes in arterial blood flow velocity, and increases in 24-h proteinuria. Cardiorenal inflammation, oxidative stress, and cellular senescence were also inhibited by fenofibrate. In salt-loaded SHRSPs, we observed severe morphological mitochondrial alterations, reduced SDH activity, and down-regulation of genes regulating mitochondrial fatty-acid oxidation (i.e. PPARα, SIRT3, and Acadm). These changes were counteracted by fenofibrate. In vitro, a direct protective effect of fenofibrate on mitochondrial membrane potential was observed in albumin-stimulated NRK-52E renal tubular epithelial cells. CONCLUSION: The results suggest that the cardiorenal protective effects of fenofibrate in young male salt-loaded SHRSPs are explained by its capacity to preserve mitochondrial function.

Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.

Mesenchymal stromal cells (MSCs) are renoprotective and drive regeneration following injury, although cellular targets of such an effect are still ill-defined. Here, we show that human umbilical cord (UC)-MSCs transplanted into mice stimulate tubular cells to regain mitochondrial mass and function, associated with enhanced microtubule-rich projections that appear to mediate mitochondrial trafficking to create a reparative dialogue among adjacent tubular cells. Treatment with UC-MSCs in mice with cisplatin-induced acute kidney injury (AKI) regulates mitochondrial biogenesis in proximal tubuli by enhancing PGC1α expression, NAD+ biosynthesis and Sirtuin 3 (SIRT3) activity, thus fostering antioxidant defenses and ATP production. The functional role of SIRT3 in tubular recovery is highlighted by data that in SIRT3-deficient mice with AKI, UC-MSC treatment fails to induce renoprotection. These data document a previously unrecognized mechanism through which UC-MSCs facilitate renal repair, so as to induce global metabolic reprogramming of damaged tubular cells to sustain energy supply.Mesenchymal stromal cells drive renal regeneration following injury. Here, the authors show that human mesenchymal stromal cells, when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.

The Role of Angiotensin II in Parietal Epithelial Cell Proliferation and Crescent Formation in Glomerular Diseases.

Crescentic glomerulonephritis (GN) is a devastating disease with rapidly progressive deterioration in kidney function, which, histologically, manifests as crescent formation in most glomeruli. We previously found that crescents derive from the aberrant proliferation and migration of parietal epithelial cells (PECs)/progenitor cells, and that the angiotensin (ang) II/ang II type-1 (AT1) receptor pathway may participate, together with the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor 4 axis, in the development of those lesions. Herein, we elucidated sequential events and cellular and molecular interactions occurring during crescentic lesion onset and evolution. By analyzing kidney biopsy specimens of patients with extracapillary GN, divided according to the grade of glomerular lesions, we found that the accumulation of macrophages expressing matrix metalloproteinase-12 started manifesting in glomeruli affected by early-stage lesions, whereas AT1 receptor expression could not be detected. In glomeruli with advanced lesions, AT1 receptor expression increased markedly, and the up-regulation of SDF-1, and its receptor C-X-C chemokine receptor 7, was documented on podocytes and PECs, respectively. In vitro studies were instrumental to demonstrating the role of ang II in inducing podocyte SDF-1 production, which ultimately activates PECs. The present findings support the possibility that angiotensin-converting enzyme inhibitor treatment might limit PEC activation and reduce the frequency and extension of crescents in extracapillary GN.

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.

Therapy with a Selective Cannabinoid Receptor Type 2 Agonist Limits Albuminuria and Renal Injury in Mice with Type 2 Diabetic Nephropathy.

BACKGROUND/AIMS: A critical involvement of the endocannabinoid/cannabinoid receptor system in diabetes and its complications has been recognized. Experimental evidence suggested that activation of the cannabinoid receptor type 2 (CB2), which is expressed in the kidney by podocytes and inflammatory cells, had a protective role in early streptozotocin-induced type 1 diabetes in mice. No experimental evidence is so far available on the effects of CB2 agonists in type 2 diabetes. In this study, we investigated the effects of a CB2 agonist given at a phase of overt disease on renal functional and structural changes in BTBR ob/ob mice, a model of type 2 diabetic nephropathy. METHODS: BTBR ob/ob mice received, from 10 to 21 weeks of age, vehicle, the selective CB2 agonist HU910, or lisinopril used as standard therapy for comparison. BTBR wild-type mice served as controls. RESULTS: Treatment with CB2 agonist reduced progressive albuminuria of BTBR ob/ob mice to a similar extent as ACE inhibitor. The antiproteinuric effect of CB2 agonist was associated with the amelioration of the defective nephrin expression in podocytes of diabetic mice. CB2 agonist limited mesangial matrix expansion, fibronectin accumulation and sclerosis. Glomerular infiltration of Mac-2-positive monocytes/machrophages was attenuated by CB2 agonist, at least in part due to the drug's ability to reduce MCP-1 chemotactic signals. Renoprotective effects of CB2 were similar to those achieved by ACE inhibitor. CONCLUSION: These results suggest that CB2 agonism is a potential option to be added to the available therapeutic armamentarium for type 2 diabetic nephropathy.

Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease.

The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease.