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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.

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.

More effective treatments for diabetic nephropathy remain a major unmet clinical need. Increased oxidative stress is one of the most important pathological mechanisms that lead to kidney damage and functional impairment induced by diabetes. Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase and critically regulates cellular reactive oxygen species (ROS) production and detoxification. Honokiol is a natural biphenolic compound that, by activating mitochondrial SIRT3, can carry out anti-oxidant, anti-inflammatory and anti-fibrotic activities. Here, we sought to investigate the renoprotective effects of honokiol in BTBR ob/ob mice with type 2 diabetes. Diabetic mice were treated with vehicle or honokiol between the ages of 8 and 14 weeks. Wild-type mice served as controls. Renal Sirt3 expression was significantly reduced in BTBR ob/ob mice, and this was associated with a reduction in its activity and increased ROS levels. Selective activation of SIRT3 through honokiol administration translated into the attenuation of albuminuria, amelioration of glomerular damage, and a reduction in podocyte injury. SIRT3 activation preserved mitochondrial wellness through the activation of SOD2 and the restoration of PGC-1α expression in glomerular cells. Additionally, the protective role of SIRT3 in glomerular changes was associated with enhanced tubular Sirt3 expression and upregulated renal Nampt levels, indicating a possible tubule-glomerulus retrograde interplay, which resulted in improved glomerular SIRT3 activity. Our results demonstrate the hitherto unknown renoprotective effect of SIRT3 against diabetic glomerular disease and suggest that the pharmacological modulation of SIRT3 activity is a possible novel approach to treating diabetic nephropathy.

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.

Inadequate production of erythropoietin (EPO) leads to anemia. Although erythropoiesis-stimulating agents can be used to treat anemia, these approaches are limited by high costs, adverse effects, and the need for frequent injections. Developing methods for the generation and transplantation of EPO-producing cells would allow for the design of personalized and complication-free therapeutic solutions. In mice, the first EPO source are neural crest cells (NCCs), which ultimately migrate to the fetal kidney to differentiate into EPO-producing fibroblasts. In humans however, it remains unknown whether NCCs can produce EPO in response to hypoxia. Here, we developed a new protocol to differentiate human induced pluripotent stem cells (hiPSCs) into NCCs and showed that cthese cells can produce functional EPO that can induce human CD34+ hematopoietic progenitor differentiation into erythroblasts in vitro. Moreover, we showed that hiPSC-derived NCCs can be embedded in clinical-grade atelocollagen scaffolds and subcutaneously transplanted into anemic mice to produce human EPO, accelerate hematocrit recovery, and induce erythropoiesis in the spleen. Our findings provide unprecedented evidence of the ability of human NCCs to produce functional EPO in response to hypoxia, and proof-of-concept for the potential clinical use of NCC-containing scaffolds as cell therapy for renal and non-renal anemia.

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.

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.

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.

Background: In healthy human subjects, nephron number varies greatly between individuals and it is estimated that a normal kidney is endowed with approximately 800,000 to 1 million nephrons. Whether and to what extent a mild nephron shortage within this physiological range can increase susceptibility to CKD, has never been investigated. Methods: We studied CKD susceptibility in mice lacking Sirtuin 3 (Sirt3-/-), used as a model of mild nephron deficiency to the degree that they exhibit a 17% nephron reduction compared to wild-type littermates. Findings: In adulthood, Sirt3-/- mice do not spontaneously develop full-blown renal dysfunction, as demonstrated by comparable levels of urinary protein excretion and serum blood urea nitrogen levels, as well as a lack of signs of glomerular or tubulo-interstitial damage. However, adult Sirt3-/- mice exhibited exacerbated CKD and increased mortality following exposure to different nephrotoxic insults, namely protein overload and adriamycin. Interpretation: Our results provide evidence that even a mild reduction in nephron number is a critical determinant for increased susceptibility to CKD when the kidney is challenged by additional hits later in life. These findings suggest that any intervention to boost nephron number during active nephrogenesis may be an attractive preventive strategy for sustainably decreasing the global burden of CKD. Funding Statement: The authors stated: "No funding available for the present study." Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All procedures involving animals were performed in accordance with institutional guidelines in compliance with national (D.L.n.26, March 4, 2014), and international laws and policies (directive 2010/63/EU on the protection of animals used for scientific purposes).

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.

Thyroid hormone (TH) signaling is a universal regulator of metabolism, growth, and development. Here, we show that TH-TH receptor (TH-TR) axis alterations are critically involved in diabetic nephropathy–associated (DN-associated) podocyte pathology, and we identify TRα1 as a key regulator of the pathogenesis of DN. In ZSF1 diabetic rats, T(3) levels progressively decreased during DN, and this was inversely correlated with metabolic and renal disease worsening. These phenomena were associated with the reexpression of the fetal isoform TRα1 in podocytes and parietal cells of both rats and patients with DN and with the increased glomerular expression of the TH-inactivating enzyme deiodinase 3 (DIO3). In diabetic rats, TRα1-positive cells also reexpressed several fetal mesenchymal and damage-related podocyte markers, while glomerular and podocyte hypertrophy was evident. In vitro, exposing human podocytes to diabetes milieu typical components markedly increased TRα1 and DIO3 expression and induced cytoskeleton rearrangements, adult podocyte marker downregulation and fetal kidney marker upregulation, the maladaptive cell cycle induction/arrest, and TRα1-ERK1/2–mediated hypertrophy. Strikingly, T(3) treatment reduced TRα1 and DIO3 expression and completely reversed all these alterations. Our data show that diabetic stress induces the TH-TRα1 axis to adopt a fetal ligand/receptor relationship pattern that triggers the recapitulation of the fetal podocyte phenotype and subsequent pathological alterations.

Aims: Sirtuins, a family of NAD+-dependent deacetylases, are recognized as nondispensable regulators of aging processes. Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase that maintains mitochondrial bioenergetics, an essential prerequisite for healthy aging. In this study, using Sirt3 knockout (Sirt3-/-) mice, we sought to establish whether Sirt3 deficiency affected life span, an endpoint that has never been tested formally in mammals, and uncover the mechanisms involved in organ damage associated with aging. Results: Sirt3-/- mice experienced a shorter life span than wild-type mice and severe cardiac damage, characterized by hypertrophy and fibrosis, as they aged. No alterations were found in organs other than the heart. Sirt3 deficiency altered cardiac mitochondrial bioenergetics and caused hyperacetylation of optic atrophy 1 (OPA1), a SIRT3 target. These changes were associated with aberrant alignment of trans-mitochondrial cristae in cardiomyocytes, and cardiac dysfunction. Gene transfer of deacetylated Opa1 restored cristae alignment in Sirt3-/- mice, ameliorated cardiac reserve capacity, and protected the heart against hypertrophy and fibrosis. The translational relevance of these findings is in the data showing that SIRT3 silencing in human-induced pluripotent stem cell-derived cardiomyocytes led to mitochondrial dysfunction and altered contractile phenotype, both rescued by Opa1 gene transfer. Innovation: Our findings indicate that future approaches to heart failure could include SIRT3 as a plausible therapeutic target. Conclusion: SIRT3 has a major role in regulating mammalian life span. Sirt3 deficiency leads to cardiac abnormalities, due to defective trans-mitochondrial cristae alignment and impaired mitochondrial bioenergetics. Correcting cardiac OPA1 hyperacetylation through gene transfer diminished heart failure in Sirt3-/- mice during aging.

Background/Aims: Glomerular filtration rate (GFR) is the best index for evaluating renal function. We aimed to develop a simplified iohexol plasma clearance procedure for GFR measurement in rats without urine collection, animal catheterization or anesthesia, with limited sampling and requiring blood instead of plasma, to further reduce the sample volume and improve animal welfare. Methods: After iohexol injection (129.4 mg), samples were drawn according to 2-compartment kinetics and analyzed by high performance liquid chromatography. Healthy male Lewis rats were used to find a correction factor (CF) to obtain the ‘reference clearance' from the simplified 1-comparment model. This approach was validated using male or female (Lewis, Sprague-Dawley) rats and animals with renal mass reduction (RMR). In additional rats, different simplified approaches were evaluated. Results: Iohexol concentrations in blood and plasma strongly correlated (r = 0.9784, p < 0.0001). A CF of 0.90 enabled the calculation of the reference GFR. Validation results in male Lewis rats were 0.99 ± 0.27 for the reference GFR and 1.03 ± 0.29 ml/min/100 g for the simplified approach. Results in female Sprague-Dawley rats confirmed the suitability of the proposed method. In RMR rats, GFR was 0.14 ± 0.05 and 0.14 ± 0.04 ml/min/100 g for the reference and simplified model, respectively. Conclusion: The procedure we set up to measure GFR in conscious rats was proven to be reliable, required a small volume of blood at only 4 selected time points, without the need to collect urine or catheterize the animals, was applicable to rats from different strains and sexes, both healthy and with renal function impairment. Moreover, the procedure enables the monitoring of GFR changes over time in the same animal, thereby reducing the number of animals to be used.

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. Electronic supplementary material The online version of this article (10.1186/s13287-018-0960-8) contains supplementary material, which is available to authorized users.

In patients with diabetes, impaired activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), the plasma metalloprotease that cleaves highly thrombogenic von Willebrand factor multimers, is a major risk factor of cardiovascular events. Here, using Adamts13−/− mice made diabetic by streptozotocin, we investigated the impact of the lack of ADAMTS13 on the development of diabetes-associated end-organ complications. Adamts13−/− mice experienced a shorter life span than their diabetic wild-type littermates. It was surprising that animal death was not related to the occurrence of detectable thrombotic events. The lack of ADAMTS13 drastically increased the propensity for ventricular arrhythmias during dobutamine-induced stress in diabetic mice. Cardiomyocytes of diabetic Adamts13−/− mice exhibited an aberrant distribution of the ventricular gap junction connexin 43 and increased phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII), and with the consequent CaMKII-induced disturbance in Ca2+ handling, which underlie propensity for arrhythmia. In vitro, thrombospondin 1 (TSP1) promoted, in a paracrine manner, CaMKII phosphorylation in murine HL-1 cardiomyocytes, and ADAMTS13 acted to inhibit TSP1-induced CaMKII activation. In conclusion, the deficiency of ADAMTS13 may underlie the onset of lethal arrhythmias in diabetes through increased CaMKII phosphorylation in cardiomyocytes. Our findings disclose a novel function for ADAMTS13 beyond its antithrombotic activity.

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.

Several biologically active compounds are present in seaweeds that show a wide range of biological properties, such as antibiotic, anti-inflammatory, antioxidant, cytotoxic, antitumour and anti-obesity activities. The aim was to evaluate the anti-obesity effects of chronic treatment with Norwegian Ascophyllum nodosum phytocomplex in rats. We also investigated the effects of this phytocomplex on blood glucose and serum lipid levels, and α-amylase, lipase, alanine aminotransferase and aspartate aminotransferase activities. Five groups of male Sprague-Dawley rats were fed for 8 weeks with standard diet, highfat diet (HFD) or HFD plus three different doses of Norwegian A. nodosum phytocomplex (HFD Low, HFD Medium, HFD High). Feeding with HFD resulted in increased body weight and deposition of visceral and abdominal white adipose tissue. These effects were significantly reduced by supplementation with high doses of A. nodosum phytocomplex in the feed, which suggests a potent anti-obesity activity of this phytocomplex. Increased serum levels of triglycerides and α-amylase activity were observed in rats fed with HFD at sacrifice. These were inhibited in a dose-dependent manner by feeding with the added A. nodosum phytocomplex in HFD Medium and HFD High. Increased liver and kidney weights were also inhibited in a dose-dependent manner by added A. nodosum phytocomplex, with significant effects at the highest phytocomplex dose. Rats fed with HFD showed a changed, irregular, liver morphology, increased infiltration of inflammatory cells and increased intravascular spaces compared to controls. These effects were partially corrected by HFD High, which suggests potent anti-inflammatory activity of the A. nodosum phytocomplex.

Activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through β-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 ETAR activation and increased β-arrestin-1 expression. Activated ETAR recruited β-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1–induced migration as confirmed by Snail knockdown experiments. Silencing of β-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ETAR-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; β-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ETAR antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular β-arrestin-1 and Snail. Increased β-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 ETAR antagonism for a group of diseases still needing a specific treatment.

Bardoxolone methyl is an antioxidant inflammation modulator acting through induction of Keap1-Nrf2 pathway. Results from a recent phase IIb clinical trial reported that bardoxolone methyl was associated with improvement in the estimated glomerular filtration rate in patients with advanced chronic kidney disease and Type 2 diabetes. However, increases in albuminuria, serum transaminase, and frequency of adverse events were noted. We studied the effect of 3-mo treatment with RTA 405, a synthetic triterpenoid analog of bardoxolone methyl in Zucker diabetic fatty rats with overt Type 2 diabetes. Rats were treated from 3 mo of age with vehicle, RTA 405, ramipril, or RTA 405 plus ramipril. RTA 405 caused severe changes in food intake and diuresis with decline in body weight, worsening of dyslipidemia, and increase in blood pressure. Early elevation in serum transaminase was followed by liver injury. RTA 405 worsened proteinuria, glomerulosclerosis, and tubular damage. Ramipril was renoprotective, but when given with RTA 405 it was not able to limit its worsening effects. These data could be due to degradation products in the drug substance used, as disclosed by the company once the study was concluded. To overcome such a drawback, the company offered to test dh404, a variant of RTA 405, in Zucker diabetic fatty rats. The dh404 did not display beneficial effects on proteinuria, glomerulosclerosis, and interstitial inflammation. Rather, kidneys from three rats receiving dh404 showed the presence of a granulomatous and inflammatory process reminiscent of a pseudotumor. Altogether these data raise serious concerns on the use of bardoxolone analogs in Type 2 diabetic 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.

Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research.

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 (TMwt/wt) and mice that lack the lectin-like domain of TM (TMLeD/LeD), which is critical for its anti-inflammatory and cytoprotective properties. After Stx2/LPS, TMLeD/LeD mice exhibited more severe thrombocytopenia and renal dysfunction than TMwt/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 TMLeD/LeD than in TMwt/wt mice. More abundant fibrin(ogen) deposits were also found in brain and lungs. Under basal conditions, TMLeD/LeD mice exhibited excess glomerular C3 deposits, indicating impaired complement regulation in the kidney that could lead to local accumulation of proinflammatory products. TMLeD/LeD mice with HUS had a higher mortality rate than TMwt/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.

Diabetic nephropathy is associated with cardiovascular morbidity. Angiotensin-converting enzyme (ACE) inhibitors provide imperfect renoprotection in advanced type 2 diabetes, and cardiovascular risk remains elevated. Endothelin (ET)-1 has a role in renal and cardiac dysfunction in diabetes. Here, we assessed whether combination therapy with an ACE inhibitor and ETAreceptor antagonist provided reno- and cardioprotection in rats with overt type 2 diabetes. Four groups of Zucker diabetic fatty (ZDF) rats were treated orally from 4 (when proteinuric) to 8 mo with vehicle, ramipril (1 mg/kg), sitaxsentan (60 mg/kg), and ramipril plus sitaxsentan. Lean rats served as controls. Combined therapy ameliorated proteinuria and glomerulosclerosis mostly as a result of the action of ramipril. Simultaneous blockade of ANG II and ET-1 pathways normalized renal monocyte chemoattractant protein-1 and interstitial inflammation. Cardiomyocyte loss, volume enlargement, and capillary rarefaction were prominent abnormalities of ZDF myocardium. Myocyte volume was reduced by ramipril and sitaxsentan, which also ameliorated heart capillary density. Drug combination restored myocardial structure and reestablished an adequate capillary network in the presence of increased cardiac expression of VEGF/VEGFR-1, and significant reduction of oxidative stress. In conclusion, in type 2 diabetes concomitant blockade of ANG II synthesis and ET-1 biological activity through an ETAreceptor antagonist led to substantial albeit not complete renoprotection, almost due to the ACE inhibitor. The drug combination also showed cardioprotective properties, which however, were mainly dependent on the contribution of the ETAreceptor antagonist through the action of VEGF.

In the present study, we evaluated the effect of simultaneously blocking angiotensin II synthesis and endothelin (ET)-1 activity as a multimodal intervention to implement renoprotection in overt diabetic nephropathy. Mechanisms underlying combined therapy effectiveness were addressed by investigating podocyte structure and function and glomerular barrier size-selective properties. Uninephrectomized rats made diabetic by streptozotocin received orally placebo, lisinopril (12.5 mg/l), the ETAreceptor antagonist avosentan (30 mg/kg), or their combination from 4 (when animals had proteinuria) to 8 mo. Proteinuria, renal damage, podocyte number, nephrin expression, and glomerular size selectivity by graded-size Ficoll molecule fractional clearance were assessed. Combined therapy normalized proteinuria, provided complete protection from tubulointerstitial damage, and induced regression of glomerular lesions, while only a partial renoprotection was achieved by each drug alone. Lisinopril plus avosentan restored to normal values the number of podocytes. Single therapies only limited podocyte depletion. Defective nephrin expression of diabetes was prevented by each drug. Altered glomerular size selectivity to large macromolecules of diabetic rats was remarkably improved by lisinopril and the combined treatment. Avosentan ameliorated peritubular capillary architecture and reduced interstitial inflammation and fibrosis. The ACE inhibitor and ETAreceptor antagonist induced regression of glomerular lesions in overt diabetic nephropathy. Regression of renal disease was conceivably the result of the synergistic effect of the ACE inhibitor of preserving glomerular permselective properties and the ETAantagonist in improving tubulointerstitial changes. These findings provide mechanistic insights to explain the antiproteinuric effect of this combined therapy in diabetes.

Blockade of the renin-angiotensin system (RAS), the standard treatment for chronic proteinuric nephropathy, slows but may not halt progression of the disease, particularly when therapy is started late. Because vasopressin may also play a role in the progression of renal disease, we measured the effect of a dual V(1a) and V(2) vasopressin receptor antagonist (RWJ-676070) alone or combined with angiotensin-converting enzyme inhibition or angiotensin II type 1 receptor blockade on proteinuria and renal disease progression during overt nephropathy. Twenty-one days after renal mass reduction, a time of established injury, rats were given vehicle, RWJ-676070, enalapril, losartan, RWJ-676070 plus enalapril, or losartan in drinking water for an additional 39 days. RWJ-676070 returned the blood pressure to pre-treatment levels, which were significantly lower than those in vehicle-treated rats. Enalapril, losartan, and the combined therapies reduced blood pressure to a greater extent. RWJ-676070 afforded a partial antiproteinuric effect, which was enhanced by the addition of enalapril or losartan. Renal functional impairment, and glomerular and tubular changes were partially ameliorated by RWJ-676070; parameters significantly improved with either enalapril or losartan alone and improved to a greater extent with the combined therapies. Our findings suggest that vasopressin receptor antagonists could be of additional therapeutic value in the treatment of chronic proteinuric nephropathy.

Intrarenal complement activation leads to chronic tubulointerstitial injury in animal models of proteinuric nephropathies, making this process a potential target for therapy. This study investigated whether a C3-mediated pathway promotes renal injury in the protein overload model and whether the abnormal exposure of proximal tubular cells to filtered complement could trigger the resulting inflammatory response. Mice with C3 deficiency were protected to a significant degree against the protein overload-induced interstitial inflammatory response and tissue damage, and they had less severe podocyte injury and less proteinuria. When the same injury was induced in wild-type (WT) mice, antiproteinuric treatment with the angiotensin-converting enzyme inhibitor lisinopril reduced the amount of plasma protein filtered, decreased the accumulation of C3 by proximal tubular cells, and protected against interstitial inflammation and damage. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell-derived C3, C3-deficient kidneys were transplanted into WT mice. Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when WT kidneys were transplanted into C3-deficient mice, protein overload led to a more mild disease and abnormal C3 deposition was not observed. These data suggest that the presence of C3 increases the glomerular filtration barrier's susceptibility to injury, ultrafiltered C3 contributes more to tubulointerstitial damage induced by protein overload than locally synthesized C3, and local C3 synthesis is irrelevant to the development of proteinuria. It is speculated that therapies targeting complement combined with interventions to minimize proteinuria would more effectively prevent the progression of renal disease.

Transplantation of bone marrow mesenchymal stem cells (BM-MSC) or stromal cells from rodents has been identified as a strategy for renal repair in experimental models of acute kidney injury (AKI), a highly life-threatening clinical setting. The therapeutic potential of BM-MSC of human origin has not been reported so far. Here, we investigated whether human BM-MSC treatment could prevent AKI induced by cisplatin and prolong survival in an immunodeficient mouse model. Results showed that human BM-MSC infusion decreased proximal tubular epithelial cell injury and ameliorated the deficit in renal function, resulting in reduced recipient mortality. Infused BM-MSC became localized predominantly in peritubular areas and acted to reduce renal cell apoptosis and to increase proliferation. BM-MSC also induced protection against AKI-related peritubular capillary changes consisting of endothelial cell abnormalities, leukocyte infiltration, and low endothelial cell and lumen volume density as assessed by morphometric analysis. These findings indicate that human MSC of bone marrow origin hold potential to prolong survival in AKI and should be considered for testing in a clinical trial. Disclosure of potential conflicts of interest is found at the end of this article.

Item does not contain fulltext In mice with cisplatin-induced acute kidney injury, administration of bone marrow-derived mesenchymal stem cells (MSC) restores renal tubular structure and improves renal function, but the underlying mechanism is unclear. Here, we examined the process of kidney cell repair in co-culture experiments with MSC and cisplatin-injured proximal tubular epithelial cells (PTEC). Exposure of PTEC to cisplatin markedly reduced cell viability at 4 days, but co-culture with MSC provided a protective effect by promoting tubular cell proliferation. This effect was mediated by insulin-like growth factor-1 (IGF-1), highly expressed by MSC as mRNA and protein, since blocking the growth factor's function with a specific antibody attenuated cell proliferation of PTEC. Confirming this, knocking down IGF-1 expression in MSC by small interfering-RNA also resulted in a significant decrease in PTEC proliferation and increased apoptosis. Furthermore, in the murine model of cisplatin-induced kidney injury, administering IGF-1 gene-silenced MSC limited their protective effect on renal function and tubular structure. These findings indicate that MSC exert beneficial effects on tubular cell repair in acute kidney injury by producing the mitogenic and pro-survival factor IGF-1.

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.

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.

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.

In experimental and human renal diseases, progression is limited by angiotensin-converting enzyme inhibitors. Whether renoprotection was due to their capacity of reducing proinflammatory and profibrotic effects of angiotensin II (Ang II) or limiting proteinuria and its long term toxicity is debated. For dissecting the relative contribution of Ang II and proteinuria to chronic renal damage, the protein-overload proteinuria model was used in genetically modified mice lacking the major isoform of murine AT1 receptor (AT1A). Uninephrectomized AT1A+/+ and -/- mice received a daily injection of BSA or saline for 4 or 11 wk. AT1A-/-BSA mice acquired a renal phenotype of proteinuria and renal glomerular and tubulointerstitial lesions, albeit attenuated with respect to AT1A+/+BSA. Administration of the calcium channel blocker lacidipine to reduce BP of AT1A+/+BSA mice to levels of AT1A-/-BSA translated into comparable values of protein excretion rate and glomerular and tubulointerstitial injury in both strains. These results confirm that the toxic effect of protein trafficking on renal disease progression is not necessarily dependent on Ang II to the extent that targeted deletion of AT1A does not prevent disease progression. A role of Ang II via AT1B or AT2 receptors is still a possibility that cannot be ruled out by the present experimental approach. These findings provide a clear rationale for specifically targeting proteinuria in pharmacologic interventions of chronic nephropathies.

Investigated was the effect of high albumin concentrations on proximal tubular cell expression of fractalkine. Human proximal tubular cells (HK-2) were incubated with human serum albumin (HSA), which induced a dose-dependent increase in fractalkine mRNA associated with increased levels of both membrane-bound and soluble forms of the protein. To evaluate the role of nuclear factor κB (NF-κB) activation in HSA-induced fractalkine mRNA, HK-2 cells were infected with a recombinant adenovirus encoding the natural inhibitor of NF-κB, IkBα; a 43% reduction of fractalkine mRNA levels resulted. Similarly, when cells were infected with the recombinant adenovirus expressing dominant negative mutant of the IkB kinase 2, a 55% inhibition of fractalkine mRNA was achieved. p38 mitogen-activated protein kinase was activated by HSA and was involved in NF-κB–dependent transcription of fractalkine. In kidneys of mice with bovine serum albumin overload proteinuria, fractalkine mRNA levels were 2.3-fold greater than those of controls. Fractalkine expression was also induced in tubular epithelial cells in this model. Anti-CXCR1 antibody treatment limited interstitial accumulation of mononuclear cells. Protein overload is a promoter of fractalkine gene induction mediated by NF-κB and p38 activation in proximal tubular cells. Fractalkine might contribute to direct mononuclear cells into peritubular interstitium and enhance their adhesion property, which in turn would favor inflammation and disease progression. E-mail: gremuzzi@marionegri.it

Combining lisinopril and L-arginine slows disease progression and reduces endothelin-1 in passive Heymann nephritis. Background Despite angiotensin-converting enzyme (ACE) inhibition is a very powerful therapy, it may not be uniformly renoprotective in patients with proteinuric nephropathies who might refer late in the course of the disease. In accelerated passive Heymann nephritis (PHN), a severe rat model of human membranous nephropathy, with proteinuria and increased urinary excretion of endothelin-1 (ET-1), early treatment with an ACE inhibition limited proteinuria as well as the exuberant formation of renal ET-1, while late treatment reduced urinary proteins not to a significant extent. Since biologic effects and production of ET-1 within the kidney are counteracted by nitric oxide, we studied the effect of combining lisinopril and L-arginine, the natural precursor of nitric oxide, starting late in the disease. Methods Uninephrectomized PHN rats were divided in four groups ( N = 10) and daily given orally: vehicle; 1.25 g/L L-arginine; 40mg/L lisinopril; and L-arginine + lisinopril. Treatments started at 2 months, when rats had massive proteinuria, until 9 months. Six normal rats served as control. Results Increase in systolic blood pressure was significantly limited by L-arginine. Lisinopril alone and the combination were more effective. Renal function impairment was not affected by L-arginine, partially ameliorated by ACE inhibitor and normalized by the combined therapy. In rats given L-arginine, proteinuria levels were similar to vehicle. ACE inhibitor kept proteinuria at values comparable to pretreatment and numerically lower than vehicle. Addition of L-arginine to lisinopril was more effective, with values significantly lower than vehicle. Glomerular and tubular changes were limited by the ACE inhibitor and further ameliorated by the combined therapy. Exaggerated urinary ET-1 of PHN was reduced by 23% and 40% after L-arginine and lisinopril, respectively, and by 62% with the combination. Defective urinary excretion of cyclic guanosine monophosphate (cGMP) was partially restored by lisinopril, while normalized by the combined therapy. Conclusion Combining L-arginine with ACE inhibitors would represent a novel strategy for patients with severe nephropathy not completely responsive to ACE inhibition. Restoring the nitric oxide/ET-1 balance could be of benefit in halting renal disease progression.

Effect of combining ACE inhibitor and statin in severe experimental nephropathy. Background Angiotensin-converting enzyme (ACE) inhibitor therapy given soon after disease induction uniformly prevents proteinuria in virtually all models of disease progression. This does not necessarily apply to patients with proteinuric nephropathies, who might be referred late in the course of their disease. Here we used a severe rat model of passive Heymann nephritis (PHN), which may mimic advanced phases of human membranous nephropathy, to study the response to ACE inhibitor alone or in combination with a HMG CoA reductase inhibitor (statin) that independently of the cholesterol-lowering effect influences pathways involved in inflammatory and fibrogenic processes. Therapies started when animals had massive proteinuria and renal lesions. Methods PHN was accelerated by uninephrectomy seven days after IV injection of rabbit anti-FX1A antibody. Four months later, when massive proteinuria and renal lesions were present, the rats were divided into five groups and daily given orally: vehicle; lisinopril 40mg/L; lisinopril 400mg/L; simvastatin 2mg/kg b.i.d; or lisinopril 40mg/L plus simvastatin. Six normal rats served as controls. Animals were sacrificed at 10 months. Results By the end of the study three PHN rats died in the vehicle group, four in the group given lisinopril at 40mg/L and two in the group at 400mg/L, whereas all rats on simvastatin or combined therapy were alive. Blood pressure increased during time in PHN and was normalized by treatment with ACE inhibitor and combined therapy. Even at the high dose lisinopril failed to reduce proteinuria. Simvastatin only partially affected proteinuria. However, combining lisinopril with simvastatin had a remarkable antiproteinuric effect, such that at 10 months the urinary proteins were comparable to pre-treatment values and significantly lower than either the vehicle or lisinopril groups. Hypercholesterolemia of PHN rats was limited by combined therapy, and a positive correlation was found between serum cholesterol and proteinuria. Renal function was only partially ameliorated by simvastatin but significantly improved by combined therapy. Drug combination significantly limited glomerulosclerosis, tubular damage and interstitial inflammation, compared to vehicle or drugs alone. Up-regulation of monocyte chemoattractant protein-1 (MCP-1) mRNA in PHN kidneys was not affected by lisinopril, it was inhibited by 30% after simvastatin, and almost completely normalized by lisinopril plus simvastatin. Conclusions These data suggest that a combined ACE inhibitor and statin approach could represent a therapeutic option for patients with advanced renal disease in whom ACE inhibitors alone fail to lower proteinuria and injury to any substantial extent.