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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.

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.

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

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.

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.

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.

The contribution of microRNA (miRNA) to the pathogenesis of renal fibrosis is not well understood. Here, we investigated whether miRNA modulates the fibrotic process in Munich Wistar Fromter (MWF) rats, which develop spontaneous progressive nephropathy. We analyzed the expression profile of miRNA in microdissected glomeruli and found that miR-324-3p was the most upregulated. In situ hybridization localized miR-324-3p to glomerular podocytes, parietal cells of Bowman's capsule, and most abundantly, cortical tubules. A predicted target of miR-324-3p is prolyl endopeptidase (Prep), a serine peptidase involved in the metabolism of angiotensins and the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). In cultured tubular cells, transient transfection with a miR-324-3p mimic reduced Prep protein and activity, validating Prep as a target of this miRNA. In MWF rats, upregulation of miR-324-3p associated with markedly reduced expression of Prep in both glomeruli and tubules, low urine Ac-SDKP, and increased deposition of collagen. ACE inhibition downregulated glomerular and tubular miR-324-3p, promoted renal Prep expression, increased plasma and urine Ac-SDKP, and attenuated renal fibrosis. In summary, these results suggest that dysregulation of the miR-324-3p/Prep pathway contributes to the development of fibrosis in progressive nephropathy. The renoprotective effects of ACE inhibitors may result, in part, from modulation of this pathway, suggesting that it may hold other potential therapeutic targets.

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury. Kidney International (2012) 81, 903-918; doi:10.1038/ki.2011.473; published online 8 February 2012

Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8−/− grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8−/− than in Tir8+/+ grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8−/− dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-α than Tir8+/+ cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8−/− kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.

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.

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

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.

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

Proteinuria is one of the most potent risk factors for renal disease progression in patients with glomerular diseases. Studies in disease models have helped to delineate mechanisms leading to renal structural damage due to persistent dysfunction of the glomerular barrier to proteins, even when the primary immune or nonimmune insult to the kidney has ceased. The main focus of this review is the role of the tubular epithelial cell in the induction of interstitial inflammatory and fibrogenic reactions to ultrafiltered proteins. Antiproteinuric drugs (angiotensin–converting enzyme inhibitors, ACEi) while preserving the integrity of the glomerular permselective barrier limit both proteinuria and protein–dependent processes which contribute to tubulointerstitial injury, and in so doing ACEi halt the progression of proteinuric nephropathies toward terminal renal failure.

Progression to end-stage renal failure is the final common pathway of many forms of glomerular disease, independent of the type of initial insult. Progressive glomerulopathies have in common persistently high levels of urinary protein excretion and tubulointerstitial lesions at biopsy. Among the cellular mechanisms that may determine progression regardless of etiology, the traffic of excess proteins filtered from glomerulus in renal tubule may have functional importance by initiating interstitial inflammation in the early phase of parenchymal injury. This study analyzes the time course and sites of protein accumulation and interstitial cellular infiltration in two different models of proteinuric nephropathies. In remnant kidneys after 5/6 renal mass ablation, albumin and IgG accumulation by proximal tubular cells was visualized in the early stage, preceding interstitial infiltration of MHC-II-positive cells and macrophages. By double-staining, infiltrates developed at or near tubules containing intracellular IgG or luminal casts. This relationship persisted thereafter despite more irregular distribution of infiltrate. Similar patterns were found in an immune model (passive Heymann nephritis), indicating that the interstitial inflammatory reaction develops at the sites of protein overload, regardless of the type of glomerular injury. Osteopontin was detectable in cells of proximal tubules congested with protein in both models at sites of interstitial infiltration, and by virtue of its chemoattractive action this is likely mediator of a proximal tubule-dependent inflammatory pathway in response to protein load. Protein overload of tubules is a key candidate process translating glomerular protein leakage into cellular signals of interstitial inflammation. Mechanisms underlying the proinflammatory response of tubular cells to protein challenge in diseased kidney should be explored, as well as ways of limiting protein reabsorption/deposition to prevent consequent inflammation and progressive disease.

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney.

One of the leading causes of long-term kidney graft loss is chronic allograft injury (CAI), a pathological process triggered by alloantigen-dependent and alloantigen-independent factors. Alloantigen-independent factors, such as cold ischemia (CI) may amplify the recipient immune response against the graft. We investigated the impact of prolonged cold ischemia and the subsequent delayed graft function on CAI in a fully MHC-mismatched rat model of kidney allotransplantation. Prolonged CI was associated with anticipation of proteinuria onset and graft function deterioration (ischemia: 90d; no ischemia: 150d), more severe tubular atrophy, interstitial fibrosis, and glomerulosclerosis, and increased mortality rate (180d survival, ischemia: 0%; no ischemia: 67%). In ischemic allografts, T and B cells were detected very early and were organized in inflammatory clusters. Higher expression of BAFF-R and TACI within the ischemic allografts indicates that B cells are mature and activated. As a consequence of B cell activity, anti-donor antibodies, glomerular C4d and IgG deposition, important features of chronic humoral rejection, appeared earlier in ischemic than in non-ischemic allograft recipients. Thus, prolonged CI time plays a main role in CAI development by triggering acceleration of cellular and humoral reactions of chronic rejection. Limiting CI time should be considered as a main target in kidney transplantation.

Cell therapy with Multipotent Mesenchymal Stromal Cells (MSC) holds enormous promise for the treatment of a large number of degenerative and immune/inflammatory diseases. Their multilineage differentiation potential, immunoprivilege and capacity of promoting recovery of damaged tissues coupled with anti-inflammatory and immunosuppressive properties are the focus of a multitude of clinical studies currently underway. The recognized clinical potential of MSC repairing/immunomodulatory effects now encompasses graft-versus-host disease, hematologic malignancies, cardiovascular diseases, neurologic and inherited diseases, autoimmune diseases, organ transplantation, refractory wounds, and bone/cartilage defects among others. However, it has been suggested that both the need of extensive ex vivo culture for MSC clinical use, and their proangiogenic, anti-apoptotic and immunomodulatory properties may act together as tumor promoters, raising significant safety concerns. This paper will review the available data on in vitro MSC maldifferentiation and the ability of MSC to sustain tumor growth in vivo, with the aim to clarify whether MSC-based therapeutic approaches may carry actual risk of malignancies.

Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell-derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line-derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning.

Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction in humans. Increases in cold and warm ischemia times lead to a higher risk of early post-transplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term kidney graft loss. The protective effect of rabbit anti-rat thymocyte immunoglobulin (rATG) was evaluated in a rat model of I/R injury following syngeneic kidney transplantation. Serum creatinine concentration was evaluated at 16 h and 24 h post-transplant. Animals were sacrificed 24 h post-transplant for evaluation of histology, infiltrating leukocytes, nitrotyrosine staining, and apoptosis. rATG was effective in preventing renal function impairment, tissue damage and tubular apoptosis associated with I/R only when was given 2 h before transplantation but not at the time of reperfusion. Pretransplant rATG treatment of recipient animals effectively reduced the amount of macrophages, CD4(+), CD8(+) T cells and LFA-1(+) cells infiltrating renal graft subjected to cold ischemia as well as granzyme-B expression within ischemic kidney. On the other hand, granulocyte infiltration and oxidative stress were not modified by rATG. If these results will be translated into the clinical setting, pretransplant administration of Thymoglobuline(®) could offer the additional advantage over peri-transplant administration of limiting I/R-mediated kidney graft damage.

A number of features of the pathology occurring in spontaneously hypertensive stroke prone rats (SHRSPs), such as MRI brain signal abnormalities, the presence of high protein content in cerebrospinal fluid and vessel wall thickening, seem to indicate that this strain is a suitable model for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). To explore this hypothesis, we sought the human diagnostic hallmarks of the disease [the accumulation of granular osmiophilic material (GOM) deposits in vessel walls and NOTCH3 gene mutations] in SHRSPs. Male SHRSPs fed a permissive diet were sacrificed 3 days after the first MRI visualisation of brain abnormalities. Whole blood and kidney samples were respectively collected for molecular and electron microscopy evaluations. Automated sequence analysis of exons and intron-exon boundaries did not reveal any genetic variation in the NOTCH3 gene, and electron microscopy excluded the presence of GOM. The findings of this study exclude SHRSPs as a possible model for CADASIL.

We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Fromter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1 + parietal epithelial cells and, to a lesser extent, WT1 + podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman's capsule: NCAM + WT1 − cells, also expressing progenitor cell marker CD24, and NCAM + WT1 + and NCAM − WT1 + cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBPδ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM + progenitor proliferation via the modulation of C/EBPδ. Thus, chaotic migration and proliferation of the Bowman's capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.

Ischemia-reperfusion injury is an important cause of graft failure. Because carnitine regulates substrate flux and energy balance across membranes which may be deranged in ischemia we determined whether its use was effective in preventing kidney injury in an allogeneic transplant model. Brown Norway rats received a Lewis rat kidney transplant and were then treated with cyclosporine A to avoid rejection. The grafts were stored in Belzer solution supplemented with propionyl-L-carnitine during the cold ischemia period. Compared to rats receiving untreated kidneys but with equal cold ischemia times, the post-transplant serum creatinine values of the carnitine-treated transplants were significantly lower. Histological evaluation 16 h after transplant showed that propionyl-L-carnitine significantly inhibited tubular necrosis and neutrophil infiltration of the allografts and improved the 3 month graft survival. Treated transplants also had decreased lipid peroxidation, inducible nitric oxide synthase expression and protein nitration compared to the untreated grafts. Post-transplant serum creatinine levels were significantly reduced and graft survival was slightly prolonged in rats not receiving cyclosporine A treatment and transplanted with a kidney treated with propionyl-L-carnitine. The efficacy of propionyl-L-carnitine to modulate ischemia-reperfusion injury during transplantation suggests that its use in human transplantation is worth testing.

Salt-loaded, spontaneously hypertensive stroke-prone rats show progressive increases in blood pressure and proteinuria and accumulate acute-phase proteins in body fluids, modeling events during renal damage. The aim of this study was to assess the pathological events occurring in the kidney of spontaneously hypertensive stroke-prone rats over time and evaluate the effects of statin treatment, which is known to improve renal and cardiovascular outcomes. Kidneys of male spontaneously hypertensive stroke-prone rats euthanized at different stages of proteinuria showed progressive inflammatory cell infiltration, the accumulation of alpha-smooth muscle actin-positive cells, degenerative changes in podocytes, and severe fibrosis. These were accompanied by an imbalance in the plasminogen/plasmin and metalloprotease systems characterized by the increased renal expression of plasminogen activator inhibitor-1, tissue plasminogen activator, and urokinase plasminogen activator; the net result was an increase in plasmin and matrix metalloproteinase (MMP)-2 and a reduction in MMP-9 activity. Chronic treatment with the hydrophilic rosuvastatin had renoprotective effects in terms of morphology and inflammation and prevented the changes in plasmin, MMP-2, and MMP-9 activity. These effects were independent of the changes in blood pressure and plasma lipid levels. Treatment with the lipophilic simvastatin was not renoprotective. These data suggest that rosuvastatin may have potential utility as a therapeutic option in renal diseases that are characterized by inflammation and fibrosis.

New intervention tools for severely damaged kidneys are in great demand to provide patients with a valid alternative to whole organ replacement. For repairing or replacing injured tissues, emerging approaches focus on using stem and progenitor cells. Embryonic kidneys represent an interesting option because, when transplanted to sites such as the renal capsule of healthy animals, they originate new renal structures. Here, we studied whether metanephroi possess developmental capacity when transplanted under the kidney capsule of MWF male rats, a model of spontaneous nephropathy. We found that six weeks post-transplantation, renal primordia developed glomeruli and tubuli able to filter blood and to produce urine in cyst-like structures. Newly developed metanephroi were able to initiate a regenerative-like process in host renal tissues adjacent to the graft in MWF male rats as indicated by an increase in cell proliferation and vascular density, accompanied by mRNA and protein upregulation of VEGF, FGF2, HGF, IGF-1 and Pax-2. The expression of SMP30 and NCAM was induced in tubular cells. Oxidative stress and apoptosis markedly decreased. Our study shows that embryonic kidneys generate functional nephrons when transplanted into animals with severe renal disease and at the same time activate events at least partly mimicking those observed in kidney tissues during renal regeneration.

Platelet-derived growth factor (PDGF) has been proved to play an important role in progressive glomerular disease of systemic lupus. The present study investigated the tyrosine kinase inhibitor of PDGF receptor, imatinib, as a novel therapeutic approach for the cure of lupus nephritis in New Zealand Black/White (NZB/W)F1 hybrid mice with established disease. Two groups of NZB/W mice (N=30 each group), starting at 5 months of age, were given by gavage vehicle or imatinib (50 mg/kg b.i.d). Fifteen mice for each group were used for the survival study. The remaining were killed at 8 months. Imatinib significantly (P=0.0022) ameliorated animal survival with respect to vehicle. The drug significantly delayed the onset of proteinuria (% proteinuric mice, 7 and 8 months: 33 and 47 vs vehicle, 80 and 87, P0.05) and limited the impairment of renal function. Imatinib protected the kidney against glomerular hypercellularity and deposits, tubulointerstitial damage, and accumulation of F4/80-positive mononuclear cells and alpha-smooth actin-positive myofibroblasts. The abnormal transforming growth factor-beta mRNA expression in kidneys of lupus mice was reduced by imatinib. In conclusion, findings of amelioration of animal survival and renal manifestations in NZB/W lupus mice with established disease by imatinib suggests the possibility to explore whether imatinib may function as steroid-sparing drug in human lupus nephritis.

Short-term results of renal transplantation have improved considerably in the past 20 yr; however, similar improvements in long-term outcome have not been achieved. The primary cause of late graft loss is chronic rejection that might be treated by gene therapeutic approaches. Ideally, one would like to impair locally the contact between transplant antigen and the host immune system without compromising the generalized immune competence of the recipient. This can be achieved by local expression of the therapeutic protein in the site of interest using gene therapy. Here it is shown that chronic allograft rejection can be prevented effectively by local delivery of recombinant adeno-associated virus (AAV) vectors that encode the CTLA4Ig immunosuppressant protein to the donor kidney in a fully MHC-mismatched rat strain combination. AAV CTLA4Ig prevented progressive proteinuria and protected transplant kidneys from renal structural injury. A population of anergic T cells with regulatory activity, which eventually were responsible for the induction of tolerance, were found in recipient lymph nodes and in the graft as long as 120 d after transplantation. These data indicate that AAV-mediated CTLA4Ig gene transfer to donor graft represents a promising tool to prevent the onset of chronic rejection and circumvent the unwanted systemic adverse effects of the administration of immunomodulatory protein.

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. The model of renal injury induced in mice by the anticancer agent cisplatin was chosen. Injection of mesenchymal stem cells of male bone marrow origin remarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury. Y chromosome-containing cells localized in the context of the tubular epithelial lining and displayed binding sites for Lens culinaris lectin, indicating that mesenchymal stem cells engraft the damaged kidney and differentiate into tubular epithelial cells, thereby restoring renal structure and function. Mesenchymal stem cells markedly accelerated tubular proliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67-positive cells within the tubuli with respect to cisplatin-treated mice that were given saline. Hematopoietic stem cells failed to exert beneficial effects. These results offer a strong case for exploring the possibility that mesenchymal stem cells by virtue of their renotropic property and tubular regenerative potential may have a role in the treatment of acute renal failure in humans.

Renin-angiotensin system (RAS) inhibitors are effective in reducing renal disease progression in early diabetic nephropathy, but they provide imperfect protection at a later stage. Due to the pivotal role of transforming growth factor–β (TGF-β) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-β and angiotensin II on disease progression in diabetic rats with overt nephropathy. Diabetes was induced by streptozotocin injection in uninephrectomized rats. Diabetic rats received murine (1D11) or human (CAT-192) anti-TGF-β monoclonal antibodies alone or in combination with lisinopril, 13C4 irrelevant murine antibody, saline or lisinopril from month 4 (when animals had proteinuria) to month 8. Normal animals served as controls. Systolic BP increase was controlled by single treatments and even more by the combined therapies. 1D11 and lisinopril kept proteinuria at levels numerically lower than irrelevant antibody and saline, while CAT-192 was ineffective. The addition of either TGF-β antibody to lisinopril normalized proteinuria. Consistent results were obtained for glomerulosclerosis and tubular damage, which were abrogated by the combined therapy. Interstitial volume expansion and infiltration of lymphocytes/macrophages were limited by 1D11 and lisinopril and further reduced by their combination. The increase of type III collagen in the renal interstitium was partially attenuated by 1D11 and lisinopril while normalized by their combination. It is concluded that anti–TGF-β antibody when added to a background of chronic angiotensin-converting enzyme (ACE) inhibition fully arrests proteinuria and renal injury of overt diabetic nephropathy, providing a novel route to therapy and remission of disease for diabetic patients who do not respond to RAS inhibition. E-mail: zoja@marionegri.it

The current therapy for chronic proteinuric nephropathies is angiotensin-converting enzyme inhibitors (ACEi), which slow, but may not halt, the progression of disease, and which may be not effective to the same degree in all patients. In accelerated passive Heymann nephritis (PHN), this study assessed the effect of combining ACEi with angiotensin II receptor antagonist (AIIRA) and with statin that, besides lowering cholesterol, influences inflammatory and fibrogenic processes. Uninephrectomized PHN rats were divided into four groups ( n = 10 each) and daily given oral doses of the following: vehicle; 40 mg/L lisinopril; 100 mg/L lisinopril plus L-158,809; 0.3 mg/kg lisinopril plus L-158,809 plus cerivastatin. Treatments started at 2 mo when rats had massive proteinuria and signs of renal injury and lasted until 10 mo. Increases in BP were equally lowered by treatments. ACEi kept proteinuria at levels comparable to pretreatment and numerically lower than vehicle. The addition of AIIRA to lisinopril was more effective, being proteinuria reduced below pretreatment values and significantly lower than vehicle. When cerivastatin was added on top of ACE inhibition and AIIR blockade, urinary protein regressed to normal values and renal failure was prevented. Renal ACE activity was increased threefold in PHN, it was inhibited by more than 60% after ACEi, and decreased below control values with triple therapy. Cerivastatin inhibited ACE activity by 30%. Glomerulosclerosis, tubular damage and interstitial inflammation were ameliorated by ACEi alone or combined with AIIRA, and prevented by addition of statin. TGF-β 1 mRNA upregulation in PHN kidney was partially reduced after ACEi or combined with AIIRA and almost normalized after adding statin. Cerivastatin inhibited TGF-β 1 gene upregulation by 25%. These data suggest a possible future strategy to induce remission of proteinuria, lessen renal injury, and protect from loss of function in those patients who do not fully respond to ACEi therapy.

We evaluated the effect of blocking angiotensin II (AngII) on the development of proteinuria and glomerular injury in antithymocyte serum (ATS) glomerulonephritis. Disease was induced in Sprague-Dawley rats by a single intravenous injection of rabbit ATS. Three groups of rats were considered: group 1 (n = 13), ATS rats with no therapy; group 2 (n = 13), ATS rats treated with angiotensin-converting enzyme inhibitor (40 mg/L lisinopril in the drinking water); and group 3 (n = 13), ATS rats treated with AngII receptor antagonist (50 mg/L L-158,809 in the drinking water). Treatment started 3 hours after ATS injection and lasted 4 days. An additional group of control rats (group 4, n = 13) received preimmune serum. At day 4, ATS rats developed proteinuria (46+/-5 mg/d v control 12+/-1 mg/d; P0.01), which was prevented by both lisinopril and L-158,809 (14+/-0.2 mg/d and 15+/-1.6 mg/d, respectively, P0.01 v ATS). Systolic blood pressure was comparable in ATS rats and in controls (119+/-4 mm Hg v 120+/-2 mm Hg). Systolic blood pressure values were significantly decreased after either lisinopril or L-158,809 (104+/-3 mm Hg and 101+/-5 mm Hg, respectively; P0.01 v ATS). Serum creatinine levels were similar in all groups. Quantitation of proliferating cells and macrophages by analysis of proliferating cell nuclear antigen-positive and ED1-positive cells/glomerular cross-section showed a marked increase in proliferating cell nuclear antigen-positive cells in glomeruli of ATS rats over controls (12.6+/-0.5 cells/glomerular cross-section v 1.9+/-0.2 cells/glomerular cross-section; P0.01), which was significantly (P0.01) prevented by both treatments (lisinopril, 5.7+/-1.0 cells/glomerular cross-section; L-158,809, 4.8+/-1.5 cells/glomerular cross-section). The increase in ED1-positive cells (10+/-0.7 cells/glomerular cross-section v controls, 1.8+/-0.2 cells/glomerular cross-section; P0.01) was also significantly (P0.01) reduced by lisinopril and L-158,809 (4.1+/-0.7 cells/glomerular cross-sections and 2.6+/-0.6 cells/glomerular cross-section, respectively). Blocking of AngII activity prevented almost completely the formation of microaneurysms in ATS rats (percent of glomeruli with microaneurysms: ATS, 11.5%+/-3.5%; ATS + lisinopril, 0.4%+/-0.2%; ATS + L-158,809, 0.8%+/-0.8%; controls, 0%). Because AngII is a potent inducer of renal transforming growth factor-beta (TGF-beta), a cytokine involved in the regulation of cell proliferation, matrix deposition, and monocyte migration (which is overexpressed in the kidney of ATS rats), we then evaluated the effect of AngII inhibitors on renal gene expression of TGF-beta1 and on urinary TGF-beta1. TGF-beta1 mRNA levels in kidneys of ATS rats were 3.6-fold higher than those of controls and were reduced by 46% and 32% after treatment with lisinopril and L-158,809, respectively. Urinary TGF-beta1 excretion increased in ATS (37.3+/-6.0 ng/d v controls, 13.8+/-3.4 ng/d; P0.01) but was normalized by lisinopril and L-158,809 (7.6+/-1.9 ng/d and 6.4+/-0.4 ng/d, respectively; P0.01). Thus, in ATS, blocking AngII synthesis prevents proteinuria and reduces glomerular cell proliferation and inflammatory cell infiltration, possibly by reducing excessive renal TGF-beta synthesis. These findings may be relevant for future strategies in the treatment of human mesangioproliferative glomerulonephritis.

Remarkable progress has been made in the past two years in the investigation of immunological events and effector mechanisms which lead to glomerular inflammation and tissue damage, suggesting a number of areas in which therapeutic modulation is possible. We review recent advances in the two major areas of investigation in this field: the nephritogenic response to glomerulonephritis and local inflammatory reaction, and the novel therapeutic perspectives that have emerged from insights into the biology and pathology of the immune and inflammatory response.

The relative role of thromboxane (TxA2) and sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) in the acute renal failure induced by cyclosporine was studied in the rats. Bolus i.v. administration of 20 mg/kg of CsA but not vehicle to adult male Sprague-Dawley rats resulted in a significant fall in glomerular filtration rate from 0.85 +/- 0.10 and renal plasma flow (RPF) 2.45 +/- 0.14 ml/min/100 g body wt to values at 20 min of 0.47 +/- 0.03 and 1.01 +/- 0.12 ml/min/100 g body wt (P less than 0.01), respectively, without a fall in mean arterial pressure. This hemodynamic effect was maintained for the following 40-min period. Pretreatment of rats with the TxA2 receptor antagonist GR32191 (3 mg/kg i.v.) allowed a partial but significant preservation of GFR (0.60 +/- 0.05 ml/min/100 g body wt) and RPF (1.55 +/- 0.12 ml/min/100 g body wt). In addition, the antagonism of endogenously produced LTC4 and LTD4 with the putative receptor antagonist L-649,923 (1 mg/kg i.v.) partially prevented the fall in GFR (0.65 +/- 0.07 ml/min/100 g body wt) and RPF (1.80 +/- 0.18 ml/min/100 g body wt) at 20 min after CsA injection. The combined administration of GR32191 and L-649,923 completely abolished the CsA-induced decline in GFR (0.80 +/- 0.09 ml/min/100 g body wt) and RPF (2.40 +/- 0.12 ml/min/100 g body wt). These findings suggest that TxA2 and LTC4/LTD4 participate in mediating renal function deterioration induced by acute CsA administration in the rat.

Mycophenolate mofetil combined with a cyclooxygenase-2 inhibitor ameliorates murine lupus nephritis.BackgroundApproaches to the treatment of lupus nephritis include immunosuppressants associated with anti-inflammatory drugs, mainly steroids, which, however, cause major side effects. Mycophenolate mofetil (MMF) has been described as being less toxic than conventional immunosuppressants, and it was effective in preventing progressive nephritis in lupus mice. Our study evaluated the therapeutic effect of MMF in NZB/W F1 hybrid mice with established disease. We also examined the combination of MMF with a selective cyclooxygenase-2 (COX-2) inhibitor, DFU, based on previous findings of excessive renal production of COX-2-derived thromboxane A2 (TXA2) in lupus nephritis.MethodsFour groups of NZB/W mice (N = 30 each group), starting at five months of age, were given daily by gavage the following: vehicle, MMF 60 mg/kg, DFU 3 mg/kg, or MMF + DFU. Fifteen mice for each group were used for the survival studies, and the remaining mice were sacrificed at nine months.ResultsMMF or DFU alone partially delayed the onset of proteinuria compared with vehicle. Combined therapy was significantly (P < 0.05) more effective than single drugs. Animal survival was partially ameliorated by MMF and significantly improved by the drug combination in comparison with the vehicle (P = 0.005) and DFU alone (P < 0.03). At nine months, serum blood urea nitrogen (BUN) levels were lower in all of the treated groups than in the vehicle group. Renal damage was also limited, but to a greater extent in mice given the combined therapy. In untreated mice, renal COX-2 mRNA expression was up-regulated, and generation of TXB2, the stable breakdown product of TXA2, increased. DFU prevented the abnormal renal TXB2 production, confirming the COX-2 origin of this eicosanoid, whereas renal 6-keto-PGF1α and prostaglandin E2 (PGE2) were not affected substantially.ConclusionsThese results offer a strong case for exploring the possibility that in humans MMF combined with COX-2 inhibitors has a role in the treatment options for lupus nephritis. This combined drug therapy may be at least as effective as steroids but without the obvious nephrotoxicity of the latter.

Proximal tubular cells promote fibrogenesis by TGF - β1–mediated induction of peritubular myofibroblasts . Background In proteinuric nephropathies with increasingly severe defects of the glomerular filtering barrier, interstitial fibrogenesis is a major effector of scarring. An early event in this process is the peritubular accumulation of myofibroblasts that express α-smooth muscle actin (α-SMA) and contribute to abnormal matrix production. Common trigger factors are poorly understood. Enhanced protein trafficking may play a role by up-regulating inflammatory and fibrogenic genes in proximal tubular cells. Methods The remnant kidney model in rats was used to ( 1 ) analyze interactions between activated proximal tubular cells, peritubular cells expressing the myofibroblast marker, and inflammatory cells at time intervals (days 7, 14, and 30) after surgery, and ( 2 ) evaluate the effects of angiotensin-converting enzyme inhibitor (ACEi) on protein trafficking, fibrogenic signaling, and α-SMA expression. Results Abnormal uptake of ultrafiltered proteins by proximal tubular cells (IgG staining) occurred at an early stage (day 7) and was subsequently associated with macrophage and α-SMA+ cell accumulation into the peritubular interstitium. α-SMA+ cells clustered with macrophages into the interstitium. These changes were associated with appearance of transforming growth factor-β1 (TGF-β1) mRNA in proximal tubular cells and in the infiltrating cells with time. At day 30, focal α-SMA staining also was found in the tubular cells and in peritubular endothelial cells on semithin ultracryosections. ACEi prevented both proteinuria and abnormal protein accumulation in tubular cells, as well as the inflammatory and fibrogenic reaction with peritubular α-SMA expression. Conclusions Profibrogenic signaling from both proximal tubular cells on challenge with filtered protein and inflammatory cells is implicated as a key candidate trigger of progressive tubulointerstitial injury.

Experimental Goodpasture's syndrome in Wistar-Kyoto rats immunized with α3 chain of type IV collagen.BackgroundGlomerulonephritis and lung hemorrhage of autoimmune Goodpasture syndrome develop due to immune reactions against epitope(s) of the non-collagenous (NC1) domain of α3-chain of type IV collagen [α3(IV) NC1]. Whether thymic mechanisms have a role in the loss of tolerance to the Goodpasture epitope has not been established. We studied the renal and pulmonary effects of immunization with different forms (monomer, dimer, or hexamer) of α3(IV) NC1 collagen in Wistar-Kyoto (WKY) rats, and assessed whether the intrathymic inoculation of the antigen may protect against anti-GBM disease.MethodsWKY rats were immunized with bovine α3(IV) monomer, dimer, or hexamer, or with α3(IV) NC1 synthetic peptide. Renal function, kidney and lung immunohistology, and circulating and tissue bound antibodies to type IV collagen chains were analyzed. Effects of intrathymic inoculation of antigen on subsequent disease induction were analyzed in WKY rats given α3(IV) NC1 dimer or GBM preparation intrathymically 48hours before immunization.ResultsProteinuria, linear IgG deposition in GBM, and crescentic glomerulonephritis developed in WKY rats immunized with α3(IV) NC1 dimer or hexamer. Lesions were dose-dependent upon injections of 10 to 100 μg dimer. The α3(IV) NC1 monomer induced less severe proteinuria and no crescents. Pulmonary hemorrhage was detectable in 35% of rats immunized with 25 to 100 μg α3(IV) NC1 dimer; α3(IV) synthetic peptide (36 carboxyl terminal) did not induce disease. Rats injected intrathymically with up to 100 μg α3(IV) NC1 dimer or with GBM 48hours before immunization were not protected against subsequent development of proteinuria and glomerulonephritis.ConclusionsThese findings document that glomerulonephritis and lung hemorrhage can be elicited in WKY rats by immunization with α3(IV) NC1. Failure of the intrathymic inoculation of antigen to prevent disease suggests that immunological tolerance cannot be achieved by this intervention, in contrast to other autoimmune conditions, and may imply independent roles for cellular and humoral nephritogenic pathways in anti-GBM nephritis.

Propionyl-L-carnitine prevents renal function deterioration due to ischemia/reperfusion. Background Ischemia-reperfusion injury after organ transplantation is a major cause of delayed graft function. Prevention of post-transplant ischemia acute renal failure is still elusive. Methods The present study was designed to examine whether propionyl-l-carnitine, an acyl derivative of carnitine involved in fatty acid oxidation pathway and adenosine 5′-triphosphate (ATP) generation of mitochondria, prevented renal function deterioration and structural injury induced by ischemia-reperfusion in an ex vivo rat model of isolated perfused kidney (IPK) preparation and in vivo in a model of syngeneic kidney transplantation. Results In the model of ischemia (20 or 40min)/reperfusion (90 or 70min) in IPK, untreated kidneys showed a marked reduction of glomerular filtration rate (GFR) and renal perfusate flow (RPF) as compared to baseline, when perfusion was established by restoring effective perfusion pressure to 100mm Hg. Exposure of kidneys to propionyl-l-carnitine before establishing the ischemia insult to tissue, largely prevented renal function impairment. Pre-exposure of ischemic kidneys to propionyl-l-carnitine largely reduced the percent of lactate dehydrogenase (LDH), a cell injury marker, released into the perfusate after reperfusion as compared to untreated ischemic kidneys. Histologic findings showed very mild post-ischemic lesions in kidneys exposed to propionyl-l-carnitine as compared to untreated ischemic kidneys. Immunohistochemical detection of 4-hydroxynonenal protein adduct, a major product of lipid peroxidation, was very low in kidney infused with propionyl-l-carnitine and exposed to ischemia/reperfusion as compared to untreated ischemic kidneys. ATP levels were not affected by propionyl-l-carnitine treatment. Renal function of kidneys exposed for four hours to cold Belzer UW solution added with propionyl-l-carnitine and transplanted to binephrectomized recipients was largely preserved as compared to untreated ischemic grafts. Propionyl-l-carnitine almost completely prevented polymorphonuclear cell graft infiltration and reduced tubular injury at 16 hours post-transplant. Conclusions These data indicate that propionyl-l-carnitine is of value in preventing decline of renal function that occurs during ischemia-reperfusion. The beneficial effect of propionyl-l-carnitine possibly relates to lowering lipid peroxidation and free radical generation that eventually results in the preservation of tubular cell structure. The efficacy of propionyl-l-carnitine to modulate ischemia-reperfusion injury in these models opens new perspectives for preventing post-transplant delayed graft function.

Increased urinary excretion of thromboxane B 2 and 2,3-dinor-TxB 2 in cyclosporin A nephrotoxicity. Cyclosporin A (CsA) administration to rats is associated with a selective increase in urinary excretion of immunoreactive thromboxane B 2 (i-TxB 2 ), the stable breakdown product of TxA 2 . The exaggerated synthesis of TxA 2 may play a role in the reduction of glomerular filtration rate (GFR) observed both in animals and humans undergoing CsA treatment. The present study was designed to get further insight into the origin of the abnormal i-TxB 2 urinary excretion. Rats given orally CsA (50 mg/kg/day) for 30 days had a significant increase in the urinary excretion of both 2,3-dinor-TxB 2 and TxB 2 measured by technique of capillary column gas chromatography-negative ion chemical ionization mass spectrometry (HRGC-NICIMS). Urinary TxB 2 is more likely to reflect the renal synthesis of the parent compound, whereas 2,3-dinor-TxB 2 is considered to reflect the amount of TxB 2 formed in the circulation. Experiments in isolated perfused kidney (IPK) taken from animals given CsA for 30 days showed a lower percentage increase in urinary TxB 2 over vehicle treated animals. Moreover in IPK the ratio 2,3-dinor-TxB 2 /TxB 2 was lower than in vivo. The amount of i-TxB 2 detectable in serum of animals given CsA was not different from that of control animals. In contrast, isolated glomeruli taken from rats given CsA had an increase in their TxA 2 synthesis measured as i-TxB 2 in the supernatants. Ultrastructural studies on kidney specimens from animals given CsA showed a focal glomerular endothelial damage together with a marked infiltration of blood borne cells of monocyte-macrophage type in the glomerular tuft. In contrast, kidney specimens taken from IPK preparations were devoid of inflammatory cells. In vitro CsA did not interfere with platelet arachidonic acid (AA) metabolism as shown by a normal i-TxB 2 generation in vitro by rat platelet-rich plasma (PRP) exposed to CsA and then challenged with AA or ADP. Similarly isolated glomeruli and isolated proximal tubules from normal rats when challenged with CsA in vitro converted AA into TxA 2 normally. It is suggested that the cause of the increased urinary excretion of 2,3-dinor-TxB 2 is the consequence of intrarenal platelet and macrophage activation, probably triggered by the endothelial damage. The parallel increase in the urinary excretion of unmetabolized TxB 2 is likely to reflect a concomitant activation of resident renal cell AA metabolism induced by CsA.

Glomerular morphological changes produced by 5-hour intravenous infusion of endotoxin (40 micrograms/kg/h) were studied in 57 rabbits. During endotoxin infusion all animals developed signs of endotoxemia, anemia, and transient leukopenia and thrombocytopenia. Acute renal failure also developed in all animals. By light microscopy a marked polymorphonuclear cell infiltration was the prominent finding within 1 h from the beginning of the infusion. At hour 5 fibrin deposits and thrombi were detected in most animals. The abnormalities were reversible. By electron microscopy endothelial damage was detected few minutes after the beginning of endotoxin infusion followed by leukocyte and platelet infiltration in glomerular capillaries. Fibrin appeared at hour 5 as free strands into the lumens or in large clumps along the luminal surface of endothelial layer. Occasionally, occlusive fibrin thrombi were seen. At hour 48 only mild endothelial damage was seen, associated with few polymorphonuclear cells in the glomerular capillary lumens. These results document the sequence of renal changes in the best available animal model of hemolytic uremic syndrome. Beside endothelial damage, leukocyte infiltration in glomerular capillaries is a prominent finding suggesting a role of inflammatory cells in the development of microvascular thrombosis.

Mononuclear cell infiltration in glomeruli and renal interstitium is a prominent feature of some types of glomerulonephritis, including lupus nephritis. The mechanism(s) underlying monocyte influx into the kidney is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a chemotactic factor involved in the recruitment of monocytes/macrophages in the glomeruli of rats with mesangioproliferative as well as anti-glomerular basement membrane glomerulonephritis. In the study presented here, renal MCP-1 mRNA expression in New Zealand Black x New Zealand White (NZB/W) F1 mice, a model of genetically determined immune complex disease that mimics systemic lupus in humans, was investigated. Northern blot analysis revealed a single 0.7 kb MCP-1 transcript of very low intensity in kidneys from 2-month-old NZB/W mice that had not yet developed proteinuria nor renal damage. Message levels, which increased markedly with the progression of nephritis and in association with mononuclear cell infiltration, were 10- and 15- fold higher in 8-10-month-old mice than in 2-month-old mice. By in situ hybridization, increased expression of MCP-1 mRNA was demonstrated in glomeruli and, even more striking, in tubular epithelial cells. Western blot analysis demonstrated increased expression of MCP-1 protein in kidneys of 10-month-old NZB/W mice, consistent with MCP-1 mRNA data. When NZB/W mice were treated with cyclophosphamide up to 12 months of age, expression of MCP-1 in the renal tissue remained low, the influx of inflammatory cells did not appear, and glomerular and tubular structures remained well preserved. These data suggest that elevated MCP-1 might act as a signal for inflammatory cells to infiltrate the kidney in lupus nephritis.

The mechanism(s) by which angiotensin-converting enzyme (ACE) inhibitors prevent glomerular membrane loss of permselective function is still not understood. In male MWF rats, which develop spontaneous proteinuria with age, ACE inhibitors prevent proteinuria and increase glomerular ultrafiltration coefficient. These renoprotective effects are not associated with ultrastructural changes of capillary wall components. This study was undertaken to investigate whether ACE inhibitors modulate functional properties of glomerular basement membrane (GBM) and/or of epithelial cells, both of which have been suggested to play a role in the maintenance of the glomerular filtration barrier. The hydraulic and macromolecular permeability of the GBM were determined, by an in vitro filtration system, in untreated or lisinopril-treated rats and in Wistar rats taken as controls. By indirect immunofluorescence and immunoelectron microscopy, glomerular distribution of the tight junction protein zonula occludens- (ZO-1), a component of the slit diaphragm, was also studied. Results document that spontaneous proteinuria in MWF rats develops without significant changes in the permeability of the GBM to water and albumin, or in the ultrastructure of the podocyte foot processes, but is associated with an important alteration in the distribution of ZO-1 at the glomerular level. Lisinopril, which prevented proteinuria, also prevented glomerular redistribution of the protein. Thus, renoprotective effects of ACE inhibitors are not associated with changes in intrinsic functional properties of GBM, or ultrastructural changes of the epithelial cells, but rather with preservation of glomerular ZO-1 distribution and slit diaphragm function, which are essential for maintaining the filtration barrier.

Chronic renal diseases progress to organ insufficiency, which may require replacement therapy within one to three decades even independently of the type of initial insults. In the majority of cases, the degrees of proteinuria and interstitial leukocyte infiltration and scarring are strictly correlated with the rate of disease progression. This study tests the hypothesis that excess intrarenal protein traffic may cause lymphocyte-dependent interstitial injury that, while not fully controlled by antiproteinuric therapy, can be further inhibited by concomitant immunosuppression. A primarily nonimmune model was used to reproduce progressive renal disease due to a critical loss of nephron mass. Angiotensin-converting enzyme (ACE) inhibitor limited proteinuria, interstitial inflammation, MHC class II antigen expression, and severe lesions. Combined treatment with ACE inhibitor and a specific antilymphocyte agent, mycophenolate mofetil, dramatically attenuated macrophage and T cell infiltration, MHC-class II overexpression, dendritic cells, and all manifestations of the disease. Evidence of lymphocyte-mediated renal injury in the setting of excess protein traffic provides the basis for combining ACE inhibition and immunosuppression to halt progression of proteinuric kidney disease and minimize the need for dialysis or transplantation.

In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1β and TNFα synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.