Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.

Renal ischemia-reperfusion injury (IRI) is a severe complication of major surgery and a risk factor for increased morbidity and mortality. Here, we investigated mechanisms that might contribute to IRI-induced progression to chronic kidney disease (CKD). Acute kidney injury (AKI) was induced by unilateral IRI for 35 min in CD1 and C57BL/6 (B6) mice. Unilateral IRI was used to overcome early mortality. Renal morphology, NGAL upregulation, and neutrophil infiltration as well as peritubular capillary density were studied by immunohistochemistry. The composition of leukocyte infiltrates in the kidney after IRI was investigated by flow cytometry. Systemic blood pressure was measured with a tail cuff, and renal perfusion was quantified by functional magnetic resonance imaging (fMRI). Mesangial matrix expansion was assessed by silver staining. Following IRI, CD1 and B6 mice developed similar morphological signs of AKI and increases in NGAL expression, but neutrophil infiltration was greater in CD1 than B6 mice. IRI induced an increase in systemic blood pressure of 20 mmHg in CD1, but not in B6 mice; and CD1 mice also had a greater loss of renal perfusion and kidney volume than B6 mice ( P < 0.05). CD1 mice developed substantial interstitial fibrosis and decreased peritubular capillary (PTC) density by day 14 while B6 mice showed only mild renal scarring and almost normal PTC. Our results show that after IRI, CD1 mice develop more inflammation, hypertension, and later mesangial matrix expansion than B6 mice do. Subsequently, CD1 animals suffer from CKD due to impaired renal perfusion and pronounced permanent loss of peritubular capillaries.

ObjectivesIL-17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL-17A deficiency or treatment with a IL-17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD).MethodsIL-17A-deficient and wild-type (WT) mice underwent transient unilateral renal pedicle clamping for 45 min to induce IRI and subsequent renal fibrosis. Furthermore, a neutralizing anti-IL-17A antibody (mAb) was injected into WT mice before induction of renal IRI intravenously. On days 1, 7 and 21, inflammation, fibrosis, leukocyte infiltration and pro-inflammatory and pro-fibrotic cytokine expression were assessed in kidneys using histology, qPCR and flow cytometry.Key findingsIL-17A was significantly increased after renal IRI in WT kidneys. Levels of pro-inflammatory (MCP-1) cytokine and pro-fibrotic (collagen 11, fibronectin) transcripts were similar in the experimental groups studied. IL-17A deficiency had no effect on renal T-cell influx or the number, inflammatory phenotype, or spatial distribution of macrophages. Similarly, administration of an IL-17A blocking antibody did not attenuate inflammation.ConclusionsDespite the effects of IL-17 in other inflammation models, neither genetic IL-17A deficiency nor treatment with an IL-17A blocking antibody attenuated IRI and progression to CKD. We conclude that in severe renal IRI IL-17A is not crucially involved in disease progression.

ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

Objective: Angiopoietin-2, a protein secreted by stimulated endothelium and an antagonist of the endothelium-stabilizing receptor Tie2, contributes to the pathophysiology of septic multiple organ dysfunction. We tested the therapeutic potential of a pulmonary-endothelium-specific RNA interference-based angiopoietin-2 targeting strategy in sepsis.Design: Laboratory and animal research.Settings: Research laboratories of the Medical School Hannover, Department of Nephrology and Hypertension, Hannover and Silence Therapeutics GmbH, Berlin.Subjects: C57Bl/6 mice.Interventions: Lung-endothelium-specific angiopoietin-2 small interfering RNA was administered both before and after sepsis induction (cecal ligation and puncture or lipopolysaccharides) intravenously.Measurements and Main Results: Angiopoietin-2 small interfering RNA was highly specific and reduced angiopoietin-2 expression in the septic murine lungs up to 73.8% (p = 0.01) and enhanced the phosphorylation of Tie2 both in control and septic animals. Angiopoietin-2 small interfering RNA reduced pulmonary inter-leukin-6 transcription, intercellular adhesion molecule expression, neutrophil infiltration, and vascular leakage. Manifestations of sepsis were also attenuated in distant organs, including the kidney, where renal function was improved without affecting local angiopoietin- 2 production. Finally, angiopoietin-2 small interfering RNA ameliorated the severity of illness and improved survival in cecal ligation and puncture, both as a pretreatment and as a rescue intervention.Conclusion: The Tie2 antagonist angiopoietin-2 represents a promising target against sepsis-associated multiple organ dysfunction. A novel RNA interference therapeutic approach targeting gene expression in the pulmonary endothelium could be a clinically relevant pharmacological strategy to reduce injurious angiopoietin-2 synthesis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides are characterized by destruction of small vessels, granulomatous inflammation of the respiratory tract and necrotizing glomerulonephritis. This review describes the clinical diagnosis and therapy as well as the patho-physiology of ANCA-associated vasculitides with a specific focus on the interplay of ANCAs with activated neutrophils and the deleterious pathophysiological consequences of neutrophil-endothelium interaction.

1 injury via TNF alpha dependent inhibition of apoptosis and inflammation 2 3 Song Rong, Katja Hueper, Torsten Kirsch, Robert Greite, Christian Klemann, Michael 4 Mengel, Matthias Meier, Jan Menne, Michael Leitges, Nathan Susnik, Martin Meier, 5 Hermann Haller, Nelli Shushakova, Faikah Gueler 6 7 8 Correspondence to: 9 Prof. Faikah Gueler, 10 Department of Nephrology 11 Medical School Hannover, 12 Carl-Neuberg-Str.1, 30625 Hannover, 13 Tel: 0049-511-532 3722, Fax: 0049-511-552366 14 email: gueler.faikah@mh-hannover.de 15 16 Department of Nephrology, Hannover Medical School, Hannover, Germany 17 Phenos GmbH, Hannover, Germany 18 The Transplantation Center of the affiliated hospital, Zunyi Medical College, China 19 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, 20 Canada 21 The Biotechnology Centre of Oslo, University of Oslo 22 Institute for Diagnostic and Interventional Radiology, Medical School Hannover, Hannover, 23 Germany 24 Articles in PresS. Am J Physiol Renal Physiol (July 23, 2014). doi:10.1152/ajprenal.00372.2013

Acute kidney injury (AKI) increases the risk of morbidity and mortality after major surgery and transplantation. We investigated the effect of PKC-ε deficiency on AKI and ischemic allograft damage after kidney transplantation. PKC-ε-deficient and wild type (WT) control mice were subjected to 35 min of renal pedicle clamping to induce AKI. PKC-ε deficiency was associated with a marked improvement in survival and an attenuated loss of kidney function. Furthermore, functional MRI experiments revealed better renal perfusion in PKC-ε-deficient mice than in WT mice one day after IRI. Acute tubular necrosis and neutrophil infiltration were markedly reduced in PKC-ε-deficient mice. To determine whether this resistance to ischemia-reperfusion injury resulted from changes in local renal cells or infiltrating leukocytes, we studied a life-supporting renal transplant model of ischemic graft injury. We transplanted kidneys from H(2b) PKC-ε-deficient mice (129/SV) and their corresponding WT littermates into major histocompatibility complex-incompatible H(2d) recipients (BALB/c) and induced ischemic graft injury by prolonged cold ischemia time. Recipients of WT allografts developed severe renal failure and died within 10 days of transplantation. Recipients of PKC-ε-deficient allografts had better renal function and survival; they had less generation of ROS and upregulation of proinflammatory proteins (i.e., ICAM-1, inducible nitric oxide synthase, and TNF-α) and showed less tubular epithelial cell apoptosis and inflammation in their allografts. These data suggest that local renal PKC-ε expression mediates proapoptotic and proinflammatory signaling and that an inhibitor of PKC-ε signaling could be used to prevent hypoxia-induced AKI.

Aldosterone (Aldo) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Aldo alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells were exposed to Aldo (10 –9 mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions and activation of intracellular signaling pathways were determined. Aldo increased endothelial permeability for molecules ≤70 kDa within 60 minutes. A transient loss of cortical actin with formation of actin stress fibers and disruption of continuous adherens and tight junction strands accompanied these changes. Mineralocorticoid receptor blockade, inhibition of RhoA, or disruption of extracellular-regulated protein kinase1/2 signaling pathways attenuated the Aldo-related effects. Moreover, Aldo-induced cytoskeletal rearrangement led to rapid dephosphorylation of protein kinase B and subsequent deactivation of endothelial nitric oxide synthase. Ex vivo tracer flux experiments with Evans blue–conjugated albumin demonstrated a concordant response to Aldo in freshly isolated umbilical arteries. Furthermore, low-dose cortisol (3×10 –10 to 3×10 –9 mol/L) mimics the effect of Aldo on endothelial integrity, and Aldo, by upregulating11β-hydroxysteroid dehydrogenase type 2, might even aggravate this deleterious effect of low-dose cortisol. We suggest that these mechanisms may contribute to the vasculopathy induced by inappropriate mineralocorticoid receptor activation.

Hypertension is one of the major risk factors for chronic kidney disease. Using quantitative trait loci analysis, we identified the gene of the F-BAR protein NOSTRIN in the center of an overlapping region in rat and human quantitative trait loci that are associated with hypertension. Immunohistochemical analysis revealed a predominantly podocytic expression pattern of NOSTRIN in human and mouse glomeruli. Further, NOSTRIN colocalizes with cell–cell contact–associated proteins β-catenin and zonula occludens-1 and interacts with the slit-membrane–associated adaptor protein CD2AP. In zebrafish larvae, knockdown of nostrin alters the glomerular filtration barrier function, inducing proteinuria and leading to ultrastructural morphological changes on the endothelial and epithelial side and of the glomerular basement membrane of the glomerular capillary loop. We conclude that NOSTRIN expression is an important factor for the integrity of the glomerular filtration barrier. Disease-related alteration of NOSTRIN expression may not only affect the vascular endothelium and, therefore, contribute to endothelial cell dysfunction but might also contribute to the development of podocyte disease and proteinuria.

Renal histology in ANCA-associated vasculitis: Differences between diagnostic and serologic subgroups.Differences in renal histopathology between microscopic polyangiitis (MPA) and Wegener's granulomatosis (WG), and between anti-neutrophil cytoplasm autoantibody (ANCA) test results in patients with ANCA-associated vasculitis may provide insight into the differences in pathogenesis and raise the opportunity of classifying the vasculitides more accurately. The possible differences in histopathology are investigated in this study.We report an analysis of 173 patients with renal disease in microscopic polyangiitis or Wegener's granulomatosis. A total of 173 renal biopsies, performed at diagnosis, were scored by two observers separately, using a previously standardized protocol. Consensus on each biopsy was achieved during a central review.Normal glomeruli were more common in WG than in MPA (P < 0.001). Glomerulosclerosis was more prominent in MPA than in WG (P = 0.003). Interstitial fibrosis (P < 0.001), tubular atrophy (P < 0.001), and tubular casts (P = 0.005) were more frequently present and more severe in MPA than in WG. Presence of glomerulosclerosis was more extensive in patients with myeloperoxidase (MPO)-ANCA than with proteinase 3 (PR3)-ANCA (P = 0.022). Interstitial fibrosis (P = 0.008), tubular necrosis (P = 0.030), tubular atrophy (P = 0.013), and intra-epithelial infiltrates (P = 0.006) were more frequently present and more severe in MPO-ANCA than in PR3-ANCA.Glomerulonephritis in relation to MPA has more characteristics of chronic injury at the time of presentation than glomerulonephritis in relation to WG. This difference may be due to a delayed establishment of diagnosis in patients with MPA compared to patients with WG. Both active and chronic lesions are more abundantly present in MPO-ANCA–positive patients than in patients with PR3-ANCA–positivity, which suggests that the pathogenesis of renal disease in these ANCA subsets could be different. Our results also suggest that ANCA test results may be useful in classifying ANCA-associated vasculitides.

Background: Ischemia reperfusion injury (IRI) leads to progressive renal fibrosis and loss of renal function. In this study we tested the efficacy of a PKC alpha/beta inhibitor (PKC-I) to attenuate post-ischemic renal fibrosis and inflammation. Methods: IRI was induced in mice by transient unilateral clamping of the left renal pedicle for 45 min. Treatment with the PKC alpha/beta inhibitor (0.6mg/day) was initiated either prior to or 24h after ischemia and continued over 28 days. Renal morphology, glomerular filtration rate (GFR), renal blood flow (RBF), expression of alpha-SMA, collagen 4, and fibronectin were examined. Furthermore, expression of CTGF and PAI-1 down-stream targets of TGF-beta as well as inflammatory cell infiltration (macrophages, CD4+ T-cells) was investigated. Results: In untreated mice GFR and RBF were significantly reduced 28 days after unilateral ischemia. In contrast, treatment with the PKC inhibitor improved renal function markedly. IRI caused severe renal fibrosis and up-regulation of pro-fibrotic proteins (a-SMA, fibronectin, collagen4). PKC-I pre-treatment partially blocked up-regulation of pro-fibrotic proteins. Furthermore, up-regulation of CTGF and PAI-1 expression was significantly ameliorated by PKC-I pre-treatment (p< 0.005) and inflammatory cell infiltration was markedly attenuated. Conclusion: PKC alpha/beta inhibition reduces experimental renal fibrosis via inhibition of TGF-beta signaling and attenuates renal inflammation.

Aldosterone (Ald) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Ald alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells (HUVEC) were exposed to Ald (10 -9 mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions, and activation of intracellular signaling pathways were determined. Ald increased endothelial paracellular permeability for molecules up to 70 kDa within 60 min. A transient loss of cortical actin with formation of actin stress fibers, and disruption of continuous adherens and tight junction strands, accompanied the changes. Mineralocorticoid receptor (MR) blockade, inhibition of RhoA or disruption of extracellular regulated protein kinase (ERK) 1/2 signaling pathways attenuated the Ald-related effects. Moreover, the observed changes of the actin cytoskeleton and intercellular junction architecture were accompanied by a rapid dephosphorylation of protein kinase B (AKT) as well as deactivation of endothelial NO synthase (eNOS). Ex vivo tracer flux experiments with Evans Blue (EB)-conjugated albumin demonstrated concordant response to Ald in freshly isolated umbilical arteries. In summary, Ald leads to increased permeability of endothelial monolayer or isolated arteries. These alterations are associated with a temporal opening of Intercellular junctions and diminished eNOS activity, and depend on activation of the RhoA/ERK pathway. We conclude that these mechanisms could contribute to Ald-induced vasculopathy.

CD4+ T cells contribute to the pathogenesis of ischemia-reperfusion injury, which is the primary cause of delayed graft failure after kidney transplantation. The TIM-1:TIM-4 pathway participates in the activation/differentiation of CD4+ T cells, suggesting that it may modulate ischemia-reperfusion injury. Here, we studied the role of TIM-1 in a murine uninephrectomized renal ischemia-reperfusion injury model. Blocking the TIM-1:TIM-4 pathway with an antagonistic monoclonal antibody protected renal function and diminished reperfusion injury resulting from 30 minutes of ischemia. Histologic examination showed significantly less evidence of renal damage as evidenced by diminished tubular necrosis, preservation of the brush border, fewer cast formations, and less tubular dilation. Blocking TIM-1 also reduced the number of apoptotic cells and diminished local inflammation within ischemic kidneys, the latter shown by decreased recruitment of macrophages, neutrophils, and CD4+ T cells and by reduced local production of proinflammatory cytokines. Furthermore, TIM-1 blockade significantly improved survival after ischemia-reperfusion injury. Taken together, these data suggest that the TIM-1:TIM-4 pathway enhances injury after renal ischemia-reperfusion injury and may be a therapeutic target.