Torsten Kirsch

Objectives: IL-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). Methods: IL-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 findings: IL-17A was significantly increased after renal IRI in WT kidneys. Levels of pro-inflammatory (MCP-1) cytokine and pro-fibrotic (collagen 1α1, 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. Conclusions: Despite 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.

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. Laboratory and animal research. Research laboratories of the Medical School Hannover, Department of Nephrology and Hypertension, Hannover and Silence Therapeutics GmbH, Berlin. C57Bl/6 mice. Lung-endothelium-specific angiopoietin-2 small interfering RNA was administered both before and after sepsis induction (cecal ligation and puncture or lipopolysaccharides) intravenously. 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 interleukin-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. 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.

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.

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.

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.

In humans, dehydroepiandrosterone (DHEA), with its sulfate, is the most abundant adrenal steroid, whereas the rat adrenals are not capable of synthesizing this steroid. Circulating concentrations of DHEA sulfate lie in the millimolar range and those of DHEA in the subnanomolar range. DHEA exerts protective potential during vascular remodeling, although the underlying mechanisms of this protection are imperfectly defined. We hypothesized that physiological doses of DHEA alter signaling pathways that are of central importance for vascular integrity. We exposed human endothelial cells, vascular smooth muscle cells, and fibroblasts to DHEA (10 ⁻⁶ to 10 ⁻¹⁰ mol/L) and observed a dose- and time-dependent increase of extracellular signal-regulated kinases 1 and 2 activation. Similar results were observed in rat vascular smooth muscle cells. In addition, in rat vascular smooth muscle cells, we found altered phosphorylation and cellular translocation of the transcription factor FoxO1. Pharmacological blockade of the mineralocorticoid receptor (MR) with eplerenone or small interfering RNA-mediated MR-silencing prevented DHEA-induced extracellular signal-regulated kinase 1/2 phosphorylation and its effects on FoxO1. Of note, in a cell-based MR transactivation assay, we did not find any agonist effect of DHEA on MR activity. We conclude that DHEA induces early signaling events in vascular cells that might underlie the DHEA-mediated protection against vasculopathies. These effects are dependent on the MR, although the finding that DHEA fails to act as a direct MR agonist suggests that additional signaling proteins are involved. In this regard, DHEA may either interact with coeffectors to modify MR activity or serves as a ligand for a yet unknown receptor that might transactivate the MR.

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.