The double edge of anti-CD40 siRNA therapy: It increases renal microcapillar density but favours the generation of an inflammatory milieu in the kidneys of ApoE\u2212/\u2212 mice

Miguel Hueso,Miguel Hueso,Angela Casas,Adrian Mallén,Angela Casas,Nuria Bolaños,Joan Torras,Laura De Ramón,Josep M Cruzado,Josep M Cruzado,Cristian Varela,Estanislao Navarro

doi:10.1186/s12950-019-0228-9

Abstract

BackgroundChronic kidney disease (CKD) is associated with endothelial dysfunctions thus prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors.Purpose of the studyWe have previously reported that siRNA-silencing of CD40 (siCD40) reduced atherosclerosis (ATH) progression. Here, we have deepened on the effects of the siCD40 treatment by evaluating retrospectively, in stored kidneys from the siCD40 treated ApoE−/− mice, the renal microcirculation (measured as the density of peritubular capillaries), macrophage infiltration and NF-κB activation.MethodsKidneys were isolated after 16 weeks of treatment with the anti-CD40 siRNA (siCD40), with a scrambled control siRNA (siSC) or with PBS (Veh. group). Renal endothelium, infiltrating macrophages and activated NF-κB in endothelium were identified by immunohistochemistry, while the density of stained peritubular capillaries was quantified by image analysis.ResultsATH was associated with a reduction in renal MC, an effect reversed by the anti-CD40 siRNA treatment (3.8 ± 2.7% in siCD40; vs. 1.8 ± 0.1% in siSC; or 1.9 ± 1.6% in Veh.; p < 0.0001). Furthermore, siCD40 treatment reduced the number of infiltrating macrophages compared to the SC group (14.1 ± 5.9 cells/field in siCD40; vs. 37.1 ± 17.8 cells/field in siSC; and 1.3 ± 1.7 cells/field in Veh.; p = 0.001). NF-κB activation also peaked in the siSC group, showing lower levels in the siCD40 and Veh. groups (63 ± 60 positive cells/section in siCD40; vs. 152 ± 44 positive cells/section in siSC; or 26 ± 29 positive cells/section in veh.; p = 0.014). Lastly, serum creatinine was also increased in the siCD40 (3.4 ± 3.3 mg/dL) and siSC (4.6 ± 3.0 mg/dL) groups when compared with Veh. (1.1 ± 0.9 mg/dL, p = 0.1).ConclusionsAnti-CD40 siRNA therapy significantly increased the density of peritubular capillaries and decreased renal inflammation in the ATH model. These data provide a physiological basis for the development of renal diseases in patients with ATH. Furthermore, our results also highligth renal off-target effects of the siRNA treatment which are discussed.Graphical abstract

Highlights

Chronic kidney disease (CKD) is associated with endothelial dysfunctions prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors.Purpose of the study: We have previously reported that short interference RNA (siRNA)-silencing of CD40 reduced atherosclerosis (ATH) progression
Anti-CD40 siRNA treatment increased microcapillar density in the kidneys of treated mice We have previously shown that silencing of the costimulatory receptor CD40 with a specific siRNA ameliorated progression of experimental ATH in the ApoE−/− model mouse when compared with control animals treated with a scrambled-sequence siRNA or with vehicle (Veh. group) [15]
We hypothesized that ATH progression could be associated to a reduction in renal microcirculation, and here we show that the silencing of CD40 (siCD40) therapy impacted on the renal microcapillar density, measured as the number of platelet-endothelial cell adhesion molecule-1 (PECAM-1) positive cells per optical field at a 200x magnification

Summary

Introduction

Chronic kidney disease (CKD) is associated with endothelial dysfunctions prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors.Purpose of the study: We have previously reported that siRNA-silencing of CD40 (siCD40) reduced atherosclerosis (ATH) progression. Insults to endothelial-cells (EC) activate oxidative systems, such as the NADPH oxidase complex, that produce hydrogen peroxide which subsequently causes oxidative stress [8] This stimulates the phosphorylation of NFκB, which leads to the activation of EC, the upregulation of chemotactic factors and adhesive receptors, and to the recruitment of leukocytes [8]. In this sense, it its known that NF-κB activates many of the genes involved in the inflammatory response network that are pivotal in the pathogenesis of vascular injury [9], and that activated NF-κB could have a role in the progression of kidney diseases [10], suggesting that NF-κB activation could be the physiological link among vascular injury and the development of renal diseases

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