Activation of α7 Nicotinic Acetylcholine Receptor Ameliorates Zymosan-Induced Acute Kidney Injury in BALB/c Mice

Zymosan, a natural compound, provokes acute peritonitis and multiple organ dysfunction that affects the kidney, beside other organs via exaggerated inflammatory response. The aim of the present study is to test the role of cholinergic anti-inflammatory pathway (CAP) in alleviating acute kidney injury (AKI) induced by zymosan in BALB/c mice, using galantamine, a cholinesterase inhibitor, known to act via α7 nicotinic acetylcholine receptor (α7 nAChR) to stimulate CAP. Galantamine verified its anti-inflammatory effect by elevating acetylcholine (ACh) level, while abating the interleukin-6/ janus kinase 2 (Y1007/1008)/ signal transducer and activator of transcription 3 (Y705) (IL-6/ pY(1007/1008)-JAK2/ pY705-STAT3) inflammatory axis, with a consequent inhibition in suppressor of cytokine signaling 3 (SOCS3). This effect entails also the nuclear factor-kappa B (p65)/ high mobility group box protein-1/ (NF-κB (p65)/ HMGB-1) signaling pathway. Furthermore, the reno-curattive effect of galantamine was associated by a reduction in plasma creatinine (Cr), cystatin (Cys)-C, IL-18, and renal neutrophil gelatinase-associated lipocalin (NGAL), as well as an improved histopathological structure. Blocking the α7 nAChR by methyllycaconitine abolished the beneficial effect of galantamine to document the involvement of this receptor and the CAP in the amelioration of AKI induced by zymosan.

Assessed parameters. For detection of ZYM-induced AKI and the effect of GAL, a histopathological examination was carried out in addition to the measurement of renal biomarkers; viz., plasma creatinine (Cr), interleukin-18, cystatin (Cys)-C, as well as renal neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, to study the possible and involved mechanism(s) of how GAL may act against AKI, acetylcholine (ACh) was assessed besides the inflammatory trajectory IL-6/ pY1007/1008-JAK2/ pY705-STAT3/ SOSC3. Besides, the renal transcriptional factor, NF-κB (p65), and the expression of one of its downstream molecules, HMGB-1 that acts in a vicious cycle were measured. These parameters were further evaluated to highlight their role in kidney damage induced by ZYM.

Techniques. Colorimetric method (End point).
Plasma Cr (Biodiagnostic colorimetric kit, Cairo, Egypt) was measured according to the kit manufacturers' instructions. The principle depends on the formation of a colored complex between Cr and picrate in alkaline medium.
Western blot analysis. The kidney lysate aliquots were brought to complete protein extraction, after several processing steps and the protein concentration was determined using Bradford Protein Assay Kit (Bio Basic, Ontario, Canada). The protein concentration of each sample was loaded with an equal volume of 2x Laemmli buffer (125 mM Tris, pH 6.8; 10% glycerol, 10% SDS; 0.006% bromophenol blue; 130 mM DTT), then the mixture was boiled for 90 sec at 100 °C. Protein samples were loaded and separated by 10% SDS-PAGE (Bio-Rad, CA, USA) using mini protein electrophoresis separation unit (Bio-Rad, CA, USA). This was followed by gel electrophoresis transfer onto polyvinylidenedifluoride (PVDF) membranes using Trans-Blot Turbo instrument (Bio-Rad, CA, USA).The membranes were blocked at room temperature for 1 h using blocking buffer (20 mM Tris, pH7.5; 150 mM NaCl; 0.1% Tween 20; 3% bovine serum albumin), then the blocked blots were incubated overnight at 4 °C with the primary antibodies; anti-pY705-STAT3 (1:1000), anti-pY1007/1008-JAK2 (1:1000) (Invitrogen, CA, USA), and anti-suppressor of cytokine signaling (SOSC)3 (1:200) (Thermo Fisher Scientific, MA, USA) as well as the antibody of the loading control anti-β-actin antibody (1:1000) (Santa Cruz Biotechnology, CA, USA). The latter was probed to assure equivalent sample loading, then the blot was washed five times in a mixture of Tris-buffered saline with Tween 20. This was followed by incubating the blot membranes to HRP conjugated to anti-rabbit antibody (Dianova, Hamburg, Germany). The charge coupled device (CCD) camera-based imager was used to capture the chemiluminescent signals and image analysis software was used to read the band intensity of the studied proteins on the Chemi Doc MP imager (Bio-Rad, CA, USA). Results were expressed as arbitrary units (AU) after normalization for β-actin protein expression.
Quantitative RT-PCR analysis. Total RNA was extracted from the kidney using RNeasy mini kit (Qiagen, MD, USA). All procedures were done according to the manufacturer protocol. Any residual DNA was removed by the kit provided DNase. The concentration of the isolated RNA in each sample was measured at 260 nm using spectrophotometer and the purity of isolated RNA was assessed by absorption ratio at 260/280 nm. For cDNA synthesis, a reverse transcription system was used (Fermentas, MA, USA), where the RNA was incubated with 5X first strand reverse transcription buffer, 10 mM dNTP mixture, oligo d (t) primers, 40U/µl RNase inhibitor, and 50 U/µl MMLV-RT enzyme at 42 °C for 60 min. The PCR reactions include 10 min at 95 °C for activation of AmpliTaq DNA Polymerase, followed by 40 cycles at 94 °C for 15 sec (denaturing), 60 °C for 1 min (annealing), and 72 °C MA, USA). The quantitative PCR reaction mixture consisted of SYBR green, cDNA template, RNase free water, in addition to the sequences of forward and reverse primers for high mobility group box protein (HMGB)-1 and β-actin ( Table 1). The relative quantification was calculated from the 2 −ΔΔCT formula 24 using β-actin as the internal standard genes.
Histopathological examination. The samples were fixed with 10% PBS buffered formalin for 8 h at room temperature, embedded in paraffin, and sectioned to 4 μm thickness. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin (H&E). The severity of renal damage was semiquantitatively assessed 15,25 with few modifications. The degree of renal injury was scored based on a subjective scale ranging from 0 to 3, where 0 = absence, 1 = mild, 2 = moderate, and 3 = severe. The ranging scale was used for vacuolation of renal tubules, epithelium congestion, glomerular tuft congestion of renal blood vessels, atrophy of glomerular tuft, necrosis of epithelial lining, and renal tubules perivascular inflammatory cells infiltration. The histological evaluations were done by a single investigator in a blinded manner and the renal damage grading was measured as an average score of the score mean of each criteria.
Statistical Analysis. Results are expressed as mean ± SD. The GraphPad Prism v5.0 (GraphPad Prism, CA, USA) was used to analyze and express all the available data. For multiple comparisons, one-way analysis of variance (ANOVA), followed by Tukey post-hoc test was used. For histopathological scoring, significance was maintained only while using non-parametric Mann-Whitney U test. P < 0.05 was considered as the significance limit for all comparisons, unless otherwise stated.
The administered drugs showed extreme significant alterations in these parameters (p < 0.001). Supplementary data for western blotting analysis are available.

GAL via α7 nAChR improves renal morphological alterations in ZYM-induced AKI.
Photomicrograph sections (Fig. 4)   minor congestion of glomerular tuft. Sections of (g and h) MLA + GAL treated animals display epithelial lining cytoplasmic vacuolization with glomerular tuft dilatation and congestion, as well as necrosis of renal blood vessels of epithelial lining renal tubules. Fig. 5 [a-f] summarizes the aforementioned data represented by histopathological renal score.

Discussion
The present study highlighted the involvement of CAP, via activation of the α7 nAChR, in the reno-protective effect of GAL against a ZYM-induced kidney injury model. Post administration of GAL ratified its anti-inflammatory potential by elevating ACh level and inhibiting the IL-6/ p-JAK2/ p-STAT3/ SOCS3 pathway, besides abating the HMGB-1/ NF-κB (p65) inflammatory vicious cycle. The favorable modulatory effects of these pathways were further confirmed by the marked decrease in the kidney function tests, viz., Cr, Cys-C, IL-18, and NGAL, in addition to the improvement of histopathological alterations. Since the selective blocker, MLA, abolished GAL effects at all levels, hence it could be concluded that GAL anti-inflammatory mechanisms are mediated through α7 nAChR. These effects were summarized and displayed in Fig. 6 as a schematic pathway. Currently, vagal stimulation has been documented to have a beneficial effect in modulating several inflammatory conditions 26 . Based on a series of studies 27,28 , GAL anti-inflammatory action was reported to override that of other anti-cholinesterases, by virtue of its allosteric modulation of α7 nAChR to synergistically activate the CAP. The findings of the present study concur with this fact, where GAL elevated ACh level, besides activating the α7 nAChR. The latter effect was confirmed by using the selective α7 nAChR blocker, which abolished its favorable effects.
GAL mediated its anti-inflammatory effect via an interplay between constellations of cues. In the present work, GAL attenuated ZYM-induced IL-6, which can be owed to the elevated level of ACh; previously, ACh was reported to reduce the release, expression, and/or mRNA half-life of IL-6 29,30 . This inhibition entailed the downstream JAK2/ STAT3 cascade, for IL-6 being the initiator of this pathway 31 . In accordance to our results, activated/ phosphorylated JAK2/ STAT3 axis was responsible for the injurious effect of different AKI insults 32,33 . Hence, the ability of GAL to suppress this signal was a culprit for the improved kidney function assessed here, besides its ability to stimulate the α7 nAChR. Ample of evidence have highlighted the importance of this signal in alleviating several inflammatory conditions via stimulating the α7 nAChR. Suppression of p-JAK2/ p-STAT3 by their inhibitors 34 or the use of cholinergic agonists [34][35][36] in LPS models was the corner stone in alleviating inflammation. These authors ascribed the blunted p-JAK2/ p-STAT3 axis to the activation of α7nAChR. In a ZYM-induced AKI model, Dimitrova et al. 15 emphasized the role of this pathway in improving kidney function upon using a JAK2 inhibitor 15 , to support further the current results. ZYM-induced AKI also elevated SOCS3, which is the downstream feedback molecule that is released in attempt to brake the inflammatory pathway IL-6/ p-JAK2/ p-STAT3. Nevertheless, in the GAL treated group, SOCS3 was also decreased possibly because of the attenuated pathway and/or the anti-inflammatory potential of GAL that does not demand the production of the feedback molecule as reported herein. A similar result was documented before in an in-vitro model using nicotine 37 . The present study proved that activation of α7 nAChR was the one responsible for the inhibition of the IL-6/ JAK2/ STAT3/ SOCS3, where the GAL effect was completely blocked upon using the selective antagonist MLA.
IL-6 is known to be one of the pro-inflammatory mediators transcribed by the transcription factor NF-κB (p65). This factor was revoked by the post-administration of GAL again by virtue of curtailing the p-JAK2/ p-STAT3 axis. In harmony with our findings, previous studies 34, 36 have conveyed that upon curbing the p-JAK2/ p-STAT3 signal, the unphosphorylated STAT3 (U-STAT3) is enhanced to compete with IκBα, hence, sequestering NF-κB (p65) and limiting its activation, translocation into nucleus, and its inflammatory response. Figure 6. Schematic pathway summarizing the manipulation of GAL via the activation of α7 nAChR to mediate its anti-inflammatory effect. GAL mediated its anti-inflammatory effect by inhibiting the IL-18-IL-6/ p-JAK2/ p-STAT3/ SOCS3 and NF-κB/ HMGB-1/ IL-18 trajectories. This was associated with improvement in kidney function (Cr, Cys-C, IL-18, NGAL) and histological structure. The beneficial renocurative effect of GAL was abolished by the use of MLA in ZYM-induced AKI in BALB/c mice. SOCS3 works as negative feedback pathway to inhibit IL-6/ STAT3, while here GAL inhibited SOCS3 possibly due to the ability of GAL to directly inhibit IL-6, which does not necessitate the activation of SOCS3. ACh: acetylcholine; AKI: acute kidney injury; CAP: cholinergic anti-inflammatory pathway; Cr: creatinine; Cys-C: cystatin-C; GAL: galantamine; HMGB-1: high motility group box-1; IκB: inhibitor of κB; IL: interleukin; JAK2: janus kinase 2; MLA: methyllycaconitine; α7 nAChR: α7 nicotinic acetylcholine receptor; NGAL: neutrophil gelatinase-associated lipocalin; p65 NFκB: p65 nuclear factor kappa B; STAT3: signal transducers and activators of transcription; SOCS: suppressor of cytokine signaling; Zym: zymosan.
HMGB-1 is another NF-κB (p65)-related downstream pro-inflammatory mediator 38 that increases during sepsis and generalized inflammatory conditions 20 , as was shown in the present work. Moreover, in a positive feedback loop, HMGB-1 acts as a ligand for either TLR or RAGE to enhance the NF-κB signaling pathway [39][40][41] . Therefore, GAL-induced inactivation of NF-κB is responsible, in part, for the inhibition of HMGB-1, besides IL-6 to signify its anti-inflammatory potential. This effect relies on the increased release of ACh and the activation of α7 nAChR, where MLA obliterated the GAL curative effect.
Additionally, another inflammatory cytokine that has been inhibited by GAL is IL-18; the cytokine is an important mediator of renal tubular epithelial cell injury that was boosted in the ZYM model. In traversing routes, IL-18 upon binding to its receptor deepened the ZYM-induced inflammatory/injurious effect via activating the JAK2/STAT3/SOCS3 pathway 42 , stimulating the transcription factor NF-κB 43 , and increasing the production of IL-6 among other cytokines 44,45 , to trigger several inflammatory cascades. GAL effect can be linked to the elevated level of ACh, as proven herein and previously 29 , as well as to the activation of the cholinergic receptor confirmed by the MLA-induced limitation of GAL effect. The ability of GAL to lower SOCS3 can be linked to the suppression of IL-18 as suggested by Matsui et al. 42 ; these authors revealed that SOCS3 may be increased as a result of the increased IL-18 content and/or to regulate the activated STAT-3 pathway 44 . Additionally, upon using a p-STAT3 inhibitor (S3I-201), the authors recorded decreases in IL-18 and SOCS3, as well.
Apart from producing inflammatory cytokines, NF-κB also modulates NGAL, which is a protein that is up-regulated in several injury settings [46][47][48] and is linked to numerous cellular responses 49 . NF-κB was reported to be essential for NGAL expression 50 , an effect that can explain its inhibition in the GAL treated group as a consequence to the inactivation of NF-κB.

Conclusion
Depending on the results of the current study, GAL post-treatment improved kidney function through acting on the α7 nAChR that modulates the inflammatory pathways; viz., IL-6/ JAK2/ STAT3/ SOCS3 and NF-κB (p65)/ HMGB-1/ IL-18 that collaborate to induce AKI. This conclusion was supported by the administration of the α7 nAChR blocker MLA, which abolished the reno-curative effect of GAL. The study paves the way for upcoming experimental studies and nominates GAL as a useful therapy for future studies in AKI clinically.

Data Availability
All data generated or analysed during this study are included in this published article and its supplementary information files.