Melanin: A Naturally Existing Multifunctional Material

Melanin is a common name used for a certain type of natural dark pigments existing in living organisms, particularly in human hair, eyes, and skin. The unique free radical scavenging effect of melanine could help protecting cells and tissues from harmful UV light. While their exact molecular structures in nature are not still well defined, their multifunctional properties including electrical and ionic conductivities, antioxidation, wet adhesion, and metal ion chelation, are highlighted for the potential applications in bioorganic electronics including biomedical sensors and devices. In this mini-review, we will discuss sources, synthesis methods, structures and multifunctional properties of melanin materials in addition to current research directions on a wide range of applications.

Introduction
Melanin indicates broad ranges of natural biopolymeric pigments, whose name is originated from melanos (dark in Greek) by Swedish chemist Berzelius (1840) [1]. In nature, melanin exist in five different structural forms and functional types; eumelanin, pheomelanin, allomelanin, pyomelanin, and neuromelanin [2,3]. In Figure 1, various precursors for synthesizing these melanins are shown. Melanin, particularly eumelanin, has been unveiled to hold unique multifunctional properties including broadband UV -visible light absorption [4,5], paramagnetism, electrical conductivities [6][7][8][9][10][11], biocompatibility, biodegradability [12], radical scavenging [13], and metal ion chelating [14]. While eumelanin particles, such as sepia inks from cuttlefish, are not well characterized due to their extreme insolubility, polydopamine is a major component of its eumelanin particles whose molecular functional groups include catechol, amine, and imine. Combined with eumelanin's versatile functions, these chemically crosslinking anchors provide extreme usability from simple coating materials to complex chemical, biological, and medical sciences. The fabrication of melanin materials has been recently activated by demonstrating, for examples, composites, films, hydrogels, and membranes. Thus, the unprecedentedly wide ranges of melanin applications include sensors, batteries, supercapacitors, solar cells, drug delivery, bioimaging, diagnosis, tissue engineering, and medical implants. The purpose of this review is to present the recent research trends of melanin by elucidating molecular structures functional properties, and devices applications, and to encourage the researches of this material.
Eumelanin is a black-to-brown pigment such as in cuttlefish ink and human black hair. Eumelanin is polymerized by oxidation of L-tyrosine or L-dopa via 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylicacid (DHICA) intermediates ( Figure 2). Large quantity of eumelanin can be extracted from cuttlefish ink ( Figure 3). Unlike other melanin, eumelanin shows electrical conductivities and paramagnetism. Furthermore, eumelanin has unique properties such as radical scavenging, metal ion binding and photoelectronic properties. Because of this multifunctional property, eumelanin is the key objective materials for the recent studies to make functional devices.
Pheomelanin is a yellowish-to-reddish pigment in red hair, freckles, and feathers of birds. Pheomelanin is polymerized by oxidation of 5-S-Cys-dopamine and 5-S-Cys-dopa (L-tyrosine is polymerized with L-cysteine), the main difference from eumelanin is presence of sulfur in aromatic ring structures. Allomelanin is a dark brown-to-totally black pigment in plant. Allomelanin is a polymer devoid of nitrogen. Because it is derived from catechol by catechol oxidase, allomelanin is also named as catechol-melanin. This melanin was reported as preventer of the undesirable browning of plants.
Pyomelanin is a dark pigment in fungal metabolite (Pseudomonas and Aspergillus fumigatus), derived from homogentisic acid (HGA). The molecular structure of this melanin is similar to eumelanin. Neuromelanin is a dark pigment present in human brain, mostly sited in Substantia nigra and in lower of the Locus coeruleus. This mela nin consists of pheomelanin, which formed in the core of the granule, and eumelanin, which formed on the surface [2,3].

Synthetic melanin
Synthetic melanin is generally polymerized by oxidation of L-dopa, L-dopamine, DHI, and DHICA as precursors with the catalytic reaction of tyrosinase, peroxidase, and hydrogen peroxide, or in a basic pH condition (pH > 7.5) with oxygen as the oxidant [2,3,5,8,11,14,18,19]( Figure 5). Particularly, oxidation of pure dopamine occurs to be polydopamine, eumelanin-like polymer, which has unique wet adhesion and coating properties ( Figure 4). Unlike natural melanin, they are free of proteins, which can potentially hinder functional device applications of melanin [17,20].
Recently, melanin films are demonstrated by the electrochemical deposition of DHI precursors in a tris-hydroxymethyl-aminomethane (THAM) buffer solution by constant potential method [21]. Similarly, polydopamine films are electrochemically deposited from dopamine by the cyclic voltammetry method [22] (Figure 6). Using other precursors such as 5,6-dimethoxyindole-2-carboxylic acid (DMICA), melanin film could be fabricated on a conductive substrate by constant current method [23].

Optical properties
Melanin has interesting light absorption characteristics. Even though natural melanin and synthetic melanin have different chemical struc-tures, both present a monotonous decrease of the absorption coefficient vs. λ without marked absorption bands in the UV, visible, and near IR ranges [5,24] (Figure 8). M. A. Rosei et al. proposed that this characteristic shape is originated from a disordered structure of melanin, which is composed of elementary nano-sized two-dimensional clusters packed into stacks [25]. According to this model, π-π* electron transitions occur inside nano-sized clusters by absorbing broad range energy, and that is the reason of the absorption spectrum of melanin [4].

Electrical properties
Melanin, particularly eumelanin, has electrical conductivity because they have π-π conjugated backbone structures (Figures 7, 9). In 1970s, eume-    lanin was believed to be an amorphous semiconductor, where protonic and electronic charge carriers coexist [6][7][8][9]. Amorphous semiconductor theory predicts that the conductivity will increase sub-exponentially with water content, but experimental data of melanin conductivity shows a super-exponential increment (Figure 10). Because of this deviated trend, dielectric amorphous semiconductor model is not accepted anymore. Albertus B. Mostert et al. has reported that melanin is an electronic-ionic hybrid conductor rather than an amorphous organic semiconductor. In an electronic-ionic hybrid model, conductivity of melanin originates from its self-doping by free carriers, electrons and protons. The densities of both free carriers are increased by comproportionation between melanin functional group and water [26]. Recently, electrical conductivities of natural eumelanin and synthetic melanin were reported to be in the range of 10 -3~1 0 -13 S/cm depending on its hydration states[9,10,18,27,28].

Biocompatibility
A natural polymer, melanin, is thought to be inherently biocompatible and biodegradable. Christopher J. Bettinger et al. tested in vitro biocompatibility of melanin films by using Schwann cells (SCs) and PC12 neural cells, and in vivo biocompatibility by implanting melanin films in a mouse [12]. In Figure 11, the SCs cultured on a melanin substrate grew rapidly and exhibited more activated phenotype than the cells cultured on a collagen substrate, positive control, and on an un     [32]. coated substrate, negative control. In Figure 12, biocompatibility of melanin was also proved by in vitro PC12 cell growth rate and its neurite length in the presence of nerve growth factor (NGF). In Figure 13 shows that while silicone implants were encapsulated by severe inflammative reaction, melanin implants were simply fractured by mechanical rupturing and were gradually degraded in a peripheral nerve.
This in vivo response of nerve to melanin implants implies that melanin is a promising candidate for biodegradable coatings as well as biocompatible functional materials. Dan kai et al. demonstrated electrically conductive melanin composite nanofibers by mixing melanin, gelatin, and poly (L-lactide-co-ε-caprolactone) (PLCL). They also cultured Human cardiac myocytes (HCM) on the conductive melanin nanofiber scaffolds to test regenerative cardiac functions. While mechanical modulus decreased by adding melanin, melanin containing fibers showed improved expression of cardiac-specific proteins and enhanced cell proliferation [29]. The result implies that the concentration of melanin is not just biocompatible, but also facilitates regenerative tissue functions, particularly for electrogenetic cells. Others also demonstrated mammalian cell biocompatibility of graphene -melanin composites in order to increase electrical conductivities and biodegradability of melanosome, melanin materials [30,31]. and cadmium ions were adsorbed on the synthesized melanin particles. The synthetic melanin particle shows excellent binding capacities due to the high surface volume of the particles in solution [32].

Other properties Metal ion binding
Radical scavenging Malgorzata Rozanowska et al. used a pulse radiolysis technique for detecting radical scavenging property of melanin. Their results show that melanin can act as both oxidizing radical scavenger and deducing radical scavenger [13]. This strong anti-oxidation properties suggest melanin to be anti-oxidant additives [33,34].

Sensors
Macro Araujo et al. made pH-responsive drug release devices by using Sepia officinalis ink. By changing pH, structural conformations of melanin are rearranged because DHI and DHICA monomers, having carboxylic acid (pKa ≒ 4.5) and phenolic group (pKa ≒ 9) respectively, are directly responsible for the chemical and surface physical properties [35]. Furthermore, melanin film was used as a pH senor of an extended gate field effect transistor (EGFET) by Marina Piacenti da Silva et al. [36]. The two hydroxyl groups in DHCIA, quinone imine and hydroxyl group of carboxylic acid [37], act as potential binding sites for H + ions. These bindings make a melanin film as the active layer in EGFET pH sensors with good sensitivity. Tong-Fei Wu et al. also reported the water-soluble dopamine-melanin films for the applications of ultra-sensitive and ultra-fast humidity sensors [38] (Figure 16). Maria D. Rubianes et al. used carbon nanotubes paste electrodes (CNTPE) and modified the electrodes with melanin to detect dopamine quantitatively in presence of high excess of ascorbic acid [39].

Batteries
Young Jo Kim et al. fabricated melanin electrodes with PTFE, extracted from Sepia officinalis, as sodium-ion energy storage devices utilizing the ability to form homogeneous nano-particles and the conductivities by hydration-dependent hybrid electronic-ionic carriers. Due to the low energy density compared to inorganic electronic materials, melanin anode needs to be improved by altering the chemical functionality of protomolecules and by increasing the surface areas for maximizing the specific sodium-ion loading capacity. This demonstrates the potential use of melanin anode [23]. In addition, they suggested the possible uses of eumelanin half-cell of reversible Mg 2+ ion charge-discharge, by utilizing the structural stability of melanin in water and their relatively high density of catechol groups [40] (Figure 17).

Other applications
Weifu Dong et al. reported the poly(vinyl alcohol) (PVA) / melanin composites with enhanced thermal stability above pure PVA ( Figure  18). Compared with natural melanin, synthetic melanin obtained from oxidation of dopamine exhibited higher efficiency on increasing the decomposition onset temperature by 80-110 ℃. By addition of synthetic  melanin, the low-temperature mass loss on the first step of PVA was disappeared and the decomposition onset temperature was shifted to 310-340 ℃ [41]. Recently, Ming Xiao and Tong-Fei Wu et al. fabricated the melanin films, which show the bio-inspired structural coloration by self-assembly of synthetic melanin nanoparticles ( Figure 19). Reflective colors were controlled by the change of film thickness. Green and red color were shown at 338 ± 9 and 444 ± 15 nm in thickness, respectively [42,43].

Conclusion
This mini-review provides an overview of recent findings including the synthesis and molecular structures of melanin, their multi-functionalities such as optical, electrical, and biocompatible properties. Although their uses in the real world are still in the stage of emerging embryo, the device applications of multifunctional melanin will be actively expanded such as from sensors to biomedical implants.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis

Background Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets. Methods To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate. Results We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite. Conclusions All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.

Plain English summary
The main role of RBCs in the human body is to transport O 2 from lungs to different tissues and CO 2 back to lungs. Recently studies propose that RBCs, under certain conditions, can produce NO and inhibit platelet activation. Nitric Oxide/soluble Guanylate Cyclase/cyclic guanosine monophosphate/protein kinase G (NO/sGC/ cGMP/PKG) pathway is one of the most powerful mechanisms responsible for platelet inhibition. However, the activation of platelet sGC/cGMP/PKG by NO released from RBCs was not directly proven.
Therefore, the main aim of our work was to test whether RBCs can release NO that can stimulate platelet sGC. Incubating RBCs with purified sGC and platelets under normal and hypoxic conditions, did not induce sGC activation indicating that NO is not released from RBCs under tested conditions. Rather, externally added NO onto whole blood was scavenged by RBCs, thereby preventing sGC activation in platelets. Our data strongly indicate that RBCs do not release biologically active NO that is sufficient to activate platelet sGC, which is a highly sensitive NO receptor.

Background
Red blood cells (RBCs) and platelets represent the major cell population in mammalian blood. Functional interactions between RBCs and platelets are known for a long time from clinical observations. The bleeding time is prolonged in patients with anemia independent on their platelet count and could be corrected by RBCs transfusion, and bleeding defects are directly connected with platelet function but not with the blood coagulation system [1][2][3]. On the other hand, RBCs transfusion could increase platelet activation and might be dangerous in treatment of coronary artery diseases [4]. These clinical observations clearly pointed out that RBCs, by still unidentified mechanism(s), play some important role in platelet activation. However, recently in several papers [5][6][7][8][9] platelet inhibition by RBCs-derived NO was described. In all these papers, platelet inhibition by deoxygenated RBCs, that can reduce nitrite to NO was shown. Importantly, in none of these papers direct activation of platelet soluble Guanylate Cyclase (sGC) by RBCs-derived NO was shown. In papers where the inhibition of platelet function by nitrite/RBCs was described [5][6][7][8][9], platelet activation was assessed by aggregometry and flow cytometry, which is not sufficient to confirm whether the inhibition was due to the activation of NO/cGMP pathway in platelets. Several publications described direct NO production from RBCs by endothelial NO synthase (eNOS) [10,11], but other studies did not find any functionally active NOS in RBCs [12]. The direct activation of platelet sGC by RBCs was never shown before. Hence, the connection between RBCs, eNOS and hemoglobin (Hb) to platelet sGC activation and platelet inhibition cannot be proven.
Hb and RBCs are involved in the modulation of NO signaling and metabolism. Since the discovery of NO as a signaling molecule, numerous studies proposed the formation of the nitrosyl-heme adduct of Hb (NO-Hb), a source of bioavailable NO. The nitrosylation of conserved cysteine of Hb β-chain (β93Cysteine) by NO and the formation of S-Nitrosohemoglobin (SNO-Hb) were proposed as putative mechanisms for Hb functioning as NO carrier [13,14]. These interactions between NO and Hb are superseded by the fast reaction between NO and oxyhemoglobin (oxyHb). According to mathematical modelling and experimental data, most of NO in the blood is annihilated by the fast irreversible reaction of oxyHb with NO, which yields oxidized Hb (methemoglobin, metHb) and nitrate [15]. On the other hand, deoxyhemoglobin (deoxy-Hb) may act as a nitrite reductase, promoting the synthesis of NO from inorganic nitrite [16,17].
The high affinity NO receptor, sGC mediates most of the biological functions of NO, especially at low NO concentrations [18,19]. Although different reactions between NO and Hb were established, the mechanisms of NO/SNO release from Hb are not well understood. It is less clear how NO can be released from RBCs under physiologic conditions of overwhelming excess of free Hb. It remains to be proved that RBCs-derived NO can activate platelet sGC.
Endogenous NO is a well-known and potent inhibitor of platelet activation (reviewed in [20,21]). NOdependent inhibition of platelets is solely mediated by sGC [22]. Intact platelet sGC is highly sensitive to endogenous NO and even nanomolar NO concentrations are sufficient for cGMP generation [23]. In platelets, Protein Kinase G (PKG), which phosphorylates multiple substrates responsible for platelet inhibition, is the major downstream target of cGMP [21]. For evaluation of platelet sGC activity, we developed a highly sensitive method, based on NO/sGC/cGMP/PKG dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in human platelets, which has been used in numerous studies [20]. Most studies that report NO release from RBCs had been performed using blood vessel relaxation experiments or with isolated washed RBCs. However, both approaches were not sufficient to answer the question whether RBCs can produce biologically active NO/SNO. Vasorelaxation is a complex process controlled not only by NO, but also by a number of other vasodilators. Moreover, the experiments with isolated RBCs never demonstrated that NO or other reactive nitrogen species generated by RBCs result in activation of sGC.
In this report, we directly addressed this question by determining the sGC-dependent signaling in intact platelets and the activation of purified sGC. Our data clearly showed that RBCs, under the tested conditions, cannot activate platelet sGC or purified sGC, but in contrast, they act as a potent NO scavengers.

Preparation of human platelets, white blood cells (WBCs) and RBCs
Human platelets were prepared as reported previously [26]. Blood was collected from healthy volunteers, and WBCs were isolated by Leucosep tubes (Greiner, Frickenhausen, Germany) according to the manufacturer's instructions. WBC concentration was adjusted to 6×10 6 /ml. RBCs were collected after separation of PRP and washed thrice with HEPES buffer; the supernatant from the last wash was used as control to confirm the absence of free Hb. For experiments with RBCs, suspensions which did not contain free hemoglobin were used.

Platelet aggregation
Aggregation in whole blood was measured by Multiplate (Roche Diagnostics, Mannheim, Germany), in PRP and washed platelets by Apact4004 (LabiTec, Ahrensburg, Germany) aggregometers. Citrated whole blood was preincubated with sodium nitroprusside (SNP) and aggregation was initiated by ADP (10 μM). PRP and washed platelets were preincubated for 1 min with SNP or iloprost and aggregation was initiated by ADP.

FACS analysis
FACS analysis was performed using a Becton Dickinson FACS Calibur with CELL Quest software, version 3.1f (Becton Dickinson, Heidelberg, Germany). Platelets in the whole blood were identified by CD41 staining. Washed platelets concentration was adjusted to 3×10 8 /ml and for washed platelets/RBC mixture 4.5×10 9 /ml washed RBCs were added to platelet suspension. All samples after stimulation were directly fixed with 3 % formaldehyde. The samples were incubated with PAC-1-FITC, or CD41-FITC antibodies and analysed by FACS. For phospho-VASP, the samples were permeabilized with 0.2 % Triton X-100.

Western blot analysis
Samples were lysed with Laemmli buffer and the proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane as described [27]. Membranes were incubated with primary antibodies overnight at 4°C. Goat anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase were used as secondary antibodies followed by ECL detection. Blots were analysed densitometrically using NIH Image J software for uncalibrated optical density.

Experiments with deoxygenated washed platelets and RBCs
HEPES buffer was degassed under vacuum and bubbled with argon. This buffer was used for washing and resuspension of washed platelets and RBCs. RBCs and platelets were further bubbled with argon and kept under argon in closed gas-tight tubes. Then, RBCs were washed 3 times with argonsaturated HEPES to wash out free hemoglobin from lysed RBCs. In some experiments deoxy-RBCs were incubated with DEA-NO and/or with NaNO 2 prior addition to platelet suspension. For experiments with purified sGC, preparations of deoxy-RBC were also transferred into an anaerobic glove chamber (Model 110 V, Coy Laboratory Products, Inc.) and equilibrated for additional 2 h in anaerobic environment.

Spectral analysis of Hb
To confirm the Hb status of RBCs, UV spectra were taken for oxy-Hb, and deoxy-Hb after incubation with NO donor. 10 μl of RBC were lysed in hypoosmotic media (HEPES buffer diluted by water to 50 mOsm), and the visible absorption spectra in the 450-700 nm range were immediately collected on a UV-vis Spectrophotometer (SPECS SSP-715, Spectroscopy Systems, Russia).

sGC activity assay
Full-length human sGC was expressed in insect Sf9 cells as described previously [28]. Recombinant sGC was purified using anion exchange and Ni-affinity chromatography as detailed previously [29]. Anaerobic sGC was prepared in an anaerobic train as described previously, [19] and transferred into the anaerobic glove chamber. Enzymatic sGC activity was assayed using a modified [α-32 P]GTP to [ 32 P]cGMP conversion assay [30] with buffers prepared anaerobically and equilibrated in the anaerobic chamber overnight. To measure NO-induced activity, 0.01 μg sGC in 10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM DTT, 3 mM MgCl 2 , was incubated with indicated RBCs or buffer prior to exposure to the mixture containing NO donor (DEA-NO) and GTP. To determine if RBCs generate any bioavailable NO from nitrite, anaerobic RBCs were incubated at 37°C for 15 s or 5 min with indicated amount of nitrite, prior to the addition to 0.1 μg sGC. The cGMP-forming reaction was initiated by 1 mM GTP/[α-32 P]GTP (~100,000 cpm). The reaction was stopped by zinc carbonate precipitation and processed following the protocol established previously [30].

Data analysis
All experiments were performed at least thrice, and data are expressed as means ± SEM. Differences between groups were analyzed by ANOVA followed by Bonferroni's test and Student's t-test as appropriate. p < 0.05 was considered statistically significant.

RBCs prevent NO donor-mediated platelet response
Inhibition of platelet aggregation by RBCs-generated NO was reported in several publications [5,7,11,31]. To test whether RBCs influence platelet aggregation, we measured ADP-triggered aggregation in whole blood, PRP, and washed platelets. In whole blood, very high (50 and 100 μM) concentrations of Sodium nitroprusside (SNP) were required for partial inhibition of platelet aggregation (25 ± 4 and 32 ± 6 %, respectively). On the contrary, in PRP and washed platelets, 0.5 μM SNP was sufficient to achieve similar degrees of inhibition. Activation of cAMP/Protein Kinase A (PKA) signaling by iloprost (5 nM) completely inhibited platelet aggregation in all the three preparations ( Fig. 1a). Corroborating the aggregation studies, NO donor (DEA-NO) significantly inhibited TRAP-6 induced integrin (αIIbβ3) activation in PRP and washed platelets, but not in whole blood or in washed platelets coincubated with washed RBCs. Iloprost, used as a positive control in these experiments, completely prevented integrin αIIbβ3 activation (Fig. 1b). To prove that the platelet inhibitory response was mediated by PKG and PKA activation, we measured VASP phosphorylation on the same sets of experiments. DEA-NO only slightly (1.7 ± 0.3 fold) increased VASP phosphorylation in the whole blood samples, whereas in PRP and washed platelets the increase of VASP phosphorylation was more than 25-fold and comparable with the iloprost-induced response. Coincubation of washed platelets with washed RBCs completely prevented DEA-NO-induced VASP phosphorylation (Fig. 2a). It was recently [31] reported that in RBCs, activation of eNOS by L-arginine has an inhibitory effect on platelet aggregation. However, this study did not determine whether the inhibition is due to the activation of sGC/cGMP/ PKG pathway in platelets. To address this issue, we incubated the whole blood with L-arginine or NOS inhibitor (L-NAME) and measured platelet VASP phosphorylation by FACS analysis. Neither inhibition, nor stimulation of NOS in whole blood affected VASP phosphorylation (Fig. 2b).

Presence of RBCs prevents NO-induced PKG activation in platelets
In the next series of experiments, we examined whether longer incubation of RBCs with platelets activates platelet PKG. We also assessed the RBC concentration necessary to inhibit NO-dependent activation of PKG. Since VASP phosphorylation by PKG is the primary parameter evaluated in this set of studies, we first tested by Western blotting whether the key elements of the NO/cGMP pathway downstream of eNOS are detectable in RBCs. The lysate from 10 8 /ml platelets exhibited a strong signal for sGC, PKG and VASP. In contrast, these proteins were not detected in RBCs (10 9 /ml) (Fig. 3a).
Next, we observed that incubation of RBCs with platelets in suspension for up to 30 min does not increase the phosphorylation of VASP. Corroborating aggregation experiments, the strong phosphorylation of VASP induced by DEA-NO was completely abolished by RBC (Fig. 3b). The titration of RBCs demonstrated that even 10 8 RBCs/ml, which is~50-fold lower than the physiological RBC count, were sufficient to prevent NOinduced phosphorylation of VASP in platelets (Fig. 3c).  Fig. 1 NO-dependent platelet inhibition is blunted in the presence of RBCs. a ADP-induced platelet aggregation measured in the whole blood, platelet rich plasma (PRP) and washed platelets (WP). Aggregation in whole blood was measured by Multiplate, in PRP and WP by Apact 4004 aggregometer. For whole blood, 300 μl of citrated whole blood and 300 μl saline were added to the test cell together with indicated concentrations of SNP. After three minutes of incubation at 37°C, aggregation was initiated by ADP (10 μM). Aggregation curves were recorded for 6 min. PRP and WP samples were preincubated for 1 min with indicated concentrations of SNP or iloprost and aggregation was initiated by 10 μm of ADP and monitored for 3 min. Data of platelet aggregation are presented as area under the curve (AUC). b FACS analysis of integrin αIIbβ3 activation (Pac-1 binding) measured in the whole blood, PRP, washed platelets (WP), and WP with washed RBCs (10 9 /ml). Samples were prepared as described under "Materials and Methods". Data are presented as mean ± SEM, n = 4; + p < 0.05, compared to control, *p < 0.05 compared to Whole blood sample preincubated with DEA-NO affected PKG activation in platelets (Fig. 3d), which fits in line with the results from whole blood (Fig. 2b).

Neither inhibition, nor stimulation of NOS in RBCs
sGC is a very sensitive receptor for NO, which evolved to respond to nanomolar concentrations of NO [32]. While oxyhemoglobin is a very efficient NO scavenger, the affinity of oxygen-free hemoglobin for NO is comparable to sGC. It was previously shown that deoxygenated RBCs can produce S-nitroso-L-cystein (L-SCNO) and S-nitrosoglutathione (GSNO) [33]. Therefore, we tested whether deoxygenated RBCs affect NO-dependent activation of purified sGC and platelet sGC/PKG pathway. Exogenously added L-CSNO and GSNO are strong activators of purified and platelet sGC (data not shown). However, presence of deoxygenated RBCs had no effect on purified sGC activity and only inhibited DEA-NOinduced sGC activation (Fig. 3e). Similar results were observed, if RBCs were incubated with DEA-NO prior to the addition to sGC (data not shown).
Because of the high NO scavenging properties of free Hb [34], special care was taken to assure that the RBC suspensions did not contain free Hb as a result of haemolysis. In all our experiments, we used supernatant from RBCs as a control for the absence of free Hb. In contrast to the addition of RBCs, supernatant from RBCs added to the platelet suspension had no effect on DEA-NOinduced VASP phosphorylation. Also, RBCs and supernatant of RBCs had no influence on PKA-induced VASP phosphorylation stimulated by iloprost (Fig. 4a). Platelet cGMP levels are affected not only by the elevation of cGMP through sGC activation, but also by the inhibition of cGMP-degrading phosphodiesterases. Phosphodiesterase 5 (PDE5) is the main PDE responsible for cGMP degradation in platelets [21]. Therefore, we tested whether RBCs may cause inhibition of PDE5 in platelets. The PDE5 inhibitor sildenafil induced similar VASP phosphorylation in platelet suspension with and without added RBCs (Fig. 4b) indicating that platelet PDE5 was not affected by RBCs.

Neither deoxygenation, nor addition of nitrate promote RBC-induced sGC activation
The nitrate-nitrite-NO reductive pathway is an alternative source for NO, which plays a significant role in bacteria and plants [35]. In mammals, the oxygen-independent nitrite reductive pathway was implicated in hypoxic vasodilation [36][37][38] and control of blood pressure. Some studies showed that RBCs promote nitrite/NO-dependent vasodilation and inhibition of platelet aggregation under hypoxic conditions [7,39,40]. We recently showed that incubation of platelets with carbonic anhydrase purified from bovine RBCs in the presence of nitrite increases platelet cGMP content and induces VASP phosphorylation [41]. Therefore, it was important to test whether intact RBCs containing Hb in different status (oxyHb, deoxyHb, NO-Hb), with or without nitrite, and normal or deoxygenated platelets, can activate platelet sGC. Incubation of platelets with different concentrations of nitrite had no effect on PKG-dependent phosphorylation of VASP (Fig. 5a). These results on human platelets are consistent with a report on mouse platelets that nitrite triggers VASP phosphorylation only in the presence of PDE5 inhibitor [42]. Next, we co-incubated deoxygenated RBCs loaded with NO (deoxy RBC + DEA-NO) with deoxygenated platelets in the presence of increasing nitrite concentrations. Even under these conditions, we found no activation of platelet PKG (Fig. 5b). We tested all possible combinations of RBCs containing Hb in different status (oxyHb, deoxyHb, NO-Hb), with or without nitrite, and normal a FACS analysis of VASP phosphorylation at S 239 measured in the whole blood, PRP, washed platelets, and WP with washed RBCs (10 9 /ml). b VASP phosphorylation at S 239 measured in the whole blood in the presence of L-arginine or NOS inhibitor L-NAME (both 200 μM, 10 min preincubation). NO donor (DEA-NO, 5 μM, 2 min) was used as positive control. Samples were prepared as described under "Materials and Methods". Data are presented as mean ± SEM, n = 4; + p < 0.05, compared to control or deoxygenated platelets, and found no evidence of activated platelet PKG (Fig. 5a, b, and other data not shown). To confirm that intact RBCs in the presence of nitrite do not produce NO, we incubated deoxygenated RBCs with nitrite and measured the activity of purified sGC. Anaerobic RBCs preincubated with 100 μM nitrite did not elevate purified sGC activity (Fig. 5c), which is consistent with strong NO-scavenging effect of RBCs. The same results were observed when the experiments were performed under aerobic conditions or with  , 30 min). c Indicated amount of RBCs (10 μl) were added to platelet suspension for 10 min, and then stimulated with DEA-NO (1 μM, 1 min). When indicated, the same volume (10 μl) of supernatant from the last wash of RBCs was added to the platelets. For the bar graphs, immunoblots were scanned and quantified by the Image J program. The intensity of the VASP Ser239 signal was normalized to the total VASP signal, which was designated as 1 in control samples. Results are means ± SEM, n = 4, + significant difference from control samples. d Platelets (3x10 8 /ml) were coincubated with RBC (5x10 8 /ml, 10 μl) and treated with L-Arginine, L-NAME, or L-NMA for 10 min. When indicated, samples were treated with DEA-NO (10 μM, 1 min). Total VASP blots served as loading control. Shown are representative blots of three independent experiments. e Basal and NO-induced (1 μM DEA-NO) purified sGC activity measured in anaerobic conditions after incubation with buffer or indicated amounts of RBC. Data are presented as mean ± SEM, n = 4 10 μM nitrite (data not shown). To confirm the Hb status of RBCs, UV spectra were taken from each sample (Fig. 5d). Each spectrum corresponds to wellcharacterised Hb pattern of oxyHb (RBC), deoxyHb (RBC + Argon), NO-Hb (RBC + Argon + DEA-NO) and visible amount of metHb after 30 min incubation with DEA-NO [43].
Endothelial cell-derived NO is the main source of platelet sGC activation In blood vessels, endothelial cells or circulating lymphocytes express NOS isoforms and may potentially be the source of NO for activation of platelet sGC/cGMP/PKG pathway. To evaluate the contribution of endothelial cells and lymphocytes, we co-incubated washed platelets with washed WBCs, or human umbilical vein endothelial cells (HUVECs) in the presence of L-arginine or L-NAME. Incubation of platelets with WBCs in the presence of L-arginine did not activate platelet PKG, however, NOS activity of WBCs was slightly increased by Larginine and inhibited by L-NAME. It is likely that the amount of NO produced by WBCs in our experimental setting was not sufficient for activation of platelet sGC. In contrast, NO producted by HUVECs, which can be inhibited by L-NAME, strongly stimulates platelet sGC (Fig. 6) suggesting that endothelial cell-derived NO is the main source of platelet sGC activation. Our in vitro data are in good agreement with in vivo mouse models, where it was shown that eNOS of endothelial cells exerts a direct antithrombotic effects on platelets [44].

Discussion
The key role of RBCs in hypoxic vasodilation is well known. RBCs trigger sGC activation in the smooth muscle cells of the vessel wall that leads to vessel dilation. Few publications report that RBCs release NO during hypoxia, thus activating sGC. Several mechanisms were proposed to argue the NO carrying properties of RBCs/Hb. In one mechanism, Hb nitrosyl-heme adduct (NO-Hb) is regarded as a form of NO storage and carrier. NO released from NO-Hb under hypoxic conditions results in vasodilation [36]. According to another proposed mechanism, S-nitrosylated Cys93 of the Hb βchain is an equally important source of NO/SNO [45]. However, later studies on mice with mutated β93Cys demonstrated that Cys93-Hb is not important for RBCinduced vasodilation [13,14]. Finally, NO reduced from nitrite by deoxyhemoglobin was considered as an explanation for hypoxic vasodilation [39,40,46]. However, this hypothesis was strongly criticized by others [15]. Strong NO scavenging properties of oxyhemoglobin in hypoxic condition is considered to be an important requirement for effective nitrite/RBCs-dependent NO generation or release. However, the link between RBCs and sGC activation is clearly missing because direct activation of sGC by NO or SNO released from RBCs is not experimentally shown. Nevertheless, until now, NO released from RBCs is considered as one of the established a Supernatant from RBC (10 μl) washed by HEPES buffer, or RBCs (1x10 8 /ml, 10 μl) were added to platelets for 10 min, then stimulated with DEA-NO, or iloprost. b RBCs (10 8 /ml, 10 μl), or supernatant from RBC (10 μl) were added to platelets for 10 min, then stimulated with DEA-NO, or with sildenafil. Total VASP blots served as loading control. Shown are representative blots of 4 independent experiments. For the bar graphs, immunoblots were scanned and quantified by the Image J program. The intensity of the VASP Ser239 signal was normalized to the total VASP signal, which was designated as 1 in control samples. Results are means ± SEM, n = 4, + significant difference from control samples mechanisms for RBC-induced hypoxic vasodilation and platelet inhibition. As RBCs-induced vasodilation might be controlled by other NO-independent mechanisms, we focused our study on the effects of RBC on platelet and purified sGC.
The main aim of our study was to establish whether RBCs could promote sGC activation by releasing NO/ SNO under any possible conditions (in whole blood, under hypoxia, or even loaded with NO). To answer the question whether RBCs can activate sGC, we used our well-established model of NO-dependent sGC/cGMP/ PKG activation in platelets and NO-mediated activation of purified sGC. Both these systems are highly sensitive to endogenous NO or SNO (GSNO, CysNO) and can be  [47]. This high concentration of NO receptor and downstream signaling molecules makes platelets a very sensitive model for detection of NO-dependent sGC activation. PKA and PKG phosphorylate VASP at serines 157 and 239, respectively. In platelets, PKA and PKG can equally phosphorylate both sites. We argue that if RBCs-released NO/SNO is sufficient to induce vasodilation, platelet sGC must be activated in the RBCs/platelet suspension. Only one paper [7] showed more than 3 times increase of platelet cGMP concentration after 5 min incubation with 0.1 μM nitrate, and addition of RBCs further increases cGMP more than 5 times. In this paper, cGMP was measured by the enzyme-linked immunoassay from Cayman Chemicals. It is important to mention that such a high cGMP increase (comparable with NO donors effect) should undoubtedly activate platelet PKG, which was not shown in this manuscript. We specifically addressed this question and used several cGMP determination assays from different suppliers, however we were not able to reproduce their results. Apparently, nitrite itself, and especially RBCs, interfere with cGMP assays and, in some cases, might give erroneous increase of platelet cGMP. After summarizing results of eight experiments from different assays, we did not detect any statistically significant cGMP accrual in platelets incubated with nitrite and RBCs. It should be mentioned that direct measurement of cGMP levels in platelets, is complex and prone to artefacts, therefore we did not include these data. This question has been described in details in our recent review [48]. VASP phosphorylation at Ser239 mediated by PKG, which is even more sensitive than determination of cGMP itself [49] is now accepted as one of the most commonly used standards for evaluation of sGC/cGMP/PKG system activation, especially in platelets. Therefore, in all our experiments we used VASP Ser239 phosphorylation as a readout of PKG activation mediated by cGMP increase. We also tested whether RBCs activate purified sGC, which is sensitive to low NO/SNO concentrations [29]. The experiments presented in the current report clearly demonstrated that RBCs did not directly stimulate purified sGC, under normoxic or hypoxic conditions or in the presence of nitrite. Moreover, RBCs at physiological concentration, completely inhibited sGC activation even by micromolar concentrations of NO donor (Fig. 3b-e). Washed RBCs do not express detectable amount of sGC, PKG, and VASP (Fig. 3a). Therefore, in the experiments with RBCs/platelet suspension, the phospho-VASP signal reflects the activation of sGC in platelets. These experiments also show lack of platelet sGC activation in the presence of RBCs and nitrite. Endothelial cell-derived NO is the only source of platelet PKG activation. Western blot analysis of VASP S239 phosphorylation in platelets (3x10 8 /ml). Washed platelets were coincubated with WBCs (7x10 6 /ml) for 10 min, or added to the confluent HUVECs cultured in 24 well plate for 10 min then L-arginine or L-NAME (200 μM each, 10 min) were added to the samples. After 10 min, incubation, WBCs were separated from platelets by centrifugation and suspensions of non-adhered platelets were collected from HUVECs for Western blot analysis. As a control washed platelets and WBCs (7x10 6 /ml) alone were stimulated with DEA-NO (1 μM, 1 min). In addition, WBCs were incubated with the same concentration/time with L-Arginine and L-NAME. Shown are representative blots of four independent experiments. Total VASP served as loading control. For the bar graphs, immunoblots were scanned and quantified by the Image J program. The intensity of the VASP Ser239 signal was normalized to the total VASP signal, which was designated as 1 in control samples. Results are means ± SEM, n = 4, + significant difference from control samples We should emphasize that thorough washing of RBCs is required because of the strong NO-scavenging by free Hb released from lysed RBCs. In about 1/3 of our experiments (these data were excluded) the supernatant of RBCs, which was always used as a control for free Hb contamination, inhibited NO-mediated VASP phosphorylation. It should be noted that in all papers describing inhibition of platelet activation by RBCs [5][6][7]50], the results were assessed by aggregometry. In such experimental setting, RBCs-induced effects other than NO/ cGMP-mediated platelet inhibition cannot be ruled out.
The experiments performed with whole blood (Fig. 2b) or with RBC/platelet suspension (Fig. 3d) demonstrated that stimulation/inhibition of NOS had no effect on platelet sGC activation. Our studies demonstrated that L-arginine-stimulated NO synthesis in WBCs is sufficient for sGC activation in WBCs, but not of platelet sGC (Fig. 6). However, these data do not exclude that under other experimental conditions WBC-produced NO could activate platelet sGC as well.
RBCs and platelets are the major cell populations in blood, however until now little is known about interactions between these two cell types. RBCs are involved in thrombus formation, but it is not clear which functional role they play in the development of thrombosis [51]. There are no indications in the literature concerning direct platelet/RBCs interactions. In vivo, circulating platelets are continuously exposed to activating and inhibiting factors. RBCs are one of the sources of platelet activating factors like ATP, ADP and Hb released from injured RBCs, which scavenges NO. Hemolysis of RBCs which leads to increase of free Hb, ADP and ATP in plasma is the main reason of platelet hyperreactivity in sickle cell disease patients [52,53]. On the other hand, endothelial cells derived NO and prostacyclin are the major platelet inhibitory factors. A well-regulated equilibrium between these two opposing processes is important for the normal hemostasis and an impairment of this equilibrium will promote thrombotic or bleeding disorders. Our experiments support the clinical data [1][2][3] that RBCs play only stimulatory role in platelets and they are not involved in platelet inhibitory pathways.

Conclusions
All our data demonstrated that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of platelet. In all tested conditions, RBCs were not able to activate platelet or purified sGC.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine Chemistry

text:
Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways.

Introduction
Hydrogen sulfide (H 2 S) is an endogenous gaseous physiological molecule, produced in mammalian tissues from L-cysteine mainly by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) along with cysteine aminotransferase (CAT) [1][2][3]. Recent study by K. Jung et. al. has shown that in ureteral obstruction (UO)-induced kidney fibrosis, the levels of CBS and CSE, and the H 2 S concentration are decreased in kidney, whereas sodium hydrosulfide (NaHS, a H 2 S producer) diminished the suppressing effect of UO on CBS, CSE and H 2 S. In the meantime, treatment with NaHS also reduced the activation of the transforming growth factor-β1 (TGF-β1) signaling caused by UO, suggesting an inverse relationship between the H 2 S level and the in kidney fibrosis [4], opening the possibility for H 2 S as a potential therapeutical target for kidney fibrosis, and our study aimed to investigate the mechanism of antifibrotic effect of H 2 S.
The role of TGF-β signaling in kidney fibrosis is attributed to its ability to induce epithelialto-mesenchymal transition (EMT) [5]. EMT is a kind of phenotypical change in epithelial cells at which circumstances they lose cell-cell basement membrane contacts and structural polarity [6]. As a result, the epithelial cells become spindle-shaped and morphologically similar to mesenchymal/myofibroblast cells [6]. The abnormal induction of EMT in kidney has been shown to contribute to tubulointerstitial fibrosis, the final common path to renal fibrosis [7].
Apart from Smads pathway, there exists evidence supporting that TGF-β1 plays its role through ERK pathway. In several types of cells, such as pancreatic cancer cells and mammary epithelial cells, ERK is activated in TGF-β1-induced EMT and Erbin, a member of LAP family, inhibits TGF-β1-induced EMT by suppressing ERK activation [8]. β-catenin/TCF/LEF of the Wnt signaling pathway has also been shown to mediate EMT [9]. Wnt-independent β-catenin transactivation was observed due to loss of E-cadherin and consequent release of free β-catenin, mimicking Wnt signaling [10]. There is an apparent redundancy of the transcription factors involved in TGF-β1-mediated EMT, suggesting that none of these factors orchestrates EMT on its own. Rather, they may act synergistically when combined [11]. Furthermore, the observations that β-catenin nuclear translocation being Smads-dependent and that LEF/TCF signaling together with the reduction of TGF-β1-induced α-SMA expression in β-catenin null cells, strongly suggested an interaction between TGF-β1 and Wnt/β-catenin signaling [12,13].
Given the effects of NaHS in the UO-induced kidney fibrosis, we hypothesized that NaHS may exerts its anti-fibrotic effect by influencing EMT process through both Smad-dependent and Smad-independent pathways like MAPK pathway and Wnt/catenin pathway, of which the latter is our interest and focus. In this study, we investigated the effect of NaHS on TGF-β1-induced EMT in human proximal tubular epithelial cells (HK-2 cells) and the underlying mechanisms related to ERK and Wnt/catenin pathways.

Materials and Methods Reagents
NaHS (Sodium hydrosulfide) was purchased from Sigma (USA). To prepare stock solution, NaHS is dissolved in PBS (135 mM NaCl, 2.7 mM KCL, 1.5 mM KH 2 PO4, 8 mM Na 2 HPO4) to the concentration of 1M. Recombinant Human transforming growth factor beta 1 (TGF-β1) is purchased from R&D Systems (USA). The inhibitor U0126 is purchased from Cell Signaling Technology and reconstituted with DMSO to 10 mM stock solution according to the product instruction. The inhibitor XAV939 is purchased from Sigma and reconstituted with DMSO to 10 mM stock solution according to the product instruction.
The primary antibodies to phospho-ERK1/2, ERK1/2, phospho-Catenin and Non-phospho (Active) β-Catenin were purchased from Cell Signaling Technology (USA). The primary antibodies to α-SMA and E-cadherin were purchased from Epitomics (USA). The primary antibodies to TGF-β receptor I, CBS and GAPDH were purchased from Santa Cruz Biotechnology (CA, USA). Antibody to Fibronectin was purchased from Sigma (USA). The secondary AlexaFluor488-conjugated anti-rabbit antibody for Immunofluorescent staining was purchased from Invitrogen. BCA Protein Assay Kit was purchased from Shanghai Biocolor BioScience & Technology Company (Shanghai, China). β-catenin siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Cell Lines and Cell Culture
Human proximal tubular epithelial cells (HK-2) were purchased from American Type Culture Collection (Rockville, MD, USA). HK-2 cells were cultured in RPMI 1640 containing 2000 mg/ L NaHCO 3 , 10% FBS (GIBCO BRL, Co. Ltd, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a 5% CO 2 incubator at 37°C. When reached 90% confluence, the cells were trypsinized and subcultured by a 1:3 split ratio in new culture flasks. For nuclear translocation experiment, cells were seeded at proper density on cell slides in 24-well plates.

Western blot analysis
HK-2 cells were harvested, rinsed twice with ice-cold PBS and re-suspended in RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM PSFM, 1 mM EDTA, 1% Triton X-100, 0.1% SDS). The liquid supernatant was collected after centrifugation at 10,000×g at 4°C for 15 min and the protein concentration was detected by BCA Protein Assay. After the addition of 5× Loading buffer, protein was boiled at 95°C for 10 min, which would be loaded and separated by equal amounts in 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween (TBST) at room temperature for 2 hours and then incubated with primary antibodies at 4°C overnight followed by incubated with horseradish peroxidase (HRP)conjugated secondary antibody at room temperature for 2 hours. The enhanced chemiluminescent (ECL) substrate was used to visualize the protein blots and β-actin or GAPDH used as loading control.

Immunofluorescent staining
HK-2 cells cultured on cell slides were fixed in 4% formaldehyde for 30 minutes, permeabilized in 0.1%Triton X-100 for 10 minutes and blocked in 5% bovine serum albumin (BSA) in PBS buffer for 1 hour at room temperature. The primary antibody for non-phosphorylated β-catenin (1:500, Cell Signaling Technology, USA) was incubated overnight at 4°C. The secondary AlexaFluor488-conjugated anti-rabbit antibody (Invitrogen, Carlsbad, CA) was used at a 1:150 dilution for 1 hour at RT. All antibodies were diluted with PBST containing 5% bovine serum albumin. Images were obtained using a Leica TCS SP5 confocal system confocal microscopy (Leica, Wetzlar, Germany)

RNA interference
HK-2 cells were transfected with β-catenin siRNA or negative control siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction.

Cell migration
Comparative migration assay were conducted using a conventional 24-well Transwell plates of 6.5-mm diameter with polycarbonate membrane filters containing 8-μm pores (6.5 mm Transwell (#3422), Corning, NY, USA). Pretreatment of 100 uM NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for 36 hours. Then, HK-2 cells were detached with trypsin-EDTA and resuspended in media without FBS to obtain cell suspension of 2×10 5 / ml. 500 μL growth medium (1640 with 20% FBS) was added to the lower wells of the chambers. A volume of 100 μL containing 2×10 4 cells was seeded to the upper section of the chamber, and then incubated for 24 h in a 5% CO 2 incubator at 37°C. The non-migrated cells were removed from the upper side of the membranes by cotton swabs. Cells that had gone through the membrane were fixed in 4% formaldehyde for 20 minutes and stained with 0.1% crystal violet solution. Three randomly microscope fields in each slide were chosen to calculate the invaded cells.

Statistical analysis
All data were shown as mean ±SEM. Statistical differences were obtained by one-way ANOVA followed by Tukey's multiple comparison test. Differences with values of p<0.05 were considered significant.

NaHS attenuated TGF-β1-stimulated EMT in HK-2 cells
As shown in Fig 1, the HK-2 cell morphology has changed from normal to spindle-shape with TGF-β1 treatment compared to that of the untreated cells, and this change was attenuated by pretreatment of NaHS (Fig 1A). These changes are accompanied by the increase of the expression of α-SMA and fibronectin, and the decrease of the expression of E-cadherin in the TGF-β1 treated cells (Fig 1B & 1C), whereas NaHS pretreatment could down-regulate both α-SMA and fibronectin and could up-regulate E-cadherin (Fig 1B & 1C).

NaHS increased the expression of Cystathionine β-synthase (CBS)
Cystathionine γ-lyase (CSE) and CBS generate H 2 S [2]. We studied whether these enzymes are the mediators of the effect of NaHS in EMT. As shown in Fig 2, the expression of CBS is decreased significantly in TGF-β1 treated HK-2 cells. NaHS up-regulated CBS. However, we failed to detect the expression of CSE.
3. NaHS decreased the TGF-β1-induced overexpression of TGF-β receptor type II (TβR II) and TGF-β receptor type I (TβR I) TGF-β signaling cascade starts with TGF-β1 binding to TGF-β receptor type II (TβR II) and then the complex phosphorylates TGF-β receptor type I (TβR I) [5]. We studied effect of NaHS on TβR II and TβR I. As shown in Fig 3, NaHS lowered both the TβR II and TβR I expression levels in renal tubular epithelial cells.

NaHS decreased TGF-β1-induced overexpression of phosphorylated ERK1/2
TGF-β can also activate ERK1/2 [14], thus we studied whether NaHS affect the activation level of ERK1/2. As shown in Fig 4, the ERK1/2 was activated by TGF-β1 and the phosphorylation level reach peak at 30 minutes ( Fig 4A). HK-2 cells were pretreated by NaHS for 12 hours and then treated by TGF-β1 for half an hour. This significantly attenuated the ERK activation ( Fig  4B & 4C). Results suggested that NaHS may inhibit TGFβ1-induced EMT via ERK-dependent signal pathway.

NaHS decreased TGF-β1-induced overexpression of nonphosphorylated β-catenin
Under normal conditions, β-catenin is phosphorylated at its GSK3β sites and degraded. Upon stimulus, this GSK3β mediated phosphorylation is inhibited and non-phosphorylated β-catenin can translocate to the nucleus. Both total β-catenin and non-phosphorylated β-catenin expression levels were increased in HK-2 cells after TGF-β1 treatment (Fig 5). Pretreatment with NaHS for 12 hours decreased both non-phosphorylated β-catenin and β-catenin expression levels. NaHS at the concentration of 100 μM showed the best suppressing effect towards non-phosphorylated β-catenin (Fig 5).

NaHS inhibited nuclear translocation of non-phosphorylated β-catenin
β-catenin is a nuclear transcription factor and a key element in Wnt/catenin pathway. The nuclear translocation of β-catenin is crucial for mediating EMT [15]. As shown in Fig 6, TGF-β1 promoted the nuclear translocation of non-phosphorylated β-catenin and pretreatment with NaHS diminish this effect, indicating that NaHS may suppresse EMT by inhibiting the nuclear translocation of non-phosphorylated β-catenin.
7. The anti-EMT effect of NaHS on HK-2 cells was blocked by the inhibitors of ERK1/2 and β-catenin As shown above, NaHS affected the activation of ERK1/2 and β-catenin (Figs 4, 5 and 6). We further explored the importance of ERK1/2 and β-catenin in the anti-EMT effect of NaHS, by blocking them with their respective inhibitors. We excluded the influence of U0126 and XAV939 themselves on the normal growth and establishment of EMT model in HK-2 cells (Fig 7C). When blocking ERK1/2 using U0126, the pharmacological inhibitor of ERK1/2, the expression level of non-phosphorylated β-catenin was decreased even in the presence of TGF-β1 (Fig 7A). This resulted loss of β-catenin activation, suggests crosstalk between ERK1/2 and β-catenin. We then blocked both ERK1/2 and β-catenin signaling pathways using U0126 and XAV939, the pharmacological inhibitor of β-catenin, and then detected whether NaHS could still attenuate EMT. As shown in Fig 7, in the presence of U0126 or XAV939, NaHS (100μM) failed to decrease the expression of fibronectin, α-SMA and TβR I or increase the expression of E-cadherin in HK-2 cells compared to the group treated with NaHS (100μM) without the inhibitors (Fig 7B & 7D).

NaHS attenuates the TGF-β1-induced EMT through Wnt/catenin pathway
In addition, we knocked down the expression of β-catenin at protein level by transfecting βcatenin siRNA into HK-2 cells. Negative siRNA was used as control. Western blotting confirmed the β-catenin knock down (Fig 8A). With β-catenin significantly being knocking down, NaHS (100μM) failed to decrease the expression of fibronectin, α-SMA and TβR I or increase the expression of E-cadherin, compared to negative siRNA group (Fig 8B & 8C). This result indicates that Wnt/catenin pathway may be the mechanism by which NaHS attenuates TGF-β1 induced EMT.

NaHS inhibits the migration of HK-2 cells induced by TGF-β1
We further evaluated the effects of NaHS on TGF-β1-induced mobility increase of HK-2 cells using transwell migration assay. As shown in Fig 9, TGF-β1 increased the mobility and invasive capacity of HK-2 cells. Treatment with NaHS significantly reduced the number of invaded cells and inhibited the migration of HK-2 cells (Fig 9).

Discussion
Study by K-J Jung et. al. had linked H 2 S to TGF-β signaling in unilateral UO-induced kidney fibrosis in mice [4]. However, the molecular events underlying the effect of H 2 S on EMT remain unknown. As a significant step towards fibrogenesis, EMT presents the morphological changes associated with downregulation of E-cadherin and upregulation of most common mesenchymal markers such as vimentin, fibronectin, α-smooth muscle actin (α-SMA) [16]. In our study, we find out that under the pretreatment by NaHS, overexpression of α-SMA and fibronectin stimulated by TGF-β1 was attenuated, meanwhile, the loss of E-cadherin induced by TGF-β1 was significantly reversed (Fig 1). This effect of H 2 S inhibiting the process of EMT, can also be seen in human breast cancer and alveolar epithelia cells as well [17,18], thus establish the anti-fibrotic role of NaHS in renal tubular cell line.
H 2 S is synthesized endogenously in mammals from the sulfur-containing amino acid l-cysteine by the action of several distinct enzymes [1][2][3], though only CBS was detected in HK-2 cells in our study (Fig 2). The other enzymes were not detected by Western blotting probably due to their low concentrations in HK-2 cells or the tissue-specific enzymatic pathways for H 2 S production. Further study can be carried out to determine whether a particular enzyme is the dominant in the entire renal tissue.
To investigate the molecular mechanism underlying the inhibition effect of H 2 S towards EMT in renal tubular epithelial cells, we first detected the levels of TβR II and TβR I expression under the treatment of NaHS. Results showed that NaHS could reduce TGF-β1-induced overexpression of both TβR II and TβR I (Fig 3) in the traditional TGF-β/Smad signaling which is a pathway most important to and highly depended by EMT [5]. In contrast, L. Sun et al. have shown that NaHS selectively inhibited TβR II in myocardium [19], suggesting that the effect of NaHS on TGF-β signaling may be tissue-specific. Further down the pathway, the phosphorylation of Smad2 and Smad3 were shown to be significantly reduced by the pretreatment with NaHS in myocardial collagen remodeling in spontaneously hypertensive rats and in TGF-β1-stimulated cultured cardiac fibroblasts [19], and in unilateral UO animal model [20].
Apart from TGF-β /Smads pathway, TGF-β receptors can also activate non-Smad signal pathways, including ERK pathway and others. Many studies showed that ERK1/2 could be phosphorylated when EMT occured [21], while NaHS abolished the increased phosphorylation of mitogen-activated protein kinase [20]. In serum-starved HK-2 cells co-treated by NaHS and TGF-β1, we found that NaHS significantly attenuated the TGF-β stimulated overexpression of phospho-ERK1/2 in cell lysates (Fig 4), though it did not affect the levels of phospho-p38/p38 and phospho-JNK/JNK (data not shown). When cells were treated by U0126 together with NaHS, the anti-EMT effect of NaHS was abolished (Fig 7), suggesting that the anti-EMT effect of NaHS in HK-2 cells relies on ERK1/2 signaling.
Many studies have shown there may exists crosstalk between TGF-β/Smad and Wnt/β-catenin pathways by interactions between intracellular proteins of the two pathways [22]. When βcatenin disassociate from the adherens junction complex, the interaction between Smad3 and Smad4, which are the intermediates in TGF-β signaling, and β-catenin is stimulated by TGF-β1 in the cytoplasm [13]. C.S. Kim et al. have reported that expression of β-catenin was increased in 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal induced EMT and established the relationship between Wnt/β-catenin signaling and renal tubular EMT [23]. B. Zhou et al. have shown that TGF-β1 induced convergence of β-catenin-dependent and canonical Smad3 signaling is critical during the development of TGF-β-induced EMT. And they found that ICG-001, a specific inhibitor of β-catenin/CBP interactions, prevented TGF-β1-induced EMT and α-SMA activation, demonstrating a novel requirement of CBP-dependent β-catenin signaling in  were pretreated with or without NaHS (100μM) for 12 hours and then they were treated by TGF-β1 for the following 36 hours. Blue color stands for nucleus stained by DAPI and green color stands for nonphosphorylated β-catenin. (E) Graphical representation of the cells in which non-phosphorylated β-catenin translocated into nucleus. The values of mean ± SEM (n = 3) were gained from three randomly microscope fields in each slide. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey's multiple comparison test.
doi:10.1371/journal.pone.0147018.g006 The expression of non-phosphorylated β-catenin under the condition of ERK signal was blocked. ERK1/2 inhibitor U0126 (10μM) was given to HK-2 cells for 1 hour and then cells were incubated with TGF-β1 for 36 hours. (B) Effects of NaHS (100μM) on the expression of fibronectin, E-cadherin,TβR I and α-SMA in the presence of TGF-β1 and U0126 or the β-catenin inhibitors XAV-939. Pretreatment by NaHS (100μM) is given to HK-2 cells for 12 hours and then U0126 (10μM) or XAV-939 (10μM) were given to the cells for 1 hour before they were incubated with TGF-β1 for the following 36 hours. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey's multiple comparison test.
the stimulation of the EMT target gene α-SMA by TGF-β [24]. In this study, we demonstrated, for the first time, that both total β-catenin and non-phosphorylated β-catenin expression levels were increased after being treated by TGF-β1 and the pretreatment with NaHS attenuated this change (Fig 5). Furthermore, NaHS reversed the nuclear translocation of non-phosphorylated β-catenin caused by TGF-β1 in HK-2 cells (Fig 6). In addition, when we blocked ERK1/2 signaling using U0126, the overexpression of the non-phosphorylated β-catenin stimulated by TGF-β1 perished, indicating that there exist a relationship between ERK and Wnt/catenin signaling pathway. The cells were transfected with β-catenin siRNA or control siRNA for 6 hours and the culture medium was replaced by fresh medium. Western blot assay was used to detect the expression level of non-phosphorylated β-catenin. (B) After transfected with β-catenin siRNA or control siRNA for 6 hours, HK-2 cells were incubated with NaHS (100μM) for 12 hours and then with TGF-β1 for the following 36 hours. The expression level of fibronectin, E-cadherin, TβR I and α-SMA were examined by western blot assay. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey's multiple comparison test. Generally speaking, our research gave evidence that hydrogen sulfide can inhibit the TGF-β1-induced EMT in renal tubular epithelial cells via both ERK-dependent and Wnt/catenindependent pathways. This may potentially indicate a new therapeutic approach to renal fibrosis.


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Increased APRIL Expression Induces IgA1 Aberrant Glycosylation in IgA Nephropathy

Supplemental Digital Content is available in the text

INTRODUCTION
P rimary IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. In China, it accounts for 58.2% of biopsy proved primary glommerulonephriti. 1 Approximately 30% to 40% of the patients with IgAN will progress to renal replacement therapy within 20 years after presentation. 2,3 The pathogenesis of the initiation and progression for IgAN remains obscure. Recently, increasing evidence has indicated that aberrantly glycosylated IgA1 molecules, mainly circulating galactose-deficient IgA1, are the trigger factors for mesangial deposition and subsequent kidney injury in IgAN. However, regarding the factors and mechanisms that induce the production of galactose-deficient IgA1, limited scientific data are available.
A genome-wide association study in IgAN revealed a locus containing the TNFSF13 gene, 4 which encodes a proliferationinducing ligand (APRIL) that shows high homology (48%) with B-cell activation factor (BAFF), 5 to be a genetic susceptible locus for IgAN. In addition, a genetic variant of TNFSF13 was reported to be associated with serum IgA levels in both patients with IgAN and in the general population. 4,6 Recently, McCarthy et al reported a transgenic mouse overexpressing BAFF, which showed high similarity to human IgA nephropathy in the presence of commensal flora. Upon overexpression of BAFF, the mice showed high circulating levels of polymeric aberrantly glycosylated IgA, mesangial deposition of IgA, and findings of hematuria and proteinuria. 7 These studies suggested the potential involvement of APRIL in IgA production and disease pathogenesis of IgAN.
APRIL is a member of tumor necrosis factor super family (TNF). In addition to influencing the survival and proliferation of human B-cells, it is an important factor that drives human Bcells to induce Ig heavy chain class switch to IgA. [8][9][10][11] After binding to their shared receptors, including TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B-cell maturation antigen), APRIL activates the downstream nuclear factor-kB (NF-kB) and then stimulates immunoglobulin production by peripheral blood Bcells. 12 In APRIL-deficient mice, a selective deficiency in IgA was observed, 13 which indicated an important role of APRIL in the IgA production process.
In the present study, to explore the underlying mechanism of APRIL in IgA nephropathy, we detected the expression of APRIL in patients with IgAN and further evaluated its effect on glycosylation of IgA1 molecules.

Study Population
One hundred and sixty-six IgAN patients diagnosed in Peking University First Hospital between January 2014 and August 2014, and who gave their consent to donate 10 mL of venous blood, were enrolled in this study. At the same time, 77 healthy volunteers whose age and gender matched with patients were also recruited. Plasma (EDTA anticoagulated) samples were collected from all individuals in this study, for patients on the morning of renal biopsy and for controls on the day of recruitment. The plasma samples were divided into aliquots and stored in À808C for the subsequent measurement of APRIL protein levels. In addition, peripheral blood mononuclear cells (PBMCs) isolation and RNA extraction were performed in 27 IgAN patients and 21 healthy controls, who were also enrolled in July and August 2014, to determine the expression of APRIL mRNA, as well as the expression of BCMA, TACI mRNA.
For patients with IgAN, diagnosis was based on the granular deposition of IgA in the glomerular mesangium by immunofluorescence detection, and the deposition of electrondense material in the mesangium using the ultrastructural examination. Patients with Henoch Schonlein Purpura, systemic lupus erythematosus and chronic hepatic diseases were excluded by detailed clinical and laboratory examinations. Clinical information, including 24-hour urine protein excretion and blood pressure, were collected from medical records. The estimated glomerular filtration rate (eGFR) was evaluated using the Modified Glomerular Filtration Rate Estimating Equation for Chinese patients. 14 For the evaluation of pathological lesions, Oxford classifications, 15,16 were scored by 1 pathologist and independently checked by another pathologist, both of them blind to the clinical data.
The Medical Ethics Committee of Peking University First Hospital approved the study protocol and informed written consent was obtained from all individuals.

Plasma APRIL Detection
Plasma APRIL levels were detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's specifications (eBioscience, San Diego, CA).

Assay for IgA1 and Gd-IgA1
Total IgA1 and total Gd-IgA1 levels in plasma and in cell culture supernatant were determined by ELISA, as previously reported; 17 however, there was a minor change in Gd-IgA1 standard compared with the previous report. 18 In IgA1 detection, native IgA1 purified by normal human plasma (EMD Chemicals, Gibbstown, NJ) was used as standard for quantification of total IgA1. While for Gd-IgA1 detection, initially, the IgA1 protein was purified from plasma from a patient with multiple myeloma using an agarose-bound jacalin affinity chromatography column (Pierce Chemical Company, State of Illinois). The residual IgG was removed by a protein G column (GE, State of Connecticut). Finally, the terminal sialic acid from O-linked GalNAc was removed by neuraminidase (Roche Diagnostic Corp, Basel, CH), and galactose from O-linked GalNAc was removed by galactosidase (Sigma, State of Missouri). As the standard IgA1 myeloma protein is not entirely devoid of galactose, we expressed the results as U/mL, in which 1 unit of Gd-IgA1 was defined as 1 ng of this standard Gd-IgA1 myeloma protein. The abnormality of the glycosylation status of IgA1 molecules were expressed as adjusted Gd-IgA1 levels and calculated as total Gd-IgA1/total IgA1 levels.

B-and T-lymphocytes Isolation
Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation using Ficoll-Paque plus (GE, State of Connecticut, America). Then, B-and T-lymphocytes were isolated using CD19þ and CD3þ magnetic beads (Invitrogen, State of California), respectively, according to the manufacturer's instructions.

Total RNA Extraction and Reverse Transcription PCR (RT-PCR)
After cell isolation, total RNA was extracted from T-lymphocytes and B-lymphocytes using the commercial TRI-ZOL 1 Reagent (Invitrogen, Carlsbad, CA). cDNA was got from 1 mg total RNA using Reverse Transcription System (Promega, State of Wisconsin) and stored at À208C for the next amplification. The mRNA levels of TNFSF13, BCMA, TACI, and GAPDH were measured by semiquantitative reverse transcriptase-PCR (the primer pairs were listed in Table 1), using SYBR 1 Premix Ex Taq TM (Takara, Shiga, JP) and performed on an Applied Biosystem 7500 Real-Time PCR System. The fold change between patients and controls was expressed by the 2 -DDCT method.

Lymphocyte Culture and Treatment
Lymphocytes were isolated from venous blood as previously reported. 19 Briefly, peripheral blood mononuclear cells (PBMCs) were first isolated by density gradient centrifugation. Monocytes were then depleted by the wall-sticking method from PBMCs. Thereafter, most of the remaining cells were lymphocytes, which were cultured in the RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin at 378C in a humidified 5% CO 2 incubator and used for subsequent experiments.
During the experiment, lymphocytes were seeded into 96-well plates at density of 3 Â 10 5 /well and incubated with

Statistical Analysis
Statistical analyses were performed using SPSS software (version 16.0; SPSS, Chicago, IL). For continuous variables, data with a normal distribution was expressed as the mean AE SD and compared by an independent-samples t test, whereas other data were expressed as the median (first quartile and third quartile) and analyzed by the Mann-Whitney U test. Categorical variables were summarized as absolute frequencies or percentages and were compared using a x 2 test. A 2-tailed P-value <0.05 was considered statistically significant.

Clinical and Pathological Manifestations of Patients With IgAN
The clinical and pathological feature of IgAN patients in the present study ( Table 2) was in accordance with previously reported IgAN cohorts. The average 24 hours proteinuria and eGFR in our patients and the grading of the pathological lesions by Oxford classification are also shown in Table 2.

Patients With IgAN Had High Levels of APRIL
To evaluate whether APRIL is involved in IgA nephropathy, we at first compared the plasma levels of APRIL in patients with IgAN and healthy controls. We found that plasma levels of APRIL were significantly higher in patients with IgAN than in the healthy controls (APRIL: 0.15 [0.00, 2.41] vs 0.00 [0.00, 0.82] ng/mL, P ¼ 0.009 Figure 1). Indeed, plasma APRIL levels were very low in some individuals. In our study population, 41.57% (69/166) IgAN patients and 57.14% (44/77) healthy controls had undetectable (below the limit of quantification) plasma APRIL levels when detected by ELISA. More individuals with undetectable plasma APRIL levels were in the control group than in the IgAN group, which was in accordance with our finding that patients with IgAN had higher APRIL levels in their plasma, suggesting APRIL's involvement in IgAN.
APRIL is widely produced by many cell types, such as T-lymphocytes, B-lymphocytes, and dendritic cells. Therefore, we isolated T-and B-lymphocytes and detected the expression of TNFSF13 mRNA and APRIL protein in them. Our results showed that compared with healthy controls, upregulated mRNA expression of TNFSF13 in IgAN patients was only observed in B-lymphocytes (2.56 AE 2.84 vs 1.06 AE 0.72, P ¼ 0.003, Figure 2A), but not in T-lymphocytes (4.30 AE 7.41 vs 1.92 AE 1.50, P ¼ 0.371, Figure 2B). Thus, in IgAN, Blymphocytes may contribute to elevated plasma APRIL levels.

Plasma APRIL Levels Correlated With Severity of IgAN
After the identification of increased APRIL levels in patients with IgAN, we further explored its association with clinical findings and pathological lesions in patients with IgAN.
As some patients with IgAN presented with undetectable plasma APRIL levels, we initially classified them into the low value group (n ¼ 69). We classified the other patients into 2 groups according to the median value of their plasma APRIL levels (median value: 2.02 ng/mL): middle value group (below median, n ¼ 48) and high value group (above median, n ¼ 49). We found that patients with high APRIL levels had significantly higher levels of proteinuria and lower levels of eGFR compared with patients with lower APRIL levels ( Table 3). The results indicated that increased plasma APRIL levels correlated with more severe clinical manifestations in IgAN.

Plasma APRIL Levels Correlated With Adjusted Gd-IgA1 Levels in IgAN
APRIL contributes to the production of IgA, which is the characteristic molecule of mesangial deposition in IgAN. Currently, increased Gd-IgA1 molecules are widely accepted as the initial factor for IgAN. Therefore, after the identification that patients with IgAN had increased plasma APRIL levels, we investigated whether plasma APRIL correlated with Gd-IgA1 levels in IgAN. In patients with detectable APRIL, the plasma APRIL levels showed a strong positive correlation with adjusted Gd-IgA1 levels (r ¼ 0.570, P ¼ 0.021, Figure 3A), whereas for those with undetectable APRIL, their adjusted Gd-IgA1 levels were significantly lower than others (undetectable APRIL group vs detectable APRIL group: 0.36 AE 0.10 vs 0.53 AE 0.16, P ¼ 0.005, Figure 3B). The correlation between APRIL and Gd-IgA1 suggested that APRIL might be involved in the mechanism of increased Gd-IgA1 production in IgAN.

APRIL Upregulates Gd-IgA1 Secretion From Lymphocytes
To evaluate whether APRIL served as the causal factor for increased Gd-IgA1 in IgAN, we further investigated the influence of APRIL on the production of Gd-IgA1 in an in vitro assay. Interestingly, we found that the responses to APRIL in lymphocytes were different between those derived from patients with IgAN and those from healthy controls. After APRIL treatment, in the supernatant of lymphocytes isolated from patients with IgAN, the adjusted Gd-IgA1 levels increased about 1.20 AE 0.15 fold, which was significantly higher than those from healthy controls (IgAN vs controls: 1.20 AE 0.15 fold vs 1.00 AE 0.17 fold, P ¼ 0.014, Figure 4A). Our in vitro assay findings implied a role for APRIL in Gd-IgA1 production in patients with IgAN. More importantly, the findings suggested that not only APRIL, but also lymphocyte abnormality, might also contribute to elevated Gd-IgA1 in IgAN, because APRIL increased Gd-IgA1 production in lymphocytes from IgAN patients, but not from healthy controls.
mRNA Levels of TACI and BCMA, the Receptors for APRIL In circulation, CD19 þ B lymphocytes are the major cells for IgA1 production. To explore the causal factor of the lymphocytes abnormality in patients with IgAN, in terms of APRIL induced Gd-IgA1 production, we detected the mRNA expression of TACI and BCMA, encoding the receptors for APRIL, in CD19 þ B lymphocytes. In patients with IgAN, the mRNA expression of TACI and BCMA in B-lymphocytes showed a trend of upregulation, although statistical significance was not reached (BCMA: 0.88 AE 0.90 vs 0.53 AE 0.39, P ¼ 0.107, Figure 4B; TACI: 1.70 AE 1.20 vs 1.26 AE 0.78, P ¼ 0.137, Figure 4C).

DISCUSSION
IgA molecules, as the predominant deposits in glomeruli of patients of IgAN, have attracted the most attention in research into the pathogenesis of the disease. 20 APRIL is a cytokine that stimulates B-lymphocyte proliferation and IgA production. 11,21 Following previous report that patients with IgAN had elevated APRIL levels, 7 in the present study, we at first validated the increased APRIL expression in IgAN, and further found that in patients with IgAN, the elevated APRIL levels together with marginal increases in the expressions of its receptors, BCMA and TACI, in B lymphocytes, inducing increased production of aberrantly glycosylated IgA1 molecules, which ultimately contributed to IgAN pathogenesis.
Consistent with a previous report, 7 we also observed elevated plasma APRIL levels in our patients with IgAN. We then investigated which kind of cells contributed to this significant increase. APRIL is expressed and secreted by multiple cells, including monocytes, macrophages, and lymphocytes; 22 therefore, we investigated its mRNA expression in T-and B-lymphocytes, because these 2 cells account for a large proportion of peripheral circulating cells and are involved frequently in autoimmune diseases. We observed upregulated APRIL expression in B-lymphocytes, but not in T-lymphocytes. The sample exhibited highest APRIL protein level (Figure 1) was the same as the one showed highest mRNA expression level  in B-APRIL ( Figure 2), but it was not comparable in T-APRIL in mRNA and protein levels. It might imply that B -lymphocytes play more important role in deciding the plasma APRIL level. We also detected the expression of BAFF, which is a highly homologous member with APRIL in TNFSF family, in T-and B-lymphocytes, and we found that the upregulated BAFF expression was also observed in B-lymphocytes, but not in T-lymphocytes (Supplementary Figure 1, http://links.lww.com/ MD/A825), which was consistent with the previous study that the expression of APRIL always positively correlated with that of BAFF. Although no information was available regarding other cells contributing to the elevated plasma APRIL levels, our results implied that, at least in part, B-lymphocytes contribute to the elevated plasma APRIL levels in IgAN, which suggested an abnormality of B-lymphocytes in IgAN. In the B-lymphocytes of IgAN, in addition to the significantly upregulated APRIL expression, the expressions of the receptors for APRIL also showed marginal elevation. Therefore, we hypothesized that APRIL and its downstream signaling should be activated in B-lymphocytes of IgAN patients.
Although abnormal APRIL expression in B-lymphocytes had not been reported before in IgAN, studies on BAFF, which has similar functions to APRIL (e.g., induction of germline Ca gene expression, AID expression, and IgA class switching in a CD40-independent manner 23 ), could be used as a reference. Recent studies reported the upregulated expression of BAFF in tonsillar mononuclear cells (TMC) in patients with IgAN after capsaicin or CpG-ODN stimulation. 24,25 Moreover, they found that upregulation of BAFF caused aberrant IgA1 O-glycosylation by suppressing C1GALT1 and Cosmc expression. Similarly, in our study, we proved that APRIL could also induce the production of Gd-IgA1. Taken together, these findings indicated that activation of the APRIL/BAFF system in B-lymphocytes might play an important role in the overproduction of pathogenic aberrant glycosylated IgA1 molecules in IgAN. However, the underlying mechanism of how APRIL induces Gd-IgA1 production remains unknown here. Future studies will focus on the signaling action and effector gene expression (such as C1GALT1 and Cosmc) downstream of APRIL-TACI/ BCMA. In addition, the expression of BCMA and TACI in IgAN patients was needed to verify in more patients, and to be validated in an independent cohort.
In our patients with IgAN, we observed a positive correlation between plasma APRIL levels and Gd-IgA1 levels, which validated our finding that APRIL could induce production of aberrant glycosylated IgA1 molecules in an in vitro cell culture model. In addition, we found that patients with higher levels of APRIL presented with more severe clinical manifestations of IgAN. In the pathogenesis of IgAN, increasing evidence suggests that aberrant O-glycosylation of IgA1 acts as a ''trigger.'' Moreover, our previous study attributed O-glycosylation abnormality of IgA1 as an independent predictor for long-term renal outcome in IgAN. 17 Therefore, the association between APRIL and IgAN severity observed in the present study suggested the important role of APRIL in IgAN and supported APRIL as a causal factor for promoting the production of Gd-IgA1 in IgAN.
In summary, the present study revealed increased APRIL levels and the association of APRIL with disease severity of IgAN, which suggested the involvement of APRIL in IgAN. Furthermore, we proved that APRIL, in cooperation with its receptors in B-lymphocytes, could induce the production of Gd-IgA1, which represent, at least in part, the mechanism of APRIL's action in the pathogenesis of IgAN.