Micro RNA‐30b (inhibitor) nanoparticles suppressed the lipopolysaccharide (LPS)‐induced acute kidney injury

Abstract

The main aim of present study is to evaluate the effect of miR‐30b on the function of human proximal tubular epithelial cell line HK‐2 cells. For this purpose, miRNA was loaded in an ionically cross‐linked polysaccharide nanoparticle. The authors have demonstrated the influence of miR‐30b mimic and inhibitor in HK‐2 cell killing effect. Lipopolysaccharide (LPS) significantly increased the level of inflammatory cytokines of TNF‐α, IL‐1β and level was further increased with the treatment of PAg‐miR mimic consistent with the cell viability assay. Interestingly, PAg‐miR inhibitor significantly downregulated the expression of inflammatory cytokines and thereby reduced the inflammation in the body. Western blot analysis showed that LPS induced severe apoptosis of HK‐2 cells and the apoptosis was further promoted by the PAg‐miR (mimic). In contrast, PAg‐miR (inhibitor) alleviated the apoptosis of HK‐2 cells as indicated in the significantly reduced levels of Bax and c‐Caspase‐3 proteins. Overall, miR‐30b promoted LPS‐induced HK‐2 cell inflammatory injury by inducing the apoptosis and by releasing inflammatory cytokines, as well as by impairing autophagy process.

1 Introduction

Acute kidney injury (AKI) is characterised by numerous clinical events, which leads to the loss of kidney function (such as reduced glomerular filtration rate) [1]. AKI is the direct result of toxicity of several small molecules such as anticancer drugs‐cisplatin or doxorubicin affects the physiological functions of glomerulus apparatus [2]. In general, 7% of hospitalised patients develop AKI and 30% of patients in intensive care unit (ICU). Especially, it affects vast percentage of paediatric patients with sepsis‐related AKI [3, 4]. Moreover, AKI is considered as a risk factor for the chronic kidney damage and other cardiovascular complications. The main cause of AKI was attributed to the release of proximal tubular epithelial products such as inflammatory cytokines (TNF‐α and IL‐6 etc.), which later on infiltrate into the kidney interstitum and causes more damage [5, 6].

Micro RNA's (miRNA) are endogenous and non‐coding RNAs with an average length of 18–25 nucleotides which are involved in the regulation of gene expression and affect the physiological functions in the body [7]. The miRNA acts by binding to the 3′untranslated regions (3′UTR) of target mRNAs and results in the functional programs. Literature is the evidence that miRNA plays an important role in the regulation of inflammatory reactions and inflammatory diseases and, therefore, plays an important role in the pathology of several vital organs [8]. To be specific, miRNA‐30b is reported to contribute to the inflammatory cytokine‐mediated β‐cell dysfunction and actively involved in the melanoma cells immunosuppression [9]. In addition, miR‐30b involved in the inhibition of autophagy of hepatic ischaemia‐reperfusion and its role in AKI is not studied well until today [10, 11]. Therefore, present study is an attempt to study the role of miR‐303b in the treatment of AKI.

Gene therapy though holds great clinical potential for the treatment of major diseases, its instability in the body fluids and rapid elimination from the circulation hampers its clinical success [12]. An ideal gene carrier should possess high biocompatibility, high condensation ability, and excellent systemic circulation stability [13]. Due to abundant amine group and high positive charge, polyethyleneimine (PEI) offers high gene condensation ability and possesses strong proton sponge effect. However, PEI owing to high positive charge leads to severe local and systemic toxicity. Besides, PEI/miRNA complex might be unstable in the circulatory environment [14]. Therefore, a negatively charged polymer is reported to confer the stability to the PEI complex. It is expected that the negatively charged polymer might form strong complex with the positively charged PEI and forms a stable particle [15]. For example, PEI/DNA complexed with sodium alginate is reported to improve the transfection efficiency and tumour tissue accumulation and higher therapeutic efficacy. Therefore, in the present study, we have employed sodium alginate to complex with PEI/miRNA to form a stable particle [16]. Furthermore, it has been reported that physical or chemical reaction between polysaccharides with abundant carboxy group and metal ions are relatively more stable in blood circulations [17]. Therefore, we have cross‐linked the PEI/miRNA/Alginic acid complex with calcium (Ca²⁺) salt to form a stable particle.

In this study, kidney cells (human proximal tubular epithelial cells, HK2) were used to study the effect of miRNA‐30b. Physical characterisation of PEI/miRNA/Alg/Ca2+ nanoparticle was studied by particle size distribution and particle morphology. The role of miRNA‐30b mimic and miRNA‐30b inhibitor in LPS‐inflammation‐induced cell was studied in terms of cell viability. The apoptosis induction and release of inflammatory cytokines were studied by Western blot analysis and RT‐PCR‐based gene expression analysis.

2 Materials and methods

2.1 Preparation of miRNA‐30b‐loaded ionic cross‐linked polysaccharide NP

Hyperbranched PEIs (PEI25k, Mw 25 kDa) were mixed with either miRNA‐30b (mimic or inhibitor; purchased from GenePharma, Shanghai, China) at a weight ratio of 2.75:1 and incubated for 30 min. The polysaccharide alginic acid (Sigma Aldrich, China) was added to the above PEI/miRNA complex in a weight ratio of 1:1 and incubated for 30 min. Finally, calcium chloride was added to the PEI/miRNA/Alg mixture in a proportion of 0.005 to the weight ratio. The unconjugated ingredients were removed by centrifugation at 10,000 rpm for 5 min. The nanoparticles were collected and stored until further use.

The particle size distribution of nanoparticle was studied by ZetaSizer (Malvern Instruments, UK). The experiment was performed in triplicate after diluting the nanoparticle dispersion in ultrapure water. The morphology of nanoparticle was studied using transmission electron microscope (TEM). The nanoparticles were diluted and observed under the electron microscope after counter staining with 2% phosphotungistic acid (PTA).

2.2 Cell culture

The human proximal tubular epithelial cell line HK‐2 (ATCC, MD, USA) was cultured in DMEM medium supplemented with 10% foetal bovine serum and 1% antibiotic mixture. The cells were maintained at 37°C under humidified conditions. As part of initial study, the HK2 cells were treated with 1–10 µg/ml of lipopolysaccharide (LPS, sigma Aldrich, China) for 6 h. The LPS‐treated HK2 cells were exposed with PAg‐miR (inhibitor and mimic) and further incubated for 48 h. After the incubation period, cells were extracted and continued for subsequent experiments.

2.3 Cell viability analysis

The cell viability of HK2 cells were determined by MTT assay. In brief, 1 × 10⁴ cells were seeded in a 96‐well plate (each well) and incubated for 24 h (80% confluence cells was used). The cells were exposed with LPS for 6 h and then cells were treated with either PAg‐miR (inhibitor and mimic) for 48 h. The cells were washed carefully and MTT solution (10 µl of 5 mg/ml) was added and incubated for 3 h at 37°C. The cells were added with DMSO to dissolve the formazan crystals and absorbance was read at 570 nm using a microplate reader.

2.4 Quantitative reverse‐transcriptase PCR

The cells were exposed with LPS for 6 h, and then cells were treated with PAg‐miR (either inhibitor or mimic) for 48 h. The cells were extracted suing Triazol reagent, and the RNA concentration was evaluated by NanoDrop 2000 Spectrophotometer (Thermo Fischer Scientific). A Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark) was used to synthesise the cDNA from RNA samples which was then quantified using ExiLERATE SYBR Green Master Mix Kit (Exiqon). GAPDH was used as an internal control. The mRNA expression levels of Bcl‐2, Bax, c‐Caspase‐3, ATG5, and LC3‐II/I were evaluated using its respective primers. Complementary DNA (cDNA) was amplified and quantified on CFX96 system (BIO‐RAD, USA). The expression of mRNA was calculated based on Ct, and relative expression levels 2^{−(CtmRNA – CtGAPDH)} after normalisation with reference to the quantification of GAPDH RNA expression and expression levels of mRNA of respective markers.

2.5 Western blot analysis

The cells were exposed with LPS for 6 h, and then cells were treated with PAg‐miR (either inhibitor or mimic) for 48 h. The cells were extracted using a stripping buffer. The cells were lysed using a lysis buffer and proteins are collected and quantified using BCA protein assay kit. The proteins are separated on a 10% SDS‐PAGE gel and transferred to nitrocellulose membrane and then blocked for 1 h using 5% skim milk. After blocking, membranes were incubated with primary antibodies, including IL‐1β, TNF‐α, b‐actin, Bax, c‐Caspase‐3, ATG5, and LC3‐II/I, respectively, and incubated overnight at 4°C. Next day, membranes are washed and incubated with secondary antibody for 1 h at room temperature. Secondary antibodies (Abcam) included goat anti‐rabbit IgG (ab6721, 1:5000) and goat anti‐mouse IgG (ab6789, 1:5000). Signals of proteins were captured using a Bio‐Rad ChemiDoc™ XRS system (Bio‐Rad, Hercules, CA, USA).

2.6 Apoptosis assay – flow cytometer

The apoptosis assay was carried out by flow cytometer after staining the cells with Annexin‐V and PI. The cells were exposed with LPS for 6 h, and then cells were treated with PAg‐miR (either inhibitor or mimic) for 48 h. The cells were scrapped and centrifuged at 1200 rpm for 5 min. The pellets were re‐dispersed in 50 µl of PBS. The cells were stained with 2.5 µl of Annexin and 2.5 µl of PI and incubated for 15 min in dark conditions. The volume was made up to 1000 µl with PBS and analysed using the BD FACS flow cytometer.

2.7 Statistical analysis

Student's t ‐test was used to determine statistical differences between two groups. P < 0.05 was considered significant. All the experiments were repeated three times.

3 Results and discussion

3.1 Formulation of miRNA‐30b‐loaded ionic cross‐linked polysaccharide NP

AKI is the direct result of toxicity of several small molecules such as anticancer drugs‐cisplatin or doxorubicin affects the physiological functions of glomerulus apparatus. miRNA‐30b is reported to contribute to the inflammatory cytokine‐mediated β‐cell dysfunction and actively involved in the melanoma cells immunosuppression. Therefore, present study is an attempt to study the role of miR‐303b in the treatment of AKI. Due to the abundant amine groups, PEI25k exhibited high DNA complexation ability and strong proton sponge effect, which endows PEI/miRNA complexes with the capacity to disrupt endo‐lysosomes and be internalised into cytoplasm. However, PEI/miRNA complexes were unstable during the systemic circulation in vivo, which would hinder their accumulation at tumour site. To solve this problem, PEGylation and molecular modification were two of the commonly used strategies; however, it results in low efficiency of gene loading and transportation. Therefore, negatively charged polysaccharides, such as sodium alginate, dextran sulphate, and hyaluronic acid, could shield the PEI/miRNA complexes (via electrostatic interactions) [18]. It could be inferred that both the biocompatibility and stability in blood could be improved by shielding PEI/miRNA complexes with polysaccharides. The polysaccharides with abundant carboxyl groups could be cross‐linked by metal ions, such as calcium ions. It was hypothesised that the stability of PEI/miRNA complexes in systemic circulation might be enhanced through Alginate shielding and Ca²⁺ cross‐linking strategy, while the transfection efficiency of PEI/miRNA complexes would not be weakened (Fig. 1 a).

Fig. 1.

Physico‐chemical properties of the nanoparticles

(a) Schematic presentation of miRNA‐loaded polysaccharide nanoparticles, (b) Particle size distribution, (c) Morphology analysis

Alginic acid was added as a shielding or protective agent. The negatively charged Alginic acid will rapidly interact with the positively charged PEI/miRNA complex. The average particle size of PEI/miRNA/Alg was 165.25 ± 1.26 nm with a polydispersity index (PDI) of 0.145 and an average surface charge of −18.2 ± 0.39 mV. The negatively charged nanoparticles will allow the enhanced circulation in the blood compartment. Though alginic acid formed complex with PEI/miRNA, it is reported to be unstable in the systemic circulation. The abundant carboxylic group of alginic acid could be utilised to cross‐link with the Ca²⁺ which are reported to be highly stable in the physiological conditions. Therefore, we have cross‐linked PEI/miRNA/Alg with Ca²⁺ that resulted in a stable particle. The average particle size of PAg‐miR was in the range of 135.46 ± 2.65 nm with a surface charge of −15.26 ± 0.12 mV (Fig. 1 b). The decrease in the particle size was attributed to the cross‐linking of particle which constrained the particles. The decrease in the surface charge was attributed to the consumption of free carboxylic group during the cross‐linking process. Overall, a small particle size of <150 nm will be beneficial for systemic applications [19]. PAg‐miR showed good stability in cell culture medium upon 7‐day incubation. The particle size on day 1 was 136.4 ± 1.26 nm and increased to 149.6 ± 1.64 nm on day 7 indicating its excellent stability. The particle morphology of PAg‐miR was analysed through TEM. The particles were uniformly distributed in the TEM grid without any aggregation indicting the stability of nanoparticles and dispersity index (Fig. 1 c).

3.2 Lipopolysaccharide‐induced HK‐2 cells inhibition

The effect of LPS on the cell viability of HK‐2 cells was observed by MTT assay (Fig. 2 a). Results clearly showed the cell viability of HK‐2 cells significantly decreased with the higher dose of LPS indicating the potent toxic effect of LPS. LPS is, therefore, used in the generation of kidney inflammatory model. Hereafter, LPS at a dose of 5 µg/ml was standardised for all subsequent experiment. Then, we have analysed the protein expression of inflammatory cytokines including IL‐1β and TNF‐α. As shown (Fig. 2 b), LPS at a dose of 5 µg/ml induced a multifold increase in the expression of IL‐1β and TNF‐α compared to that of control. These results clearly indicate the inflammation inducing potentials of LPS in the body.

Fig. 2.

HK‐2 cell inhibition

(a) LPS induced cell viability of HK‐2 cells by MTT assay, (b) LPS induced mRNA levels of TNF‐α and IL‐1β detected by quantitative reverse‐transcriptase PCR (qRT‐PCR), (c) LPS induced mRNA levels of Bax, caspase‐3, ATG5 and LC3‐II/I

3.3 Lipopolysaccharide‐induced HK‐2 cells apoptosis and autophagy

We have evaluated the LPS‐induced apoptosis process by RT‐PCR. As seen (Fig. 2 c), LPS significantly increased the protein expression of apoptosis proteins such as Bax or caspase‐3 and significantly decreased the expression the anti‐apoptotic factor Bcl‐2. At the same time, miRNA expression of ATG5 and LC3‐II was significantly increased compared to that of control. Results clearly indicate that LPS induces both apoptosis and autophagy [20, 21].

3.4 Mir‐30b (PAg‐miR) influenced the cell viability and inflammatory cytokine release

At first, we studied the influence of miR‐30b (mimic and inhibitor) on the cell viability of HK‐2 cells by MTT assay. As seen (Fig. 3 a), LPS showed a significant reduction in the cell viability. More than 40% of cells were killed by LPS exposed at 5 µg/ml. Interestingly, PAg‐miR mimic further reduced the cell viability with >60% of cells killed. The results clearly showed the influence of miR‐30b mimic in HK‐2 cell killing. The cell killing effect was revered when exposed with PAg‐miR inhibitor with only 25% of cells killed. It is worth noting that cell viability was significantly higher compared to that of LPS‐treated HK‐2 cells. Next, we have evaluated the protein expression of inflammatory cytokines of TNF‐α and IL‐1β by Western blot analysis (Fig. 3 b). As shown, LPS significantly increased the level of inflammatory cytokines of TNF‐α, IL‐1β and level was further increased with the treatment of PAg‐miR mimic consistent with the cell viability assay. Interestingly, PAg‐miR inhibitor significantly downregulated the expression of inflammatory cytokines and thereby reduced the inflammation in the body. The reversal effect was mainly attributed to the miRNA‐30 b inhibitor. These results collectively indicate that miRNA‐30b mimic inhibits the HK‐2 cell viability and induce the release of cytokines while miRAN‐30b inhibitor reversed the effect in the kidney cells. Several reports showed that miRNA‐30b involved in the pathogenesis of various carcinomas. For example, Zheng et al. demonstrated that miRNA‐30b was responsible for the cytokine‐induced B‐cell dysfunction [9]. Gaziel et al. showed that miR‐30b was responsible for the immunosuppression of melanoma cells [10]. Naqvi et al. reported that miR‐30b regulated the phagocytosis in myeloid inflammatory cells [22]. Here in the present study, we have showed that miRNA‐30b is responsible for the release of inflammatory cytokines and induces cell death.

Fig. 3.

Influence of miR‐30b on cell viability

(a) Cell viability of HK‐2 cells after the incubation of LPS and LPS + PAg‐miR mimic and inhibitor, (b) Western blot analysis of HK‐2 cells, (c) Analysis of expression of protein levels of TNF‐α and IL‐1β

3.5 PAg‐miR in apoptosis of HK‐2 cells

The influence of PAg‐miR (mimic and inhibitor) on the apoptosis of HK‐2 cells was studied by flow cytometer after staining with Annexin V and PI. Results showed that >50% of cells underwent apoptosis as shown by flow cytometer histogram (Fig. 4). The incubation of PAg‐miR (mimic) further induced the apoptosis consistent with the cell viability assessment. However, PAg‐miR (inhibitor) significantly reversed the trend and >75% of cells were viable and fewer cells (<25%) were in the apoptosis phase. As shown, PAg‐miR (inhibitor) significantly reversed the trend of cell apoptosis as equal to that of non‐coated PEI/miRNA polyplex indicating that coating of polyplex with a stable polymer did not have any effect on the therapeutic efficacy. In terms of systemic stability, PAg‐miR is expected to possess a greater stability than that of non‐coated polyplex, an experiment subjected to futuristic study. Results clearly suggested the mechanism of action of mimic and inhibitor of miRNA‐30b [23].

Fig. 4.

Apoptosis analysis of HK‐2 cells after staining with Annexin V and PI and detected by flow cytometer. The HK‐2 cells were exposed with LPS for 6 h and then cells were treated with either PAg‐miR (inhibitor and mimic) for 48 h

3.6 PAg‐miR was involved in the apoptosis and autophagy of HK‐2 cells

The role of PAg‐miR (mimic and inhibitor) on apoptosis and autophagy was studied in LPS‐induced HK‐2 cells. Western blot analysis showed that LPS induced severe apoptosis of HK‐2 cells, and the apoptosis was further promoted by the PAg‐miR (mimic). Consistent with the flow cytometer analysis, PAg‐miR (inhibitor) alleviated the apoptosis of HK‐2 cells as indicated in the significantly reduced levels of Bax and c‐Caspase‐3 proteins (Fig. 5 a). The increase and decrease in the proapoptotic factors are consistent with the previous analysis. Furthermore, protein expression of ATG5 and LC3‐II/I was increased by LPS and alleviated by PAg‐miR (mimic), while it was promoted by PAg‐miR (inhibitor) (Fig. 5 b).

Fig. 5.

Apoptosis and Autophagy Studies

(a) Role of miRNA‐30b mimic and inhibitor in the apoptosis proteins Bax and caspase‐3, (b) Influence of miRNA‐30b mimic and inhibitor in the autophagy process by its protein analysis ATG5 and LC3‐II/I

The results clearly indicate that miRNA‐30b acts as a dual functionality. The double function of miRNA‐30b on apoptosis and autophagy depends on the cell types. For example, it was anti‐apoptotic on glioma cells while promoted the apoptosis on gastric carcinoma [24, 25]. In this study, we have observed that overexpression of miRNA‐30b promoted the cell apoptosis, while it impaired the cell autophagy. Similarly, miRNA‐30b was reported to impair autophagy in epithelial cells by regulating ATG5 and LC3‐I/LC3‐II, respectively. Likewise, our study shows that the miRNA‐30b regulated the autophagy mechanism by inhibiting the ATG5 and LC3‐II/I. Therefore, it would be wise to conclude that miRNA‐30b induce the cell death of HK‐2 cells by inducing the cell apoptosis and by impairing autophagy process [26].

4 Conclusions

The main aim of present study is to evaluate the effect of miR‐30b on the function of human proximal tubular epithelial cell line HK‐2 cells. For this purpose, miRNA was loaded in an ionically cross‐linked polysaccharide nanoparticle. We have demonstrated the influence of miR‐30b mimic and inhibitor in HK‐2 cell killing effect. LPS significantly increased the level of inflammatory cytokines of TNF‐α, IL‐1β and level was further increased with the treatment of PAg‐miR mimic consistent with the cell viability assay. Interestingly, PAg‐miR inhibitor significantly downregulated the expression of inflammatory cytokines and thereby reduced the inflammation in the body. Western blot analysis showed that LPS induced severe apoptosis of HK‐2 cells and the apoptosis was further promoted by the PAg‐miR (mimic). In contrast, PAg‐miR (inhibitor) alleviated the apoptosis of HK‐2 cells as indicated in the significantly reduced levels of Bax and c‐Caspase‐3 proteins. Overall, suppression of miR‐30b alleviated the LPS‐induced HK‐2 cell by inhibiting the apoptosis and by inhibiting inflammatory cytokines, as well as by impairing autophagy process.

Regardless of either miR‐30b mimic or inhibitor, PAg‐miR system did not have any effect on the cellular uptake.

It is well known that cellular uptake of nanoparticles will be influenced only in the presence and absence of targeting ligands (regardless of encapsulated component). We have performed new cellular uptake experiment using CLSM and flow cytometer. CLSM experiment showed that no change in cellular internalisation was observed between either nanoparticle systems. As shown in Fig. 6, The intensity of red fluorescence was similar in HK2 cells treated with PAg‐miR (inhibitor) and PAg‐miR (mimic). The uptake was further confirmed by flow cytometer. As seen, no significant difference in cellular uptake was observed between either nanoparticle systems or the flow cytometer histogram stood at the same level. We have included the statement in the manuscript.

Fig. 6.

Cellular uptake of PAg‐miR (inhi) and PAg‐miR (mim) in HK‐2 cells.

(a) Cellular uptake was analysed by CLSM microscope, (b) The uptake of PAg‐miR (inhi) and PAg‐miR (mim) was evaluated by flow cytometry