MD2 blockade prevents oxLDL-induced renal epithelial cell injury and protects against high-fat-diet-induced kidney dysfunction

 There is a strong epidemiological link between obesity, a growing worldwide concern, and kidney disease

• Emerging evidence indicates that the pathogenic basis of obesity-related kidney disease may be attributed to Toll-like receptor 4 (TLR4) of the innate immune system.
• In the present study, the authors hypothesized that renal epithelial cell injury in response to oxidized low-density lipoprotein (oxLDL) requires myeloid differentiation factor 2 (MD2), a co-receptor of TLR4, and is MD2-dependent in the high-fat diet (HFD) mouse model.

Obesity, a growing worldwide concern, is an independent risk factor of kidney disease, of which there is a strong epidemiological link.

• The pathogenic mechanism remains unclear, but the obesity-linked risks of hypertension, type 2 diabetes and cardiovascular diseases could account in part for the nephropathy.

Reagents and Kits

• OxLDL was purchased from Yiyuan Biotechnology (Guangzhou, China).
• Antibodies for transforming growth factor-β (TGF-β; ab92486), collagen IV (COL-4; ab6586), SQSTM1/p62 (ab56416) and Bax (ab32503) were purchased from Abcam (Cambridge, MA, USA.
• The mammalian target of rapamycin (mTOR), phosphorylated mTOR, Beclin1 (3495s), LC3B (3868S), poly(ADP-ribose)polymerase-1 (PARP-1), caspase 3, Bcl-2(3498s), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 5174s) and the horseradish-peroxidase-conjugated secondary antibody
• Active/cleaved Caspase 8 (NB100-56116), a novel compound 30,(E)-2,3-dimethoxy-4'methoxychalcone (L6H21), was synthesized and structurally characterized using MS and 1H NMR analyses as described by us.

Cell culture

• The rat proximal tubular epithelial cell line NRK-52E was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China).

Mouse maintenance

• Male C57BL/6 mice (8 weeks old, weighing 18-20 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China)
• All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee
• Mice were fed the HFD or normal control diet (Ctrl) for 4 months.
• Ctrl diet contained 10 kcal% fat, 20 kcal% protein and 70 kcal% carbohydrate
• After 4-month HFD feeding, mice were sacrificed using sodium pentobarbital anesthesia

Histological, histochemical, and immunofluorescence analyses

• Kidneys were fixed in 4% paraformaldehyde solution, embedded in paraffin and sectioned at 5 μm. After dehydration, sections were stained with hematoxylin and eosin (H&E) for evaluation of general morphology, Masson's trichrome for connective tissue detection or periodic acid-schiff for deposition of polysaccharides/glycogen.
• The stained sections were viewed under the Nikon microscope under magnification 400×.

Western blot analysis

• Cells were lysed, and protein concentrations determined using the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Immunoprecipitation

• Cell lysates were with incubated with anti-MD2 antibody (1:200) at 4°C overnight, and antigen-antibody complexes incubated for 1 h with protein G-Sepharose beads to pull down the complexes.

Quantitative real-time qPCR

• RNA was isolated from cells using TRIZOL

Knockdown of TLR4 and MD2 using siRNA

• The small interfering RNA (siRNA) targeting the nucleotides ofTLR4 or MD2 and its control siRNA containing negative scrambled sequences were obtained from Gene Pharma LTD.

Autophagy detection in culture cells

• Western blot detection for beclin 1 and LC3B
• Cell surface localization was evaluated by fluorescence microscopy
• The product was additionally evaluated by the Premo Autophagos sensor kit using green fluorescent-labeled LC3 B (LC3B-GFP)
• For the former, LC3BP was evaluated in fluorescence.

Measurement of apoptotic rate oxLDL

• cell apoptosis and necrosis were determined using Annexin V-EGFP/PI double staining and flow cytometry.
• Briefly, 1×105 NRK-52E cells were harvested, resuspended in 100 μl of a binding buffer containing 5 μl (5 μl) of the Apoptosis Detection Kit (Yeasen), and incubated for 48 h at room temperature in the dark.

OxLDL injures renal epithelial cells through TLR4

• The treatment of NRK-52E cells with oxLDL increased the protein expression of TGF-β and COL-4 mRNA levels 2.5-fold over control but not in cells with TLR-4 knockdown.
• OxLDl stimulated profibrotic responses, activated autophagy and induced apoptotic cell death in NRK52E cell lines. All of these injurious activities were dependent on TLR.

OxLDL-induced renal epithelial cell injury is dependent on MD2

• Two approaches of MD2 blockade were used: (1) a small molecule inhibitor, L6H21, developed and characterized by us as an MD2 selective inhibitor with anti-inflammatory properties, and (2) knockdown of MDII using siRNA.

Effects of L6H21 on oxLDL-induced renal cell injury

• OxLDL activated TLR4 in an MD2-dependent manner
• Presently, MD2 was downregulated in NRK-52E cells using targeted siRNA sequences prior to the treatment protocol
• The efficiency of MD2 knockdown was ~65% as indicated by Western blot analysis (Fig. 3D and Supplementary Fig. S4)
• It was found that the protection of the cells by MD2 may involve the intrinsic apoptotic pathway rather than the extrinsic pathway
• MD2 knocking down reversed oxLD-induced Bax-down and S5K-induced apoptosis

MD2 blockade in mice prevents renal dysfunction in response to HFD

• HFD-induced kidney dysfunction was associated with morphological abnormalities when evaluated on H&E histological sections, particularly increased vacuoles in renal tubular epithelium and intraglomerular inclusions (Fig. 4E).
• Evaluation of kidney sections stained with Masson's trichrome indicated that HFD induced increased connective tissue content, prominently localized in interstitial spaces surrounding renal tubules and within glomeruli in the interstitium surrounding blood vessels.
• Additionally, accumulations of polysaccharides/glycogen were detected in PAS-stained kidney sections from HFD mice with either oral treatment with L6H21 or MD2 knockout.

Discussion

• The key pieces of evidence supporting the conclusion are: the MD2 selective inhibitor (L6H21) abrogated the oxLDL-induced formation of MD2-TLR4 dimerization in the renal tubular epithelial cell line NRK-52E, MD2 blockade and MD2 knockout in HFD mice protected against development of kidney dysfunction and renal tissue injury
• MD2 likely is a predominant pathogenic factor in obesity-induced chronic kidney diseases
• As a crucial co-receptor for TLR4, Md2 serves to recognize and bind microbial ligands on the cell surface, resulting in inflammation and injury in kidney.
• Identification of the mechanism by which MD2 mediates oxLD-induced renal injuries should be noted and investigated in future studies

Reference

10.1016/j.jnutbio.2019.04.003