Bone marrow-derived exosomes promote inflammation and osteoclast differentiation in high-turnover renal osteodystrophy

Figures & data

Table 1. Primers used for qRT–PCR.

Marks	Forward/reverse	Sequence	Gene accession no
TNF-α	Forward	5-ATG GGC TCC CTC TCA TCA GT-3′	NM_012675.3
	Reverse	5′-GCT TGG TGG TTT GCT ACG AC-3′
IL-1β	Forward	5′-GTC CTC TGC CAA GTC AGG TC-3′	NM_031512.2
	Reverse	5′-CAG GGA GGG AAA CAC ACG TT-3′
IL-6	Forward	5′-CCC ACC AGG AAC GAA AGT CA-3′	NM_012589.2
	Reverse	5′-ACT GGC TGG AAG TCT CTT GC-3′
NF-κB1	Forward	5′-CTG AGT CCC GCC CCT TCT AA-3′	NM_001276711.1
	Reverse	5′-CTC CAC CAG CTC TTT GAT GGT-3′
I-κB	Forward	5′-TGA GTA CCT GGA CTT GCA GAACG-3′	NM_030867.2
	Reverse	5′-TGT AGA TGC CTC TCC AAG GATGG-3′
MMP-9	Forward	5′-GAT CCC CAG AGC GTT ACT CG-3′	NM_031055.2
	Reverse	5′-GTT GTG GAA ACT CAC ACG CC-3′
CTSK	Forward	5′-TCC TCA ACA GTG CAA GCG AA-3′	NM_031560.2
	Reverse	5′-CCA GCG TCT ATC AGC ACAGA-3′
GAPDH	Forward	5′-AGT GCC AGC CTC GTC TCA TA-3′	NM_017008.4
	Reverse	5′-ACC AGC TTC CCA TTC TCA GC-3′

Figure 1. Increased osteoclasts and bone loss in adenine-induced ROD model. (A) Bone histomorphometry data. BV/TV, bone volume/tissue volume; BFR/BS, bone formation rate; Oc.S/BS, osteoclast surface per millimeter bone perimeter; N.Oc/BS, osteoclast number per millimeter bone perimeter; OS/BS, osteoid surface/bone surface; MAR, mineral apposition rate; MLT, mineralization lag time. n = 12-15 per group, ***p < 0.001 or as indicated, student’s t-test. (B) Representative micro-computed tomography images of distal femurs of NC and ROD rats.

Table 2. Serum biochemical parameters of rats.

Parameters	NC (n = 12)	ROD (n = 15)
BUN (mmol/L)	7.77 ± 1.35	13.63 ± 3.09***
Cr (μmol/L)	57.32 ± 12.99	178.37 ± 25.27***
Ca (mmol/L)	2.55 ± 0.21	2.25 ± 0.20**
P (mmol/L)	2.30 ± 0.22	3.48 ± 0.51***
PTH (pg /mL)	54.63 ± 21.00	1025.40 ± 201.16***

Data represent the mean ± SD. NC: normal control group; ROD: renal osteodystrophy group; BUN: blood urea nitrogen; Cr: creatinine; Ca, calcium; P phosphate; PTH: parathyroid hormone. **p < 0.01, ***p < 0.001.

Figure 2. Increased proinflammatory cytokines in BM supernatant and BMMs of ROD rats. (A) ELISA was used to detect the concentrations of IL-1β, IL-6, and TNF-α in the BM supernatant of rats. (B) BMMs from NC or ROD rats were cultured for 24 h followed by RT–PCR of IL-1β, IL-6, and TNF-α. (C) ELISA was used to detect the concentrations of IL-1β, IL-6, and TNF-α in the BMM supernatants of rats in the two groups. (D) BMMs from NC or ROD rats were cultured for 24 h followed by RT–PCR of NF-κB₁ and IκB. Data represent the mean ± SD, n = 6 per group, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 3. Effect of ROD-Exos on the proinflammatory activity of BMMs. (A) Transmission electron microscope images of Exos. (B) Western blot for exosome-associated proteins TSG101 and CD9. (C) The size distribution of Exos was measured by dynamic light scattering. (D, E) BMMs were treated with PBS, NC-Exos or ROD-Exos for 36 h followed by qRT–PCR of IL-1β, IL-6, TNF-α, NF-κB₁, and IκB. Data represent the mean ± SD, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05. (F) PKH26-labeled BM-Exos were cultured with BMMs for 12 h, and uptake was observed using laser confocal microscopy. Blue, DAPI-stained nuclei; red, PKH26-labeled Exos.

Figure 4. Effect of ROD-Exos on osteoclast differentiation of BMMs. (A) Primary BMMs (1 × 10⁵/mL) were cultured with M-CSF and RANKL for 4 days followed by TRAP staining. TRAP⁺ cells with three or more nuclei (black arrows in panel A) were defined as osteoclasts. Magnification: 100×. (B) Normal primary BMMs (1 × 10⁶/mL) were cultured with M-CSF and RANKL and treated with PBS, NC-Exos or ROD-Exos for 6 days followed by TRAP staining. The number of TRAP⁺ cells with three or more nuclei (black arrows in panel B) was counted. Magnification: 200×. (C) BMMs were cultured with M-CSF and RANKL and treated with PBS, NC-Exos or ROD-Exos for 3 days followed by qRT–PCR of osteoclast-specific genes (MMP-9 and CTSK). Data represent the mean ± SD. n = 6 per group (A); n = 3 per group (B, C). ***p < 0.001,**p < 0.01, *p < 0.05.

Figure 5. Profiles for differentially expressed miRNAs. (A) Heatmap of differentially expressed miRNAs between NC-Exos and ROD-Exos. n = 5. (B) The circos plot showing the miRNA-mRNA-pathway network. Red represents upregulated miRNAs, green represents downregulated miRNAs, and black represents target mRNAs. (C) qRT–PCR showing upregulation of miR-9a-5p expression in ROD-Exos. Data represent the mean ± SD, n = 6, **p < 0.01.

Data availability statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.