Exogenous miR-29a Attenuates Muscle Atrophy and Kidney Fibrosis in Unilateral Ureteral Obstruction Mice

Abstract

Renal fibrosis leads to end-stage renal disease, but antifibrotic drugs are difficult to develop. Chronic kidney disease often results in muscle wasting, and thereby increases morbidity and mortality. In this work, adeno-associated virus (AAV)-mediated overexpressing miR-29a was hypothesized to counteract renal fibrosis and muscle wasting through muscle-kidney crosstalk in unilateral ureteral obstruction (UUO) mice. miR-29a level was downregulated in the kidney and skeletal muscle of UUO mice. The secretion of exosome-encapsulated miR-29a increased in cultured skeletal muscle satellite cells and HEK293 renal cells after stimulation with serum from UUO mice. This result was confirmed by qPCR and microRNA deep sequencing in the serum exosomes of mice with obstructed ureters. A recombinant AAV-miR-29a was generated to overexpress miR-29a and injected into the tibialis anterior muscle of the mice 2 weeks before UUO surgery. AAV-miR-29a abrogated the UUO-induced upregulation of YY1 and myostatin in skeletal muscles. Renal fibrosis was also partially improved in the UUO mice with intramuscular AAV-miR-29a transduction. AAV-miR-29a overexpression reversed the increase in transforming growth factor β, fibronectin, alpha-smooth muscle actin, and collagen 1A1 and 4A1 levels in the kidney of UUO mice. AAV-green fluorescent protein was applied to trace the AAV route in vivo, and fluorescence was significantly visible in the injected/uninjected muscles and in the kidneys. In conclusion, intramuscular AAV-miR-29a injection attenuates muscle wasting and ameliorates renal fibrosis by downregulating several fibrotic-related proteins in UUO mice.

Keywords: MuRF1; TGF-β1; TGF-β3; YY1; kidney fibrosis; αSMA.

Figure 1.

miR-29a-3p levels increased in the serum exosome of UUO mice. (A, B) Serum exosome size and concentration were measured by NanoSight Instruments in 7d UUO mice and sham mice (n = 5/group). (C) Expression and bar graph of Tsg101 in 7d UUO mice and sham mice (n = 9/group). (D) Heat map graph showing that miR-29a increased in serum exosomes from UUO mice compared with controls at 7 and 14 days. (E) Expression of miR-29a-3p/miR-103 (n = 9/group). 7d UUO, 7 days unilateral ureteral obstruction. Color images are available online.

Figure 2.

Provision of AAV-miR29a attenuated UUO-induced muscle loss. (A) Representative frozen cross-sections from TA muscles transduced with AAV in the normal control mice. The right panels show fluorescence microscopy images for detecting GFP expression; the left panels show bright-field microscopic images of muscle cross-section. The top panels show microscopy images in mice with AAV-miR-29 transduction. The bottom panels show microscopy images in mice with AAV-GFP transduction. (B) miR-29a/U6 expression in the TA muscle (n = 9/group). (C) miR-29a/U6 expression in the uninjected TA (Uni-TA), soleus, and EDL (n = 9/group). (D) Body weights and (E) muscle weights of sham-operated and UUO cohorts (n = 9/group). (F) Expression of YY1, myoD, myogenin, MuRF1, PTEN, and GAPDH. The bar graph shows the fold change of each protein band compared with the levels in sham mice (represented by a line at onefold) (n = 6/group). (G) The representative cross-sectional area of TA muscle of sham/ctr and UUO cohorts (n = 9/group). AAV, adeno-associated virus; EDL, extensor digitorum longus; GFP, green fluorescent protein; TA, tibialis anterior; UUO, unilateral ureteral obstruction. Color images are available online.

Figure 3.

Exogenous miR-29a in skeletal muscle can attenuate fibrosis progression in the UUO kidney. (A) Masson's trichrome staining of paraffin sections from kidneys of various groups as indicated. The bar graph shows the collagen amount (n = 6). (B) Immunostaining for fibronectin and bar graph showing the amounts of fibronectin in various groups as indicated (n = 6/group). (C) TGF-β1, TGF-β3, YY1, and collagen 1A1 expression in kidney lysates from different groups of mice. The bar graph shows the fold change of each protein band compared with levels in sham mice (represented by a line at onefold). (n = 9/group). TGF-β, transforming growth factor β; UUO, unilateral ureteral obstruction. Color images are available online.

Figure 4.

Evidence of miR-29a travel from the muscle to the kidney through exosome circulation of exosomes. (A) miR-29a/miR103 expression and bar graph showing miR expression in the serum exosomes of each group of mice. (n = 6/group). (B) Expression and bar graph of miR-29a/U6 in the right and left kidneys of each group of mice (n = 6/group). (C) Representative fluorescent organ images. Mice were injected in the left TA muscle with AAV/miR-ctrl (GFP positive). The fluorescence was assessed 14 days after injection. In each pair, the left kidney received UUO ligation (Obs = obstructed kidney), and the right was not obstructed (Uno = unobstructed kidney). The left TA muscle was transduced by AAV (Inj = injected muscle) and the right muscle was not (Uni = uninjected). Bar graph showing GFP intensity from AAV-GFP transduced mice compared with non-AAV injection mice (n = 3/group). AAV, adeno-associated virus; GFP, green fluorescent protein; TA, tibialis anterior; UUO, unilateral ureteral obstruction. Color images are available online.