Myofibroblasts play a central role in renal fibrosis although the origin of these cells remains controversial. We recently reported that bone marrow-derived macrophages can give rise to myofibroblasts through macrophage to myofibroblast transition (MMT). However, several important issues remain to be addressed, including whether MMT occurs in human kidney disease and verification of the MMT process through lineage tracing. Biopsies from a cohort of 58 patients with various forms of kidney disease were examined for MMT cells that co-express macrophage (CD68) and myofibroblast (α-smooth muscle actin, α-SMA) markers. MMT cells were evident in active fibrotic lesions, but were largely absent in acute inflammatory or sclerotic lesions, suggesting that MMT cells contribute to progressive renal fibrosis. Fate-mapping studies in LysMCreTomato mice identified substantial numbers of Tomato+ myeloid cells with F4/80+ macrophage phenotype expressing α-SMA and collagen I in the unilateral ureteral obstructive model of renal fibrosis, providing direct evidence for the MMT process during the development of renal fibrosis. In addition, MMT cells had a predominant M2 phenotype in both human and mouse renal fibrosis. Finally, selective depletion of myeloid cells via diphtheria toxin in LysMCreiDTR mice largely abolished macrophage infiltration and MMT cells in the obstructed kidney and substantially reduced accumulation of α-SMA+ myofibroblasts and collagen deposition, revealing a pathogenic role for inflammatory macrophages in MMT and tissue fibrosis. In conclusion, these findings provide substantial new data to support the postulate that macrophages can directly transdifferentiate into collagen-producing myofibroblasts in human and experimental kidney disease.

Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-β (TGF-β)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-β/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-β1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-β and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-β/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.

Myofibroblasts are a main cell-type of collagen-producing cells during tissue fibrosis, but their origins remains controversial. While bone marrow-derived myofibroblasts in renal fibrosis has been reported, the cell origin and mechanisms regulating their transition into myofibroblasts remain undefined. In the present study, cell lineage tracing studies by adoptive transfer of GFP+ or dye-labelled macrophages identified that monocyte/macrophages from bone marrow can give rise to myofibroblasts via the process of macrophage-myofibroblast transition (MMT) in a mouse model of unilateral ureteric obstruction. The MMT cells were a major source of collagen-producing fibroblasts in the fibrosing kidney, accounting for more than 60% of α-SMA+ myofibroblasts. The MMT process occurred predominantly within M2-type macrophages and was regulated by TGF-β/Smad3 signalling as deletion of Smad3 in the bone marrow compartment of GFP+ chimeric mice prevented the M2 macrophage transition into the MMT cells and progressive renal fibrosis. In vitro studies in Smad3 null bone marrow macrophages also showed that Smad3 was required for TGF-β1-induced MMT and collagen production. In conclusion, we have demonstrated that bone marrow-derived fibroblasts originate from the monocyte/macrophage population via a process of MMT. This process contributes to progressive renal tissue fibrosis and is regulated by TGF-β/Smad3 signalling.

Transforming growth factor-β/Smad signaling plays an important role in diabetic nephropathy. The current study identified a novel Smad3-dependent long noncoding RNA (lncRNA) Erbb4-IR in the development of type 2 diabetic nephropathy (T2DN) in mice. We found that Erbb4-IR was highly expressed in T2DN of mice and specifically induced by advanced glycosylation end products (AGEs) via a Smad3-dependent mechanism. The functional role of Erbb4-IR in T2DN was revealed by kidney-specific silencing of Erbb4-IR to protect against the development of T2DN, such as elevated microalbuminuria, serum creatinine, and progressive renal fibrosis in mice, and to block AGE-induced collagen I and IV expression in mouse mesangial cells (mMCs) and mouse tubular epithelial cells (mTECs). Mechanistically, we identified that the Erbb4-IR-microRNA (miR)-29b axis was a key mechanism of T2DN because Erbb4-IR was able to bind the 3' untranslated region of miR-29b genomic sequence to suppress miR-29b expression at transcriptional level. In contrast, silencing of renal Erbb4-IR increased miR-29b and therefore protected the kidney from progressive renal injury in mice and prevented mTECs and mMCs from AGE-induced loss of miR-29b and fibrotic response in vitro. Collectively, we identify that Erbb4-IR is a Smad3-dependent lncRNA that promotes renal fibrosis in T2DN by suppressing miR-29b. Targeting Erbb4-IR may represent a novel therapy for T2DN.