Elevated expression of MCM6 in fibrotic kidneys and TECs
To determine the expression pattern of MCMs proteins in the kidney during renal fibrogenesis, we first examined the expression of the MCMs in patients with CKD using the nephroseq website (http://nephroseq.org). We found that the expression of MCM3, MCM5, MCM6, and MCM7 was higher in patients with CKD (Fig. 1A). To confirm the role of the MCM family in the pathogenesis of CKD, two unrelated in vivo fibrotic models—UUO and UIRI—were used to establish chronic kidney fibrosis models. We examined the mRNA level of the MCM family in fibrotic kidneys using qRT-PCR and found that the mRNA level of MCM6 was significantly upregulated in both fibrotic models (Fig. 1B). Notably, compared with that in normal kidney tissues, MCM6 was markedly increased in the tubulointerstitial area in UUO mice as observed in the immunohistochemistry results (Fig. 1C-D). The western blotting assay also showed increased MCM6 level in UUO-induced renal fibrosis, accompanied by upregulation of fibronectin and a-SMA levels (Fig. 1E-F). To confirm the renal localization of MCM6, we dually stained cortex sections for MCM6 and Lotus tetragonolobus lectin (LTL, a proximal tubular marker) and showed that LTL-positive proximal tubular cells (green) co-localized with MCM6 (red), suggesting that the increase in MCM6 mainly occurred in proximal TECs post obstruction injury (Fig. 1G). Consistently, MCM6 was also significantly increased in UIRI-induced renal fibrosis, as determined through immunohistochemistry (Fig. 1H-I) and immunoblotting analyses (Fig. 1J-K). Additionally, to determine whether the upregulation of MCM6 is secondary to tubular epithelial injury, we treated proximal TECs (NRK-52E) with TGF-β1 or H/R stimulation. We found that MCM6 expression began to increase 12 h after TGF-β1-treatment in NRK-52E cells, which preceded the accumulation of fibronectin, vimentin, and a-SMA—the three classic markers of tubular EMT (Fig. 1L-M). Similarly, MCM6 levels were significantly upregulated by H/R stimulation (Fig. 1N-O). In summary, these results indicate that renal fibrosis is associated with an increase in MCM6 expression in the tubulointerstitial area, particularly in proximal TECs.
MCM6 deficiency promoted partial EMT program in TECs
To explore the possible role of MCM6 in renal fibrosis, we disrupted the expression of MCM6 using siRNA. MCM6 expression was dramatically decreased, as determined using immunoblotting (Fig. 2A-B) and qRT-PCR analyses (Fig. 2C). TGF-β1 is a critical mediator implicated in renal TIF; therefore, we evaluated whether MCM6 expression had an effect on the partial EMT process in TGF-β1-treated TECs. As shown in Fig. 2D, TECs exhibited the loss of cell–cell contacts and their typical epithelial cell morphology and acquired the elongated spindle-shaped mesenchymal phenotype after incubation with 5 ng/mL of TGF-β1 for 48 h. MCM6 deficiency aggravated these morphological changes in TECs. Simultaneously, partial EMT induction was confirmed through the loss of E-cadherin and accumulation of fibronectin, α-SMA, and vimentin in response to TGF-β1 stimulation, whereas silencing of MCM6 further promoted the tubular EMT process (Fig. 2E-F). Immunofluorescence data further revealed notable upregulation of fibronectin and α-SMA in TGF-β1-treated TECs (Fig. 2G). Consistent with TGF-β1-induced profibrotic changes, MCM6 levels showed significant increase in the TECs under H/R stimulation accompanied by the induction of mesenchymal markers (Fig. 2H-I). These results indicate that MCM6 is involved in the tubular partial EMT process, ECM deposition, and myofibroblast activation during the progression of renal fibrosis.
In vivo knockdown of MCM6 aggravates UUO-induced renal fibrosis
To further investigate the role of MCM6 in renal fibrogenesis, AAV harboring MCM6 (AAV-sh-MCM6) was delivered into mouse kidneys via intraparenchymal injection to knock down the expression of MCM6 in renal tubules before establishing the UUO model (Fig. 3A). Renal expression of MCM6 was robustly blocked in UUO-induced mice that received AAV-sh-MCM6 as evidenced by the results of immunohistochemistry and western blotting analyses (Fig. 3B-E). Notably, HE and Masson’s trichrome staining showed that delivery of AAV-sh-MCM6 resulted in more significant tubular injury and fibrotic lesions than that induced by the delivery of negative-control AAV (AAV-sh-Ctrl). Examination of MCM6 and fibrotic markers showed that delivery of AAV-sh-MCM6 significantly reduced MCM6 expression, whereas it led to an increase in fibronectin, vimentin, and a-SMA expression in the obstructed kidneys (Fig. 3B-G). To further explore the role of MCM6 in tubular damage, we detected the expression of E-cadherin—an epithelial marker that maintains normal epithelial integrity—and found that MCM6 deficiency led to a significant loss of E-cadherin in the tubules (Fig. 3F-G). Furthermore, a-SMA-positive myofibroblasts and ECM accumulation were greatly increased in the interstitium of the obstructed kidney in AAV-sh-MCM6 group mice compared with that in the AAV-sh-Ctrl group mice (Fig. 3H). These data suggest that MCM6 plays an important role in tubular injury, partial EMT, myofibroblast activation, ECM accumulation, and eventually in renal fibrogenesis.
Renal tubular-specific knockout of MCM6 exacerbated renal fibrosis in UIRI model mice
To further confirm the role of MCM6 in renal fibrosis, AAV-sh-MCM6 virions were delivered into mouse kidneys before establishing the UIRI model (Fig. 4A). As shown in Fig. 4B-E, the injured kidney displayed typical features of renal fibrosis after UIRI, whereas knockdown of MCM6 expression further aggravated tubular damage and atrophy, severity of TIF, and area of interstitial fibrosis. When examining the mesenchymal markers, we found that MCM6 ablation remarkably decreased MCM6 in renal TECs but contributed to the accumulation of fibronectin, α-SMA, and vimentin in the tubulointerstitial area as observed using immunostaining (Fig. 4B- E). Immunoblotting data further validated the changes in these fibrosis markers, which were accompanied by decreased E-cadherin levels (Fig. 4F-G). Meanwhile, α-SMA-positive myofibroblasts and ECM accumulation greatly increased in the interstitium of AAV-sh-MCM6 group compared with that in the negative-control group (Fig. 4H). These results showed that MCM6 deficiency accentuated renal fibrosis in UIRI mice, which was consistent with the results observed in the UUO model mice. This demonstrates the important role of MCM6 in renal fibrogenesis.
In vivo overexpression of MCM6 alleviates UUO-induced renal fibrosis
To clarify the therapeutic efficacy of targeting MCM6 in the progression of renal fibrosis, AAV harboring MCM6 (AAV-OE-MCM6) was delivered into mouse kidneys via intraparenchymal injection to overexpress MCM6 in renal tubules before establishing the UUO model (Fig. 5A). Immunohistochemistry and western blotting analyses showed that kidney MCM6 expression greatly increased in the mice with AAV-OE-MCM6 (Fig. 5B-G). Notably, the obstructed kidneys displayed typical features of renal fibrosis following UUO, and overexpression of MCM6 significantly decreased the severity of TIF and the area of interstitial fibrosis (Fig. 5B-D). Immunostaining and immunoblotting results showed that the increased levels of fibronectin, vimentin, and a-SMA were prominently attenuated in UUO-induced mice, particularly in the tubulointerstitial area, after AAV-OE-MCM6 delivery (Fig. 5B- G). Overexpression of MCM6 notably inhibited the loss of E-cadherin and restored epithelial integrity, indicating that tubular MCM6 protects against tubular injury and TIF during the progression of renal fibrosis. Immunofluorescence data further revealed that a-SMA-positive myofibroblasts and ECM accumulation were dramatically decreased in the AAV-OE-MCM6 group compared with that in the negative-control group (Fig. 5H). These data further confirmed the essential roles of MCM6 such as maintaining epithelial integrity and tubular function, protecting against tubular injury or dedifferentiation, and eventually involving in fibrogenesis.
Overexpression of MCM6 suppresses renal fibrosis in UIRI mice
To further examine whether MCM6 expression was associated with renal fibrosis, AAV-OE-MCM6 virions were delivered into mouse kidneys to overexpress MCM6 before establishing the UIRI model (Fig. 6A). As shown in Fig. 6B–E, the extent of UIRI-induced tubular damage and interstitial area was substantially alleviated after the delivery of AAV-OE-MCM6. Moreover, compared with that in the negative control mice, kidney MCM6 expression was greatly elevated in AAV-OE-MCM6 mice, whereas the increase in the levels of fibronectin, vimentin, and a-SMA were reversed (Fig. 6B-G). Overexpression of MCM6 significantly inhibited myofibroblast activation and ECM deposition in UIRI-induced renal fibrosis (Fig. 6H), which was consistent with the results observed in the UUO model mice. These results further exhibited a potent effect of MCM6 on attenuating fibrotic response in renal fibrogenesis.
MCM6 modulates the activation of ERK/GSK-3β/Snail1 signaling pathway in tubular partial EMT progress
Snail1—a prominent inducer of EMT—is a highly labile protein and a critical transcriptional regulator of E-cadherin that represses epithelial-related gene expression and contributes to the mesenchymal phenotype[16]. To explore the potential cellular signaling pathways, we examined GSK-3β-mediated Snail activity and the ERK pathway, which are known to play pivotal roles in the EMT of renal tubular cells and the progression of kidney fibrosis. As shown in Fig. 7A-D, compared with that in the sham group, upregulation of phospho-ERK, phospho-GSK-3β, and Snail1 was observed in UUO mice, and these increases were further elevated in the kidneys of UUO mice following the delivery of AAV-sh-MCM6; however, these increases were markedly inhibited in the kidneys of UUO mice that were delivered AAV-OE-MCM6. Additionally, induction of phospho-ERK and phospho-GSK-3β were observed in vitro in TECs treated with TGF-β1 for 1 h, and the expression of Snail1 increased in TECs treated with TGF-β1 for 48 h. MCM6 deficiency further aggravated the increase in phospho-ERK, phospho-GSK-3β, and Snail1 expression in TECs upon TGF-β1 stimulation (Fig. 7E-H). To assess whether ERK signaling and GSK-3β activity were implicated in MCM6-induced partial EMT process, a MEK1/2 inhibitor U0126 and a GSK-3β inhibitor SB216763 were used alone or combined in TECs to block ERK signaling and GSK-3β activity. As shown in Fig. 7I-K, pretreatment with U0126 blocked the increases in phospho-ERK, phospho-GSK-3β, and Snail1 levels, pretreatment with SB216763 suppressed the increase in phospho-GSK-3β and Snail1 levels, and co-pretreatment with U0126 and SB216763 further inhibited the expression of Snail1, in MCM6-deficient TECs. Collectively, our data demonstrate the involvement of ERK/GSK-3β/Snail1 signaling in MCM6-induced tubular partial EMT and renal fibrosis.
DUSP6 is downregulated in renal fibrosis and associates with the activation of ERK signaling in MCM6-mediated partial EMT progress
Dual-specificity phosphatases (DUSPs), which belong to the family of protein tyrosine phosphatases, are expressed in various types of kidney cells at different levels and have been implicated in various kidney diseases[25]. DUSP5 and DUSP6 are negative regulators of ERK signaling[26]. Based on data from the website of nephroseq, we found that the expression of DUSP5 and DUSP6 (especially that of DUSP6) was notably decreased in various types of human CKD including IgA nephropathy, focal segmental glomerulosclerosis, membranous glomerulonephropathy, minimal change disease, and thin basement membrane disease (Fig. 8A). To clarify the expression of DUSP5 and DUSP6 in CKD, we examined their mRNA levels in fibrotic kidneys by qRT-PCR assay and found that compared with that of DUSP5, the mRNA level of DUSP6 was more significantly downregulated in mice with UUO and UIRI (Fig. 8B). Therefore, we mainly discuss the role of DUSP6 in renal fibrosis and whether its expression is mediated by MCM6. As shown in Fig. 8C-D, compared with that in normal kidney tissues, DUSP6 expression was dramatically decreased in fibrotic kidneys of mice with UUO and UIRI. And immunostaining data further confirmed this result (Fig. 8E). Moreover, TGF-β1 and H/R treatment led to a significant decrease in DUSP6 expression in TECs (Fig. 8F-G), suggesting a potential role for DUSP6 in renal fibrogenesis. To corroborate the potential role of DUSP6 in renal fibrosis mediated by MCM6, we detected the expression of DUSP6 in fibrotic kidneys of mice with MCM6 deficiency or MCM6 overexpression. And found that DUSP6 expression was further decreased in the kidneys of UUO mice injected with AAV-sh-MCM6; however, this decrease was significantly reversed in the kidneys of UUO mice injected with AAV-OE-MCM6 (Fig. 8H-K). In summary, our results reveal the dysregulation of endogenous DUSP6 levels in kidney fibrosis and suggest that DUSP6 possibly plays a role in renal fibrogenesis, which is at least in part mediated by MCM6.