Glce is significantly reduced in injured human kidneys. It is unclear whether there is an association between Glce and kidney fibrosis. To investigate this, we first examined Glce in renal biopsies from IgA nephropathy (IgA), lupus nephritis (LN), membranous nephropathy (MN), and diabetic nephropathy (DN) subjects compared with minimal-change nephrotic syndrome (MCNS) by immunohistochemistry (IHC) staining (Fig. 1a). Notably, the Glce positive rate was markedly reduced in renal biopsies from patients with different forms of chronic kidney disease compared with MCNS (Fig. 1b). In addition, the results indicated that the decrease of Glce positive rate in patients with advanced CKD was more accentuated except for the LN group (Extended Data Fig. 1a-h). We also observed that the interstitial fibrosis area in the kidney of MCNS patients is significantly less than in other several types of chronic kidney disease (Fig. 1a, c). To confirm the severity of kidney injury, we detected serum creatinine (Scr) and blood urea nitrogen (BUN) as the markers of renal functions20. Results showed that the level of Scr and BUN in the MCNS groups was significantly lower than in other groups (Fig. 1d, e). Furthermore, we found the Glce positive rate was negatively correlated with the interstitial fibrosis area (Fig. 1f), serum creatinine (Fig. 1g), and negatively correlated with blood urea nitrogen in all subjects (Fig. 1h).
Glce deficiency aggravates renal failure and renal-specific AAV-mediated Glce overexpression improves kidney fibrosis. Considering that the Glce decline was significantly associated with almost most of the common chronic kidney diseases, we performed an additional analysis in mice subjected to unilateral ureteral occlusion (UUO) surgery or Folic acid (FA) treatment. The results showed that the mRNA and protein levels of Glce were reduced in the kidney of UUO and FA-treated mice over time (Fig. 2a, b and Supplementary Fig. 2a). Furthermore, immunofluorescent results showed a significant reduction of Glce expression accompanied by an increase in one of the most well-accepted fibrosis markers α-SMA21protein in the kidneys of UUO mice and FA-treated mice (Fig. 2c). To elucidate the role of Glce in kidney fibrosis and injury, we deleted Glce in kidney tubules by cross-breeding Cdh16-Cre and Glceflox/flox mice, which was identified by tail genotyping (Supplementary Fig. 2b). Compared to control (WT/Glceflox/flox) littermates, Cdh16/Glceflox/flox (Glce−/−) mice demonstrated significant retardation of kidney growth at 8 weeks of age (Supplementary Fig. 2c). Interestingly, following UUO surgery some Glce−/− mice displayed abnormal morphology in the contralateral kidney (Fig. 2d). Then we measured serum creatinine and blood urea nitrogen to evaluate renal dysfunction. The results showed that the renal injury was more severe in Glce−/−mice at 7 days or 14 days after UUO (Fig. 2e). Histological analysis indicated significant epithelial atrophy, dilated tubules, and interstitial fibrosis in the UUO group after Glce knockdown (Fig. 2f). The ablation of Glce in tubular cells caused higher gene expression of Acta2, Col1a1, Col3a1, Fn1, Tgfb1 and increased expression of proteins including α-SMA, Vimentin, Collagen I (COL1) compared to that in WT mice after UUO (Fig. 2g, h and Supplementary Fig. 2d). Next, we overexpressed Glce in Cdh16/Glceflox/flox mouse kidney in vivo using adeno-associated viral (AAV) vectors, after which the mice have subjected to UUO or Sham treatment after 6 weeks and sacrificed at 14 days post-UUO (Supplementary Fig. 2e). To verify whether Glce protein was successfully expressed in the mice kidney, we labeled Glce protein with Alexa 594–conjugated secondary antibody (red) and observed both green fluorescent protein (GFP) encoded by AAV vectors and red signals by immunofluorescent analysis (Supplementary Fig. 2f). Interestingly, AAV-directed persistent expression of Glce in the Cdh16/Glceflox/flox mouse kidneys protects against renal fibrosis. The result suggested that Kidney morphology was improved with a decrease in serum creatinine and BUN levels in Glce−/−mice with UUO surgery after the AAV injection (Fig. 2i, j). In addition, the histological staining revealed that the AAV-mGlce group had a more normal appearance with fewer deposition of the collagen fibers in kidney tissue of mice (Fig. 2l). Further investigation manifested that overexpression of Glce prevents the higher gene expression of Acta2, Col1a1, Col3a1, Fn1, Tgfb1 and increased expression of proteins including α-SMA, Vimentin, Collagen I (COL1) compared to the empty vector group mice (AAV-ZsG1) after UUO treatment (Fig. 2k, m and Supplementary Fig. 2g).
Tubule-specific Glce deletion exacerbates EMT progress via the TGF-β signaling pathway. To determine the role of Glce in the resistance to renal fibrosis, short hairpin RNA of Glce (shGlce group) was employed to disrupt the Glce expression, and the control vector was used as negative control (NC group) in HK-2 cells. Our previous study has found that the morphological changes in a part of HK-2 cells from a cobblestone-like appearance to an elongated fibroblast-like shape by the knock-down of Glce expression. Additionally, we also observed the reduced mRNA and protein levels of Glce with increasing induction time by TGF-β treatment (Supplementary Fig. 4a-c). As we know, the microstructural changes observed in renal tubular epithelial cells mean that they might have lost their polarized morphology and acquired mesenchymal features22,23. This phenomenon suggests that Glce may affect the progression of Epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells24. The Western blotting analysis confirmed that the knockdown of Glce in HK-2 cells had profoundly increased expression of EMT marker genes at the protein level, including N-cadherin, Snail, and Slug as well as a diminution of E-cadherin25, compared with controls in response to TGF-β stimulation (Fig. 3a and Supplementary Fig. 3a). In addition, the ablation of Glce in mice kidneys caused a significant elevation of EMT markers such as Snail, Slug, and N-cadherin (Fig. 3b, c and Supplementary Fig. 3b). To further confirm that Glce participates in the renal tubular EMT process, Glce was overexpressed in HK-2 cells. As inferred, the results showed that overexpression of Glce indeed suppressed the expression of Snail, Slug, and N-cadherin at the protein level in HK-2 cells induced by TGF-β (Fig. 3d and Supplementary Fig. 3c). Moreover, we also evaluated the expression levels of EMT-related proteins in mice kidneys. The results showed that the levels of EMT markers expression decreased significantly after AAV encoding full-length mouse Glce injection (Fig. 3e, f and Supplementary Fig. 3d).
To study the potential cellular signaling pathway, we examined the activities of the TGF-β-Smad2/3 pathway, which are known to have a critical role in the EMT of renal tubular cells and the progression of kidney fibrosis26,27. Western blotting analysis showed that tubule-specific Glce deletion promotes the phosphorylation of TGFBR1 and Smad2/3. However, this process can be reversed by overexpression of Glce (Fig. 3g, h, i and Supplementary Fig. 3e, f). We next tried to explore the exact sites regulated by Glce. TGFBR1 inhibitor (SB431542) significantly attenuated TGF-β1-mediated increase in EMT markers expression both in NC and ShGlce groups, while the Smad inhibitor (SIS3) only caused a diminution in NC group HK-2 cells (Fig. 3j). Based on this result, we hypothesize that Glce might modulate EMT through an effect on the phosphorylation of the TGFB1 receptors. To address, we then designed an experimental procedure and treated the animals with a TGFBR1 inhibitor (SB431542) to validate this prediction (Supplementary Fig. 4d). Interestingly, the results showed that the function indexes of the kidney (Scr, BUN) were improved. In addition, renal tissue pathological analysis also revealed that areas of collagen deposition and tubular necrosis became smaller after treatment of SB431542 in the Glce−/− mice group with UUO surgery. But after the UUO operation, the WT mice did not show signs of improvement compared with Glce−/− mice and some individual mice even deteriorated during therapy with SB431542 (Supplementary Fig. 4e, f). Analogously, immunohistochemistry results showed that protein expression of α-SMA, and COL1 decreased in the kidneys of Glce−/− mice after UUO, while no significant changes were found in WT mice (Supplementary Fig. 4g). Western blot assay indicated that SB431542 treatment suppressed the phosphorylation of TGFBR1, Smad2/3, and the protein level of N-cadherin, Snail, and Slug in both groups of mice. As the disease progressed, obstructed kidneys of the Glce−/−mice maintained the lower expression levels of Snail1, and Cdh2 genes compared with the WT mice treated with SB431542 (Supplementary Fig. 4h-l).
The Glce binds to EGFR to suppress its activation and impact on renal fibrosis.
The previous sections have shown that Glce played vital role in the regulation of EMT via the TGFβ1/Smad pathway. However, upon further evaluation, we found there’s no direct interaction between Glce and TGFBR1 (Unpublished data). It has been reported that the enhanced activation of the EGF receptor (EGFR) associated with the development and progression of renal fibrosis and genetic or pharmacologic blockade of EGFR could inhibit renal fibrosis28. To explore whether EGFR might be a target of the Glce protein for the possible treatment of renal fibrosis, we firstly carried out the docking analysis between these two proteins. Surprisingly, docking results revealed that K264, D297, and T349 on the Glce protein (PDB code: 6HZZ) could interact with the cytoplasmic domain of EGFR (Fig. 4a). Next, to further confirm the result, a surface plasmon resonance (SPR) screen was employed to identify the promising targets. After a preliminary screen and subsequent validation, we eventually indeed found that the Glce protein had a strong binding interaction with the intracellular domain of EGFR however did not bind to the EGFR extracellular domain. As a positive control, N-Sulfated K5 polysaccharide, a classical substrate29 for the Glce enzyme was used (Fig. 4c). To examine whether the interaction between EGFR and the Glce protein really happens in the cells system, Co-IP assays was further employed. Indeed, the results suggested that the Glce interacted with EGFR in HK-2 cells. However, when EGFR was activated by EGF protein we observed that the combination of both declined obviously (Fig. 4b). Confocal immunofluorescence analysis also showed the Glce/EGFR colocalization in the HK-2 cells (Fig. 4d). More interestingly, Glce-knockdown caused an increase in the phosphorylation of EGFR in renal tubular epithelial cells (Fig. 4e). To study the effect of Glce on the MAPK pathway, we assessed the activation of the pathway in the Glce knockdown and overexpressing cell lines. The results confirmed that the Glce knockdown might activate EGFR/ERK pathways, however the overexpression of Glce did not affect EGFR phosphorylation in HK-2 cells (Fig. 4f, g, h, i). Consistently, the phosphorylation of EGFR showed a significant increase in the kidneys of UUO-treated Glce−/− mice compared with the control group. However, after AAV encoding full-length Glce injection, the activation of EGFR could be impaired obviously in Glce−/− obstructed kidneys (Fig. 4j, k). To further investigate whether the binding between EGFR and the Glce protein, the kidneys of model mice by coimmunoprecipitation analysis were performed. Compared with the Sham group overexpressing of AAV-mediated Glce, the interaction between Glce protein and EGFR was markedly decreased in the mice kidneys after UUO treatment. Moreover, the Glce overexpression in the mice with tubule-specific deletion of Glce significantly prevents EGFR activation both in WT and Glce−/− groups (Fig. 4l).
The MAPK pathways have been reported to play a vital role in the EMT of different epithelial cells in vitro30, as well as the progression of renal fibrosis in vivo31. Moreover, previous studies had shown that TGF-β-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation32. To explore the effects of Glce on pathogenesis and progression of renal fibrosis in vivo through the EGFR/ERK signaling pathway, an EGFR inhibitor erlotinib was employed following UUO surgery. Histological analysis indicated significant epithelial atrophy, dilated tubules, and interstitial fibrosis after UUO, of which Glce−/− mice performed more severe changes. After erlotinib administration for 14 days, the renal damage was significantly ameliorated (Fig. 5a). Additionally, the biochemical assays demonstrated that levels of serum creatinine and blood urea nitrogen was increased by varying degrees in UUO-treated animals, however the levels were significantly decreased in both groups that receiving erlotinib treatment (Fig. 5b). To assess the involvement of the MAPK pathway and TGF-β-induced EMT in accelerated fibrotic response in mice upon UUO in vivo, we first analyzed the expression levels of p-EGFR, EGFR, p-ERK1/2, ERK1/2 (Fig. 5c, d) and fibrosis-related protein including COL1, α-SMA, vimentin in the kidneys of both WT and Glce−/− mice. As inferred, we found a marked increase of p-EGFR, p-ERK1/2, COL1, α-SMA, and vimentin in obstructed kidneys of animals after UUO. Compared with WT mice, UUO-treated Glce−/− mice showed further increased protein expression. Consistently, enhanced expression of TGF-β1, p-Smad2/3, and its downstream EMT markers such as Snail, Slug induced by UUO surgery were markedly decreased after administration of erlotinib (Fig. 5e, f). Therefore, we concluded that the genetic deletion of Glce promoted the activation of the EGFR signal pathway. Subsequently, aberrant activation of EGFR resulted in increased expression of TGF-β1 and then exacerbated the EMT process via SMAD-dependent pathways in kidneys (Fig. 7).
Adeno-associated virus encoding mutant Glce orthotopically injected to Glce−/− mice still can ameliorate renal fibrosis. According to SPR results, the Glce protein may robustly interacted with the intracellular region of EGFR. Further study revealed that the potential EGFR binding regions in human Glce protein might mostly clustered in the classical β-sandwich domain by the molecular docking analysis. Interestingly, we noticed that the binding sites were not the active site of the Glce which is well known as a classical enzyme. To determine impact of the Glce enzymatic function on kidney fibrosis, we next chose three sites that were considered crucial for the enzyme activity including Y500, Y560, and Y578, respectively for mutational studies12. HK-2 cells with stable Glce knockdown were transfected with plasmids containing mutant genes. The protein expression level of p-EGFR, p-MEK1/2, and p-ERK1/2 was markedly higher in the Glce knockdown HK-2 cells however mutant Glce showed no effect on the EGFR/ERK pathway (Fig. 6a). The immunofluorescence examination further demonstrated that mutant Glce was expressed successfully and distributed in the cytoplasm of renal tubular cells besides Golgi (Fig. 6b). Furthermore, to understand the therapeutic potential of mutant Glce, we constructed the adeno-associated virus vectors encoding GFP as well as all three mutant sites (mutGlce) simultaneously and then injected them orthotopically into the kidney of Glce−/− mice. Empty vector plasmid (ZsG1) was utilized as a control. Immunofluorescence staining results showed that the mutant Glce protein (red) marked by a corresponding antibody was overexpressed in renal tubular epithelial cells (Fig. 6c). Compared with those injected with empty vector plasmid (AAV-ZsG1), the overexpression of mutant Glce (AAV- mutGlce) resulted in a lower level of Scr and BUN. H&E and Masson staining showed that both tubular damage and the degree of interstitial fibrosis induced by UUO were improved in AAV- mutGlce mice. (Fig. 6d, e). Additionally, the reduction in COL1, α-SMA, Vimentin, and Fibronectin as well as the elevation of Glce, and E-cadherin were further confirmed by Western blotting (Fig. 6f and Supplementary Fig. 5a). Expression of TGF-β, p-Smad2/3, p-EGFR, p-MEK1/2, and p-ERK1/2; EMT markers such as Snail, Slug, and N-cadherin were markedly lower in AAV- mutGlce knockout mice (Fig. 6g, h and Supplementary Fig. 5b, c). Consistent with the previous results, the immunofluorescence analysis also showed co-location of EGFR (yellow) and mutant Glce (red) in mice kidneys (Fig. 6i). Taken together, AAV- mutGlce mice challenged with UUO showed better tubular health and renal function, and less interstitial fibrosis.