Verteporfin inhibits the dedifferentiation of tubular epithelial cells via TGF-ß1/Smad pathway but induces podocyte loss in diabetic nephropathy.

AIMS: The dedifferentiation of tubular epithelial cells has been identified as an important trigger of renal fibrosis. The Hippo pathway is a crucial regulator of cell proliferation and differentiation. In this study, we determined the role of Hippo proteins in tubular dedifferentiation in diabetic nephropathy (DN). MAIN METHODS: In this study, we measured dedifferentiation markers and Hippo proteins in db/db mice and high glucose treated tubular epithelial cells. Then, verteporfin and knockdown of large tumor suppressor kinase (LATS) 1 and 2 were performed to uncover therapeutic targets for DN. KEY FINDINGS: Here, we found dedifferentiation and upregulated Hippo proteins in tubular epithelial cells in DN model both in vivo and in vitro. Both verteporfin and LATS knockdown could inhibit the tubular mesenchymal transition, but verteporfin showed broad inhibitory effect on Hippo proteins, especially nuclear YAP, and exacerbated podocyte loss of DN. LATS2 knockdown did not reverse the tubular E-Cadherin loss while it also induced podocyte apoptosis. Overall, intervention of LATS1 inhibited tubular dedifferentiation efficiently without affecting YAP and bringing podocyte apoptosis. Further mechanistic investigations revealed that the TGF-ß1/Smad, instead of the YAP-TEAD-CTGF signaling, might be the underlying pathway through which verteporfin and LATS1 engaged in the tubular dedifferentiation. SIGNIFICANCE: In conclusion, verteporfin is not a suitable treatment for DN owing to evitable podocyte loss and apoptosis. Targeting LATS1 is a better choice worthy of further investigation for DN therapy.

TGF-ß1 inhibits the autophagy of podocytes by activating mTORC1 in IgA nephropathy.

IgA nephropathy (IgAN) is a mesangial proliferative glomerulonephritis which often shows proteinuria, an indicator for podocyte damage. TGF-ß1 has been known to contribute to podocyte injury by inducing apoptosis, cytoskeleton relocation or cytoskeleton loss. And Decorin, a small proteoglycan known to neutralize TGF-ß1, was reported to induce autophagy in vascular endothelial cells. However, it remains unknown how TGF-ß1 and Decorin can affect podocyte autophagy in mesangial proliferative glomerulonephritis. In this study, we used in vivo and in vitro models to find out the effect of TGF-ß1 and Decorin on podocyte autophagy. P-rpS6 and p-ULK1 were detected by Western blot to show the activation of mTORC1 pathway following TGF-ß1 treatment. Also, we collected serum from IgAN patients and anti-Thy1.1 nephritis, and quantified TGF-ß1 and Decorin using ELISA. Together, we showed that TGF-ß1 could activate mTORC1 and inhibit autophagy, while Decorin has precisely the opposite effect. As the mesangial cells (MCs) proliferate, TGF-ß1 increases and Decorin decreases in the serum of IgAN and anti-Thy1.1 nephritis. This finding deepened our understanding regarding how MC proliferation could finally result in podocyte dysfunction.

Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.

Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga(3+) and Fe(3+) for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga(3+)-Fe(3+)-immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5-3.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe(3+), Ti(4+), Ga(3+), and Al(3+)). In addition, up to 92% of the 6283 phosphopeptides were uniquely enriched in either the first Ga(3+)-IMAC (41%) or second Fe(3+)-IMAC (51%). The complementary properties of Ga(3+) and Fe(3+) were further demonstrated through the exclusive enrichment of almost all of 1214 multiply phosphorylated peptides (99.4%) in the Ga(3+)-IMAC, whereas only 10% of 5069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga(3+)-Fe(3+)-IMAC to human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlap. In addition to the above-mentioned mono- and multiply phosphorylated peptides, this fractionation ability was also demonstrated on the basic and acidic phosphopeptides: acidophilic phosphorylation sites were predominately enriched in the first Ga(3+)-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe(3+)-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to cancer invasion and metastasis of lung cancer. Given the fractionation ability and ease of tip preparation of this Ga(3+)-Fe(3+)-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.