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Background: Renal allograft interstitial fibrosis/tubular atrophy (IF/TA) constitutes the principal histopathological characteristic of chronic allograft dysfunction (CAD) in kidney‐transplanted patients. While renal vascular endothelial‐mesenchymal transition (EndMT) has been verified as an important contributing factor to IF/TA in CAD patients, its underlying mechanisms remain obscure. Through single‐cell transcriptomic analysis, we identified Rictor as a potential pivotal mediator for EndMT. This investigation sought to elucidate the role of Rictor/mTORC2 signalling in the pathogenesis of renal allograft interstitial fibrosis and the associated mechanisms. Methods: The influence of the Rictor/mTOR2 pathway on renal vascular EndMT and renal allograft fibrosis was investigated by cell experiments and Rictor depletion in renal allogeneic transplantation mice models. Subsequently, a series of assays were conducted to explore the underlying mechanisms of the enhanced mitophagy and the ameliorated EndMT resulting from Rictor knockout. Results: Our findings revealed a significant activation of the Rictor/mTORC2 signalling in CAD patients and allogeneic kidney transplanted mice. The suppression of Rictor/mTORC2 signalling alleviated TNFα‐induced EndMT in HUVECs. Moreover, Rictor knockout in endothelial cells remarkably ameliorated renal vascular EndMT and allograft interstitial fibrosis in allogeneic kidney transplanted mice. Mechanistically, Rictor knockout resulted in an augmented BNIP3‐mediated mitophagy in endothelial cells. Furthermore, Rictor/mTORC2 facilitated the MARCH5‐mediated degradation of BNIP3 at the K130 site through K48‐linked ubiquitination, thereby regulating mitophagy activity. Subsequent experiments also demonstrated that BNIP3 knockdown nearly reversed the enhanced mitophagy and mitigated EndMT and allograft interstitial fibrosis induced by Rictor knockout. Conclusions: Consequently, our study underscores Rictor/mTORC2 signalling as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis progression, exerting its impact through regulating BNIP3‐mediated mitophagy. This insight unveils a potential therapeutic target for mitigating renal allograft interstitial fibrosis. [ABSTRACT FROM AUTHOR]

Summary: Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP‐β‐l‐rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP‐β‐l‐rhamnose. The mutant H357Q exhibited activity with both UDP‐β‐l‐rhamnose and UDP‐glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor‐His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism. Significance Statement: The crystal structures of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana revealed the key structural features and residues for sugar donor recognition and specificity for UDP‐β‐l‐rhamnose. UGT89C1 utilizes His21 as the only catalytic residue for its catalysis as it lacks the other catalytic residue Asp, which is highly conserved in other reported UGTs, suggesting that His21 forms a catalytic dyad with acceptor to facilitate the UGT89C1‐mediated rhamnosylation. [ABSTRACT FROM AUTHOR]

Background: Glomerular filtration rate (GFR) is a preferred indicator of allograft renal function, but direct measurement of GFR remains complicated.Purpose: To prospectively compare dynamic contrast-enhanced MR renography (DCE-MRR) with 99m Tc-DTPA-based single-photon emission computed tomography (SPECT) for determination of allograft renal function.Study Type: Prospective.Population: Seventy kidney-transplant recipients FIELD STRENGTH: A low-dose DCE-MRR with a 3.0T scanner and a 99m Tc-DTPA-based SPECT after renal transplantation were performed.Assessment: A Baumann-Rudin (BR) and a modified two-compartment model (JZ2C) were used for DCE-MRR analysis. Standard Gate's method was used for SPECT analysis. An endogenous creatinine clearance rate (CCr) constituted the reference standard.Statistical Tests: Pearson correlation test and Bland-Altman agreement analysis.Results: The reference CCr-GFR was 59.58 ± 23.72 mL/min/1.73 m2 . GFR determined by eGFR, BR, JZ2C, and SPECT was 90.22 ± 34.38, 36.78 ± 14.46, 48.99 ± 23.88, and 67.32 ± 18.44 mL/min/1.73 m2 , respectively. DCE-MRR using JZ2C had the best overall performance, with a Pearson correlation coefficient of 0.81, a bias of -10.58 mL/min/1.73 m2 , and a precision of 14.61 mL/min/1.73 m2 , as well as high accuracy (30-50% intervals: 74.3-90.0%). Although SPECT had a small bias (7.74 mL/min/1.73 m2 ), it had a poor correlation coefficient (0.38), poor precision (23.93 mL/min/1.73 m2 ), and low accuracy (64.3-72.3%) as compared with DCE-MRR using JZ2C.Data Conclusion: DCE-MRR using JZ2C is superior to 99m Tc-DTPA-based SPECT to determine allograft renal function.Level Of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:262-269. [ABSTRACT FROM AUTHOR]

Chronic allograft dysfunction (CAD) poses a significant challenge in kidney transplantation, with renal vascular endothelial‐to‐mesenchymal transition (EndMT) playing a vital role. While renal vascular EndMT has been verified as an important contributing factor to renal allograft interstitial fibrosis/tubular atrophy in CAD patients, its underlying mechanisms remain obscure. Currently, Src activation is closely linked to organ fibrosis development. Single‐cell transcriptomic analysis in clinical patients revealed that Src is a potential pivotal mediator in CAD progression. Our findings revealed a significant upregulation of Src which closely associated with EndMT in CAD patients, allogeneic kidney transplanted rats and endothelial cells lines. In vivo, Src inhibition remarkably alleviate EndMT and renal allograft interstitial fibrosis in allogeneic kidney transplanted rats. It also had a similar antifibrotic effect in two endothelial cell lines. Mechanistically, the knockout of Src resulted in an augmented AMBRA1‐mediated mitophagy in endothelial cells. We demonstrate that Src knockdown upregulates AMBRA1 level and activates mitophagy by stabilizing Parkin's ubiquitination levels and mitochondrial translocation. Subsequent experiments demonstrated that the knockdown of the Parkin gene inhibited mitophagy in endothelial cells, leading to increased production of Interleukin‐6, thereby inducing EndMT. Consequently, our study underscores Src as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis, exerting its impact through the regulation of AMBRA1/Parkin‐mediated mitophagy. [ABSTRACT FROM AUTHOR]