Tandem "One-Shot" Measurement of Residual Chemical Shift Anisotropy and Residual Dipolar Coupling using Biphasic Supramolecular Peptide Liquid Crystals.

Both solitary and tandem applications of residual chemical shift anisotropy (RCSA) and residual dipolar coupling (RDC) show great potential for the structural and configurational determination of organic molecules. A critical component of both RDC and RCSA methodologies is the alignment medium, whose availability is limited, especially for RCSA measurement. Moreover, reported RDC and RCSA acquisitions mainly rely on two experiments conducted under two different conditions, which are relatively time-consuming and easily cause experimental errors. Herein, a biphasic supramolecular lyotropic liquid crystalline (LLC) system was developed through the self-assembly of C21H43-CONH-V4K3-CONH2, which could act as an alignment medium for not only RDC but also RCSA extraction in DMSO-d6. Notably, the RCSA extraction was easily achieved via one-shot measurement from a single one-dimensional 13C NMR experiment, with no need for special instruments, devices, and correction. Relying on the biphasic LLC medium, meanwhile, RDC data were simply extracted from a single F1-coupled HSQC experiment, different from the standard protocol that requires two spectral acquisitions corresponding to the isotropic and anisotropic conditions. Collectively, the biphasic LLC medium is applicable for tandem RCSA and RDC measurements in one single sample, advancing the stereochemical elucidation of molecules of interest.

Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155.

BACKGROUND: Chronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence. METHODS: Among the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155-/- mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. RESULTS: Compared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated ß-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs. CONCLUSIONS: Our work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

tRF3-IleAAT reduced extracellular matrix synthesis in diabetic kidney disease mice by targeting ZNF281 and inhibiting ferroptosis.

It is well established that the synthesis of extracellular matrix (ECM) in mesangial cells is a major determinant of diabetic kidney disease (DKD). Elucidating the major players in ECM synthesis may be helpful to provide promising candidates for protecting against DKD progression. tRF3-IleAAT is a tRNA-derived fragment (tRF) produced by nucleases at tRNA-specific sites, which is differentially expressed in the sera of patients with diabetes mellitus and DKD. In this study we investigated the potential roles of tRFs in DKD. Db/db mice at 12 weeks were adapted as a DKD model. The mice displayed marked renal dysfunction accompanied by significantly reduced expression of tRF3-IleAAT and increased ferroptosis and ECM synthesis in the kidney tissues. The reduced expression of tRF3-IleAAT was also observed in high glucose-treated mouse glomerular mesangial cells. We administered ferrostatin-1 (1 mg/kg, once every two days, i.p.) to the mice from the age of 12 weeks for 8 weeks, and found that inhibition of the onset of ferroptosis significantly improved renal function, attenuated renal fibrosis and reduced collagen deposition. Overexpression of tRF3-IleAAT by a single injection of AAV carrying tRF3-IleAAT via caudal vein significantly inhibited ferroptosis and ECM synthesis in DKD model mice. Furthermore, we found that the expression of zinc finger protein 281 (ZNF281), a downstream target gene of tRF3-IleAAT, was significantly elevated in DKD models but negatively regulated by tRF3-IleAAT. In high glucose-treated mesangial cells, knockdown of ZNF281 exerted an inhibitory effect on ferroptosis and ECM synthesis. We demonstrated the targeted binding of tRF3-IleAAT to the 3'UTR of ZNF281. In conclusion, tRF3-IleAAT inhibits ferroptosis by targeting ZNF281, resulting in the mitigation of ECM synthesis in DKD models, suggesting that tRF3-IleAAT may be an attractive therapeutic target for DKD.