Molecular Insights into the Effects of F16L and F19L Substitutions on the Conformation and Aggregation Dynamics of Human Calcitonin.

Human calcitonin (hCT) regulates calcium-phosphorus metabolism, but its amyloid aggregation disrupts physiological activity, increases thyroid carcinoma risk, and hampers its clinical use for bone-related diseases like osteoporosis and Paget's disease. Improving hCT with targeted modifications to mitigate amyloid formation while maintaining its function holds promise as a strategy. Understanding how each residue in hCT's amyloidogenic core affects its structure and aggregation dynamics is crucial for designing effective analogues. Mutants F16L-hCT and F19L-hCT, where Phe residues in the core are replaced with Leu as in nonamyloidogenic salmon calcitonin, showed different aggregation kinetics. However, the molecular effects of these substitutions in hCT are still unclear. Here, we systematically investigated the folding and self-assembly conformational dynamics of hCT, F16L-hCT, and F19L-hCT through multiple long-time scale independent atomistic discrete molecular dynamics (DMD) simulations. Our results indicated that the hCT monomer primarily assumed unstructured conformations with dynamic helices around residues 4-12 and 14-21. During self-assembly, the amyloidogenic core of hCT14-21 converted from dynamic helices to ß-sheets. However, substituting F16L did not induce significant conformational changes, as F16L-hCT exhibited characteristics similar to those of wild-type hCT in both monomeric and oligomeric states. In contrast, F19L-hCT exhibited substantially more helices and fewer ß-sheets than did hCT, irrespective of their monomers or oligomers. The substitution of F19L significantly enhanced the stability of the helical conformation for hCT14-21, thereby suppressing the helix-to-ß-sheet conformational conversion. Overall, our findings elucidate the molecular mechanisms underlying hCT aggregation and the effects of F16L and F19L substitutions on the conformational dynamics of hCT, highlighting the critical role of F19 as an important target in the design of amyloid-resistant hCT analogs for future clinical applications.

Associations between life's essential 8 and metabolic health among us adults: insights of NHANES from 2005 to 2018.

BACKGROUND: Metabolic unhealth (MUH) is closely associated with cardiovascular disease (CVD). Life's Essential 8 (LE8), a recently updated cardiovascular health (CVH) assessment, has some overlapping indicators with MUH but is more comprehensive and complicated than MUH. Given the close relationship between them, it is important to compare these two measurements. METHODS: This population-based cross-sectional survey included 20- to 80-year-old individuals from 7 National Health and Nutrition Examination Survey (NHANES) cycles between 2005 and 2018. Based on the parameters provided by the American Heart Association, the LE8 score (which ranges from 0 to 100) was used to classify CVH into three categories: low (0-49), moderate (50-79), and high (80-100). The MUH status was evaluated by blood glucose, blood pressure, and blood lipids. The associations were assessed by multivariable regression analysis, subgroup analysis, restricted cubic spline models, and sensitivity analysis. RESULTS: A total of 22,582 participants were enrolled (median of age was 45 years old), among them, 11,127 were female (weighted percentage, 49%) and 16,595 were classified as MUH (weighted percentage, 73.5%). The weighted median LE8 scores of metabolic health (MH) and MUH individuals are 73.75 and 59.38, respectively. Higher LE8 scores were linked to lower risks of MUH (odds ratio [OR] for every 10 scores increase, 0.53; 95% CI 0.51-0.55), and a nonlinear dose-response relationship was seen after the adjustment of potential confounders. This negative correlation between LE8 scores, and MUH was strengthened among elderly population. CONCLUSIONS: Higher LE8 and its subscales scores were inversely and nonlinearly linked with the lower presence of MUH. MUH is consistent with LE8 scores, which can be considered as an alternative indicator when it is difficult to collect the information of health behaviors.

Targeting ferroptosis as a prospective therapeutic approach for diabetic nephropathy.

Diabetic nephropathy (DN) is a severe complication of diabetes mellitus, causing a substantive threat to the public, which receives global concern. However, there are limited drugs targeting the treatment of DN. Owing to this, it is highly crucial to investigate the pathogenesis and potential therapeutic targets of DN. The process of ferroptosis is a type of regulated cell death (RCD) involving the presence of iron, distinct from autophagy, apoptosis, and pyroptosis. A primary mechanism of ferroptosis is associated with iron metabolism, lipid metabolism, and the accumulation of ROS. Recently, many studies testified to the significance of ferroptosis in kidney tissue under diabetic conditions and explored the drugs targeting ferroptosis in DN therapy. Our review summarized the most current studies between ferroptosis and DN, along with investigating the significant processes of ferroptosis in different kidney cells, providing a novel target treatment option for DN.

Molecular Insights into the Differential Effects of Acetylation on the Aggregation of Tau Microtubule-Binding Repeats.

Aggregation of tau protein into intracellular fibrillary inclusions is characterized as the hallmark of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. The microtubule-binding (MTB) domain of tau, containing either three or four repeats with sequence similarities, plays an important role in determining tau's aggregation. Previous studies have reported that abnormal acetylation of lysine residues displays a distinct effect on the formation of pathological tau aggregates. However, the underlying molecular mechanism remains mostly elusive. In this study, we performed extensive replica exchange molecular dynamics (REMD) simulations of 144 µs in total to systematically investigate the dimerization of four tau MTB repeats and explore the impacts of Lys280 (K280) or Lys321 (K321) acetylation on the conformational ensembles of the R2 or R3 dimer. Our results show that R3 is the most prone to aggregation among the four repeats, followed by R2 and R4, while R1 displays the weakest aggregation propensity with a disordered structure. Acetylation of K280 could promote the aggregation of R2 peptides by increasing the formation of ß-sheet structures and strengthening the interchain interaction. However, K321 acetylation decreases the ß-sheet content of the R3 dimer, reduces the ability of R3 peptides to form long ß-strands, and promotes the stable helix structure formation. The salt bridge and Y310-Y310 π-π stacking interactions of the R3 dimer are greatly weakened by K321 acetylation, resulting in the inhibition of dimerization. This study uncovers the structural ensembles of tau MTB repeats and provides mechanistic insights into the influences of acetylation on tau aggregation, which may deepen the understanding of the pathogenesis of tauopathies.

Deciphering the influence of Y12L and N17H substitutions on the conformation and oligomerization of human calcitonin.

The abnormal aggregation of human calcitonin (hCT) hormone peptides impairs their physiological function, leading to harmful immune responses and cytotoxicity, which limits their clinical utility. Interestingly, a representative hCT analog incorporating Y12L and N17H substitutions (DM-hCT) has shown reduced aggregation tendencies while maintaining bioactivity. But the molecular mechanism of Y12L and N17H substitutions on the conformational dynamics of hCT remains unclear. Here, we systematically investigated the folding and self-assembly dynamics of hCT and DM-hCT using atomistic discrete molecular dynamics (DMD) simulations. Our findings revealed that hCT monomers predominantly adopted unstructured conformations with dynamic helices. Oligomerization of hCT resulted in the formation of ß-sheet-rich aggregates and ß-barrel intermediates. The Y12L and N17H substitutions enhanced helical conformations and suppressed ß-sheet formation in both monomers and oligomers. These substitutions stabilized the dynamic helices and disrupted aromatic interactions responsible for ß-sheet formation at residue 12. Notably, DM-hCT assemblies still exhibited ß-sheets in phenylalanine-rich and C-terminal hydrophobic regions, suggesting that future optimizations should focus on these areas. Our simulations provide insights into the molecular mechanisms underlying hCT aggregation and the amyloid-resistant effects of Y12L and N17H substitutions. These findings have valuable implications for the development of clinical hCT analogs.

Computational insights into the cross-talk between medin and Aß: implications for age-related vascular risk factors in Alzheimer's disease.

The aggregation of medin forming aortic medial amyloid is linked to arterial wall degeneration and cerebrovascular dysfunction. Elevated levels of arteriolar medin are correlated with an increased presence of vascular amyloid-ß (Aß) aggregates, a hallmark of Alzheimer's disease (AD) and vascular dementia. The cross-interaction between medin and Aß results in the formation of heterologous fibrils through co-aggregation and cross-seeding processes both in vitro and in vivo. However, a comprehensive molecular understanding of the cross-interaction between medin and Aß-two intrinsically disordered proteins-is critically lacking. Here, we employed atomistic discrete molecular dynamics simulations to systematically investigate the self-association, co-aggregation and also the phenomenon of cross-seeding between these two proteins. Our results demonstrated that both Aß and medin were aggregation prone and their mixture tended to form ß-sheet-rich hetero-aggregates. The formation of Aß-medin hetero-aggregates did not hinder Aß and medin from recruiting additional Aß and medin peptides to grow into larger ß-sheet-rich aggregates. The ß-barrel oligomer intermediates observed in the self-aggregations of Aß and medin were also present during their co-aggregation. In cross-seeding simulations, preformed Aß fibrils could recruit isolated medin monomers to form elongated ß-sheets. Overall, our comprehensive simulations suggested that the cross-interaction between Aß and medin may contribute to their pathological aggregation, given the inherent amyloidogenic tendencies of both medin and Aß. Targeting medin, therefore, could offer a novel therapeutic approach to preserving brain function during aging and AD by improving vascular health.

Upregulation of UHRF1 Promotes PINK1-mediated Mitophagy to Alleviates Ferroptosis in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a common diabetic complication. Studies show that mitophagy inhibition induced-ferroptosis plays a crucial role in DN progression. UHRF1 is associated with mitophagy and is highly expression in DN patients, however, the effect of UHRF1 on DN is still unclear. Thus, in this study, we aimed to investigate whether UHRF1 involves DN development by the mitophagy/ferroptosis pathway. We overexpressed UHRF1 using an adeno-associated virus 9 (AAV9) system in high-fat diet/streptozotocin-induced diabetic mice. Renal function index, pathological changes, mitophagy factors, and ferroptosis factors were detected in vivo. High-glucose cultured human renal proximal tubular (HK-2) cells were used as in vitro models to investigate the mechanism of UHRF1 in DN. We found that diabetic mice exhibited kidney damage, which was alleviated by UHRF1 overexpression. UHRF1 overexpression promoted PINK1-mediated mitophagy and inhibited the expression of thioredoxin interacting protein (TXNIP), a factor associated with mitochondrial dysfunction. Additionally, UHRF1 overexpression alleviated lipid peroxidation and free iron accumulation, and upregulated the expression of GPX4 and Slc7a11, indicating the inhibition effect of UHRF1 overexpression on ferroptosis. We further investigated the mechanism of UHRF1 in the mitophagy/ferroptosis pathway in DN. We found that UHRF1 overexpression promoted PINK1-mediated mitophagy via inhibiting TXNIP expression, thus suppressing ferroptosis. These findings confirmed that upregulation of UHRF1 expression alleviates DN, indicating that UHRF1 has a reno-protective effect against DN.

Co-aggregation of α-synuclein with amyloid-ß stabilizes ß-sheet-rich oligomers and enhances the formation of ß-barrels.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases with markedly different pathological features of ß-amyloid (Aß) plaques and α-synuclein (αS) Lewy bodies (LBs), respectively. However, clinical overlaps in symptoms and pathologies between AD and PD are commonly observed caused by the cross-interaction between Aß and αS. To uncover the molecular mechanisms behind their overlapping symptoms and pathologies, we computationally investigated the impact of αS on an Aß monomer and dimerization using atomistic discrete molecular dynamics simulations (DMD). Our results revealed that αS could directly interact with Aß monomers and dimers, thus forming ß-sheet-rich oligomers, including potentially toxic ß-barrel intermediates. The binding hotspot involved the second half of the N-terminal domain and NAC region in αS, along with residues 10-21 and 31-42 in Aß. In their hetero-complex, the binding hotspot primarily assumed a ß-sheet core buried inside, which was dynamically shielded by the highly charged, amyloid-resistant C-terminus of αS. Because the amyloid prion region was the same as the binding hotspot being buried, their fibrillization may be delayed, causing the toxic oligomers to increase. This study sheds light on the intricate relationship between Aß and αS and provides insights into the overlapping pathology of AD and PD.

LncRNA H19: a novel player in the regulation of diabetic kidney disease.

Diabetic kidney disease (DKD), one of the most severe complications of diabetes mellitus (DM), has received considerable attention owing to its increasing prevalence and contribution to chronic kidney disease (CKD) and end-stage kidney disease (ESRD). However, the use of drugs targeting DKD remains limited. Recent data suggest that long non-coding RNAs (lncRNAs) play a vital role in the development of DKD. The lncRNA H19 is the first imprinted gene, which is expressed in the embryo and down-regulated at birth, and its role in tumors has long been a subject of controversy, however, in recent years, it has received increasing attention in kidney disease. The LncRNA H19 is engaged in the pathological progression of DKD, including glomerulosclerosis and tubulointerstitial fibrosis via the induction of inflammatory responses, apoptosis, ferroptosis, pyroptosis, autophagy, and oxidative damage. In this review, we highlight the most recent research on the molecular mechanism and regulatory forms of lncRNA H19 in DKD, including epigenetic, post-transcriptional, and post-translational regulation, providing a new predictive marker and therapeutic target for the management of DKD.

Unveiling Medin Folding and Dimerization Dynamics and Conformations via Atomistic Discrete Molecular Dynamics Simulations.

Medin is a principal component of localized amyloid found in the vasculature of individuals over 50 years old. Its amyloid aggregation has been linked to endothelial dysfunction and vascular inflammation, contributing to the pathogenesis of various vascular diseases. Despite its significance, the structures of the medin monomer, oligomer, and fibril remain elusive, and the dynamic processes of medin aggregation are not fully understood. In this study, we comprehensively investigated the medin folding and dimerization dynamics and conformations using atomistic discrete molecular dynamics simulations. Our simulation results suggested that the folding initiation of the medin involved the formation of ß-sheets around medin30-41 and medin42-50, with subsequent capping of other segments to their ß-sheet edges. Medin monomers typically consisted of three or four ß-strands, along with a dynamic N-terminal helix. Two isolated medin peptides readily aggregated into a ß-sheet-rich dimer, displaying a strong aggregation propensity. Dimerization of medin not only enhanced the ß-sheet conformations but also led to the formation of ß-barrel oligomers. The aggregation tendencies of medin1-18 and medin19-29 were relatively weak. However, the segments of medin30-41 and medin42-50 played a crucial role as they primarily formed a ß-sheet core and facilitated medin1-18 and medin19-29 to form intra- and interpeptide ß-sheets. The findings highlight the critical role of the medin30-41 and medin42-50 regions in stabilizing the monomer structure and driving the medin amyloid aggregation. These regions could potentially serve as promising targets for designing antiamyloid inhibitors against amyloid aggregation of medin. Additionally, our study provides a full picture of the monomer conformations and dimerization dynamics for medin, which will help better understand the pathology of medin aggregation.

Deciphering the Inhibitory Mechanism of Naphthoquinone-Dopamine on the Aggregation of Tau Core Fragments PHF6* and PHF6.

The formation of neurofibrillary tangles by abnormal aggregation of tau protein is considered to be an important pathological characteristic of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. Two hexapeptides 275VQIINK280 and 306VQIVYK311 in the microtubule binding region, named PHF6* and PHF6, are known to be aggregation-prone and responsible for tau fibrillization. Previous experiments reported that naphthoquinone-dopamine (NQDA) could effectively inhibit the aggregation of PHF6* and PHF6 and disrupt the fibrillar aggregates into nontoxic species, displaying a dual effect on the amyloid aggregation. However, the underlying molecular mechanism remains mostly elusive. Herein, we performed all-atom molecular dynamics (MD) simulations for 114 µs in total to systematically investigate the impacts of NQDA on the oligomerization of PHF6* and PHF6. The conformational ensembles of PHF6* and PHF6 peptides generated by replica exchange MD simulations show that NQDA could effectively prevent the hydrogen bond formation, reduce the ability of peptides to self-assemble into long ß-strand and large ß-sheets, and induce peptides to form a loosely packed and coil-rich oligomer. The interaction analysis shows that the binding of NQDA to PHF6* is mainly through hydrophobic interactions with residue I277 and hydrogen bonding interactions with Q276; for the PHF6 peptides, NQDA displays a strong π-π stacking interaction with residue Y310, thus impeding the Y310-Y310 π-π stacking and I308-Y310 CH-π interactions. The DA group of NQDA displays a stronger cation-π interaction than the NQ group, while the NQ group exhibits a stronger π-π stacking interaction. MD simulations demonstrate that NQDA prevents the conformational conversion to ß-sheet-rich aggregates and displays an inhibitory effect on the oligomerization dynamics of PHF6* and PHF6. Our results provide a complete picture of inhibitory mechanisms of NQDA on PHF6* and PHF6 oligomerization, which may pave the way for designing drug candidates for the treatment of tauopathies.

Dissecting the Self-assembly Dynamics of Imperfect Repeats in α-Synuclein.

The pathological aggregation of α-synuclein (αS) into amyloid fibrils is the hallmark of Parkinson's disease (PD). The self-assembly and membrane interactions of αS are mainly governed by the seven imperfect 11-residue repeats of the XKTKEGVXXXX motif around residues 1-95. However, the particular role of each repeat in αS fibrillization remains unclear. To answer this question, we studied the aggregation dynamics of each repeat with up to 10 peptides in silico by conducting multiple independent micro-second atomistic discrete molecular dynamics simulations. Our simulations revealed that only repeats R3 and R6 readily self-assembled into ß-sheet-rich oligomers, while the other repeats remained as unstructured monomers with weak self-assembly and ß-sheet propensities. The self-assembly process of R3 featured frequent conformational changes with ß-sheet formation mainly in the non-conserved hydrophobic tail, whereas R6 spontaneously self-assembled into extended and stable cross-ß structures. These results of seven repeats are consistent with their structures and organization in recently solved αS fibrils. As the primary amyloidogenic core, R6 was buried inside the central cross-ß core of all αS fibrils, attracting the hydrophobic tails of adjacent R4, R5, and R7 repeats forming ß-sheets around R6 in the core. Further away from R6 in the sequence but with a moderate amyloid aggregation propensity, the R3 tail could serve as a secondary amyloidogenic core and form independent ß-sheets in the fibril. Overall, our results demonstrate the critical role of R3 and R6 repeats in αS amyloid aggregation and suggest their potential as targets for the peptide-based and small-molecule amyloid inhibitors.

SEVI Inhibits Aß Amyloid Aggregation by Capping the ß-Sheet Elongation Edges.

Inhibiting the aggregation of amyloid peptides with endogenous peptides has broad interest due to their intrinsically high biocompatibility and low immunogenicity. Here, we investigated the inhibition mechanism of the prostatic acidic phosphatase fragment SEVI (semen-derived enhancer of viral infection) against Aß42 fibrillization using atomistic discrete molecular dynamic simulations. Our result revealed that SEVI was intrinsically disordered with dynamic formation of residual helices. With a high positive net charge, the self-aggregation tendency of SEVI was weak. Aß42 had a strong aggregation propensity by readily self-assembling into ß-sheet-rich aggregates. SEVI preferred to interact with Aß42, rather than SEVI themselves. In the heteroaggregates, Aß42 mainly adopted ß-sheets buried inside and capped by SEVI in the outer layer. SEVI could bind to various Aß aggregation species─including monomers, dimers, and proto-fibrils─by capping the exposed ß-sheet elongation edges. The aggregation processes Aß42 from the formation of oligomers to conformational nucleation into fibrils and fibril growth should be inhibited as their ß-sheet elongation edges are being occupied by the highly charged SEVI. Overall, our computational study uncovered the molecular mechanism of experimentally observed inhibition of SEVI against Aß42 aggregation, providing novel insights into the development of therapeutic strategies against Alzheimer's disease.

SUMO specific peptidase 6 regulates the crosstalk between podocytes and glomerular endothelial cells in diabetic kidney disease.

There is increasing evidence that the crosstalk between podocytes and glomerular endothelial cells (GECs) exacerbates the progression of diabetic kidney disease (DKD). Here, we investigated the underlying role of SUMO specific peptidase 6 (SENP6) in this crosstalk. In the diabetic mice, SENP6 was decreased in glomerular tissues and its knockdown further exacerbated glomerular filtration barrier injury. In the mouse podocyte cell line MPC5 cells, SENP6 overexpression reversed HG-induced podocyte loss by suppressing the activation of Notch1 signaling. Notch1 intracellular domain (N1ICD) is the active form of Notch1. SENP6 upregulated the ubiquitination of N1ICD by deSUMOylating Notch1, thereby reducing N1ICD and suppressing Notch1 signaling activation in MPC5 cells. Endothelin-1 (EDN1) is a protein produced by podocytes and has been reported to promote GEC dysfunction. The supernatant from HG-treated MPC5 cells induced mitochondrial dysfunction and surface layer injury in GECs, and the supernatant from SENP6-deficient podocytes further exacerbated the above GEC dysfunction, while this trend was reversed by an EDN1 antagonist. The following mechanism study showed that SENP6 deSUMOylated KDM6A (a histone lysine demethylase) and then decreased the binding potency of KDM6A to EDN1. The latter led to the upregulation of H3K27me2 or H3K27me3 of EDN1 and suppressed its expression in podocytes. Taken together, SENP6 suppressed the HG-induced podocyte loss and ameliorated GEC dysfunction caused by crosstalk between podocytes and GECs, and the protective effect of SENP6 on DKD is attributed to its deSUMOylation activity.

Development and validation of a nomogram to estimate future risk of type 2 diabetes mellitus in adults with metabolic syndrome: prospective cohort study.

OBJECTIVES: To develop and validate the 4-year risk of type 2 diabetes mellitus among adults with metabolic syndrome. DESIGN: Retrospective cohort study of a large multicenter cohort with broad validation. SETTINGS: The derivation cohort was from 32 sites in China and the geographic validation cohort was from Henan population-based cohort study. RESULTS: 568 (17.63) and 53 (18.67%) participants diagnosed diabetes during 4-year follow-up in the developing and validation cohort, separately. Age, gender, body mass index, diastolic blood pressure, fasting plasma glucose and alanine aminotransferase were included in the final model. The area under curve for the training and external validation cohort was 0.824 (95% CI, 0.759-0.889) and 0.732 (95% CI, 0.594-0.871), respectively. Both the internal and external validation have good calibration plot. A nomogram was constructed to predict the probability of diabetes during 4-year follow-up, and on online calculator is also available for a more convenient usage ( https://lucky0708.shinyapps.io/dynnomapp/ ). CONCLUSION: We developed a simple diagnostic model to predict 4-year risk of type 2 diabetes mellitus among adults with metabolic syndrome, which is also available as web-based tools ( https://lucky0708.shinyapps.io/dynnomapp/ ).

Novel lncRNA-prader willi/angelman region RNA, SNRPN neighbour (PWARSN) aggravates tubular epithelial cell pyroptosis by regulating TXNIP via dual way in diabetic kidney disease.

OBJECTIVES: Elevated thioredoxin-interacting protein (TXNIP)-induced pyroptosis contributes to the pathology of diabetic kidney disease (DKD). However, the molecular mechanisms in dysregulated TXNIP in DKD remain largely unclear. MATERIALS AND METHODS: Transcriptomic analysis identified a novel long noncoding RNA-Prader Willi/Angelman region RNA, SNRPN neighbour (PWARSN)-which was highly expressed in a proximal tubular epithelial cell (PTEC) under high glucose conditions. We focused on revealing the functions of PWARSN in regulating TXNIP-mediated pyroptosis in PTECs by targeting PWARSN expression via lentivirus-mediated overexpression and CRISPR-Cas9-based knockout in vitro and overexpressing PWARSN in the renal cortex by AAV-9 targeted injection in vivo. A number of molecular techniques disclosed the mechanisms of PWARSN in regulating TXNIP induced-pyroptosis in DKD. RESULTS: TXNIP-NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and PTEC pyroptosis were activated in the renal tubules of patients with DKD and in diabetic mice. Then we explored that PWARSN enhanced TXNIP-driven PTECs pyroptosis in vitro and in vivo. Mechanistically, cytoplasmic PWARSN sponged miR-372-3p to promote TXNIP expression. Moreover, nuclear PWARSN interacted and facilitated RNA binding motif protein X-linked (RBMX) degradation through ubiquitination, resulting in the initiation of TXNIP transcription by reducing H3K9me3-enrichment at the TXNIP promoter. Further analysis indicated that PWARSN might be a potential biomarker for DKD. CONCLUSIONS: These findings illustrate distinct dual molecular mechanisms for PWARSN-modulated TXNIP and PTECs pyroptosis in DKD, presenting PWARSN as a promising therapeutic target for DKD.

Identification of circular RNAs and functional competing endogenous RNA networks in human proximal tubular epithelial cells treated with sodium-glucose cotransporter 2 inhibitor dapagliflozin in diabetic kidney disease.

Diabetic kidney disease (DKD) is a serious diabetes complication. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are novel anti-diabetes drugs that confer clinical renal protection. However, the molecular mechanisms involved remain unclear. Here, human proximal tubular epithelial cells (PTECs) were treated with normal glucose, high glucose, and anti-diabetes agents, including SGLT2i (dapagliflozin), metformin, and dipeptidyl peptidase-4 inhibitor (DPP-4i, vildagliptin) and microarray analysis was performed. Firstly, a total of 2,710 differentially expressed circular RNAs (circRNAs) were identified. Secondly, network pharmacology and transcriptomics analyses showed that the effects of dapagliflozin on PTECs primarily involved lipid metabolism, Rap1, and MAPK signaling pathways. Metformin mainly affected the AMPK and FOXO signaling pathways, whereas vildagliptin affected insulin secretion and the HIF-1 signaling pathway. Furthermore, circRNA-miRNA-mRNA networks, real-time reverse transcription-polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) assay revealed that the expression of hsa_circRNA_012448 was increased in PTECs treated with high glucose, whereas its expression was reversed by dapagliflozin. Finally, the hsa_circRNA_012448-hsa-miR-29b-2-5p-GSK3ß pathway, involved in the oxidative stress response, was identified as an important pathway mediating the action of dapagliflozin against DKD. Overall, our study provides novel insights into the molecular mechanisms underlying the effects of dapagliflozin on DKD.

Renal outcomes and prognostic factors in patients with type-2 diabetes and chronic kidney disease confirmed by renal biopsy.

AIM: To evaluate the renal outcomes and prognostic factors among patients with type-2 diabetes (T2D) and biopsy-confirmed diabetic nephropathy (DN), non-diabetic renal disease (NDRD) and DN mixed with NDRD (MIX). DESIGN AND METHODS: Patients with both T2D and chronic kidney disease (CKD) who underwent renal biopsy between January 2014 and December 2016 were recruited in this prospective observational study. Participants were divided into DN group, NDRD group, or MIX group according to the baseline pathological diagnosis. The primary endpoint was a composite renal event of end-stage renal disease (ESRD) or ⩾ 40% reduction in estimated glomerular filtration rate (eGFR). RESULTS: Among the 292 participants included, 153 (52.4%) belonged to the DN group, 30 (10.3%) belonged to the NDRD group, and 109 (37.3%) belonged to the MIX group. During the median follow-up of 27 months, the adverse renal events occurred in 132 (44.2%) patients. Compared with NDRD group, the multiple adjusted hazard ratios (HRs) for renal events in patients with DN and MIX groups were 3.900 (95% confidence interval [CI]: 1.103-13.788) and 2.691 (95% CI: 0.662-10.936), respectively. Baseline lower eGFR (HR: 1.159, 95% CI: 1.060-1.266), severe proteinuria (HR: 2.047, 95% CI: 1.227-3.416), lower hemoglobin (HR: 1.170, 95% CI: 1.008-1.267), and a family history of diabetes (HR: 1.138, 95% CI: 1.008-2.285) were independent predictors for adverse renal outcomes in patients with DN. CONCLUSION: In patients with T2D and CKD, pure DN and MIX group displayed a worse renal prognosis than NDRD group. Worse renal function, severe proteinuria, lower hemoglobin, and a family history of diabetes may be associated with adverse renal outcomes in patients with DN.

FoxO1-mediated inhibition of STAT1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease.

BACKGROUND: Tubulointerstitial fibrosis (TIF) plays an important role in the progression of diabetic kidney disease (DKD). Forkhead box O1 (FoxO1) is involved in the regulation of metabolism and cell apoptosis, but its function in renal TIF induced by DKD is less well understood. METHODS: Human kidney biopsies with DKD and normal controls were used to detect apoptosis and TIF induced by diabetes. A mouse model with kidney-specific overexpression of Pax2-3aFoxO1 was established to further investigate the functions of FoxO1 in vivo. The in vitro roles of FoxO1 were analyzed in HK-2 cells with 3aFoxO1-knockin (3aFoxO1-KI) or FoxO1-knockdown (FoxO1-KD) via CRISPR/Cas9. Western blot, immunohistochemistry, and chromatin immunoprecipitation were used to explore the underlying mechanisms. FINDINGS: In this study, DKD patients had increased renal TIF and apoptosis. In vivo study showed that kidney-specific overexpression of Pax2-3aFoxO1 significantly reduced the expression of p-STAT1 with resultant renal functional impairment, retarding renal TIF and apoptosis in diabetic mice. Meanwhile, We observed that FoxO1-KD in HK-2 cells aggravated the expression of p-STAT1, leading to activation of epithelial-to-mesenchymal transition (EMT) and intrinsic apoptotic pathway. Conversely, EMT and apoptosis were significantly attenuated in HK-2 cells with 3aFoxO1-KI under hyperglycemic conditions. INTERPRETATION: Taken together, these data suggest that the protection role of FoxO1 against renal TIF and apoptosis in DKD is likely in part to target STAT1 signaling, which may be a promising strategy for long-term treatment of DKD. FUND: This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 81570746 and 81770812).

Dapagliflozin Attenuates Renal Tubulointerstitial Fibrosis Associated With Type 1 Diabetes by Regulating STAT1/TGFß1 Signaling.

Tubulointerstitial fibrosis (TIF) plays an important role in the progression of renal fibrosis in diabetic nephropathy (DN). Accumulating evidence supports a crucial inhibitory effect of dapagliflozin, a SGLT2 inhibitor, on TIF, but the underlying mechanisms remain largely unknown. This study aimed to shed light on the efficacy of dapagliflozin in reducing TIF as well as its possible impact on renal function. TIF in human kidney biopsies obtained from patients with DN was quantified by histopathological staining. In vitro, HK-2 cells were incubated in high glucose with dapagliflozin or fludarabine, and epithelial-mesenchymal transition (EMT) was determined. In vivo experiments were performed in streptozotocin (STZ)-induced type 1 diabetic mice treated with dapagliflozin by gavage for 16 weeks, after which specific functional characteristics and TIF were analyzed. In both DN patients and diabetic mice, fibronectin and Col IV, as well as STAT1 protein in the kidneys were increased as compared with controls. Dapagliflozin significantly decreased blood glucose, and renal STAT1 and TGF-ß1 expression in mice. Furthermore, dapagliflozin improved renal function, and attenuated diabetes-induced TIF. In HK-2 cells, dapagliflozin, and fludarabine directly decreased aberrant STAT1 expression and reversed high glucose-induced downregulation of E-cadherin and α-SMA induction. Thus, the results demonstrate that dapagliflozin not only improves hyperglycemia but also slows down the progression of diabetes-associated renal TIF by improving hyperglycemia-induced activation of the STAT1/TGF-ß1 pathway.