A lower eGFRcystatin C/eGFRcreatinine ratio is associated with greater cardiovascular risk (higher Framingham Risk Score) in Chinese patients with newly diagnosed type 2 diabetes mellitus.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of mortality in type 2 diabetes mellitus (T2DM) patients. Shrunken pore syndrome (SPS) is defined as eGFRcystatin C/eGFRcreatinine ratio <0.70 and predicts high CVD mortality. The Framingham Risk Score (FRS) is used to estimate an individual's 10-year CVD risk. This study investigated the association between FRS and eGFRcystatin C/eGFRcreatinine ratio in T2DM patients. METHODS: Patients aged 18-80 years who were newly diagnosed with T2DM were included in this retrospective study. Ordinal logistic regression analysis was used to investigate the association between risk factors of T2DM and FRS. A Generalized Linear Model was used to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: There were 270 patients included in the study. Only 27 patients (10%) met the diagnostic criteria of SPS. Ordinal logistic regression analysis showed that SPS was not correlated with FRS risk (OR = 1.99, 95%CI = 0.94-4.23, p = 0.07), whereas eGFRcystatin C/eGFRcreatinine (OR = 0.86, 95%CI = 0.77-0.97, p = 0.01) showed a significant negative association with FRS risk. Compared with eGFRcystatin C/eGFRcreatinine>0.85, eGFRcystatin C/eGFRcreatinine≤0.85 increased FRS risk (OR = 1.95, 95%CI = 1.18-3.21, p < 0.01). After adjustment for confounding factors, increased eGFRcystatin C/eGFRcreatinine ratio was associated with decreased FRS risk when considered as a continuous variable (OR = 0.87, 95%CI = 0.77-0.99, p = 0.03). The FRS risk in patients with eGFRcystatin C/eGFRcreatinine≤0.85 is 1.86 times higher than that in patients with eGFRcystatin C/eGFRcreatinine>0.85 (OR = 1.86, 95%CI = 1.08-3.21, p = 0.03). CONCLUSIONS: In the current study, no significant association between SPS and FRS was identified. However, lower eGFRcystatin C/eGFRcreatinine and eGFRcystatin C/eGFRcreatinine≤0.85 were associated with a significantly increased CVD risk in T2DM.

Elevated serum FGF21 is an independent predictor for adverse events in hemodialysis patients from two large centers: a prospective cohort study.

Introduction: We explored the relationship and the predictive value of serum fibroblast growth factor 21 (FGF21) with all-cause mortality, major adverse cardiovascular events (MACEs) and pneumonia in hemodialysis (HD) patients.Methods: A total of 388 Chinese HD patients from two HD centers were finally enrolled in this prospective cohort study (registration number: ChiCTR 1900028249) between January 2018 and December 2018. Serum FGF21 was detected. Patients were followed up with a median period of 47 months to record the MACEs and pneumonia until death or 31 December 2022.Results: The incidence of all-cause mortality, MACEs and pneumonia in HD patients were 20.6%, 29.6%, and 34.8%, respectively. The optimal cutoffs for FGF21 to predict all-cause mortality, MACEs and pneumonia were 437.57 pg/mL, 216.99 pg/mL and 112.79 pg/mL. Multivariate Cox regression analyses showed that FGF21, as a categorical variable, was an independent predictor for all-cause mortality, MACEs and pneumonia (HR, 3.357, 95% CI, 2.128-5.295, p < 0.001; HR, 1.575, 95% CI, 1.046-2.371, p = 0.029; HR, 1.784; 95% CI, 1.124-2.830; p = 0.014, respectively). The survival nomogram, MACEs-free survival nomogram and pneumonia-free survival nomogram based on FGF21 constructed for individualized assessment of HD patients had a high C-index with 0.841, 0.706 and 0.734.Conclusion: Higher serum FGF21 is an independent predictor of all-cause mortality, MACEs and pneumonia in HD patients.

Computed tomography-determined skeletal muscle density predicts 3-year mortality in initial-dialysis patients in China.

BACKGROUND: Skeletal muscle mass and quality assessed by computed tomography (CT) images of the third lumbar vertebra (L3) level have been established as risk factors for poor clinical outcomes in several illnesses, but the relevance for dialysis patients is unclear. A few studies have suggested a correlation between CT-determined skeletal muscle mass and quality at the first lumbar vertebra (L1) level and adverse outcomes. Generally, chest CT does not reach beyond L1. We aimed to determine whether opportunistic CT scan (chest CT)-determined skeletal muscle mass and quality at L1 are associated with mortality in initial-dialysis patients. METHODS: This 3-year multicentric retrospective study included initial-dialysis patients from four centres between 2014 and 2017 in China. Unenhanced CT images of the L1 and L3 levels were obtained to assess skeletal muscle mass [by skeletal muscle index, (SMI), cm2 /m2 ] and quality [by skeletal muscle density (SMD), HU]. Skeletal muscle measures at L1 were compared with those at L3. The sex-specific optimal cutoff values of L1 SMI and L1 SMD were determined in relation to all-cause mortality. The outcomes were all-cause death and cardiac death. Cox regression models were applied to investigate the risk factors for death. RESULTS: A total of 485 patients were enrolled, of whom 257 had both L1 and L3 images. Pearson's correlation coefficient between L1 and L3 SMI was 0.84 (P < 0.001), and that between L1 and L3 SMD was 0.90 (P < 0.001). No significant association between L1 SMI and mortality was observed (P > 0.05). Low L1 SMD (n = 280, 57.73%) was diagnosed based on the optimal cutoff value (<39.56 HU for males and <33.06 HU for females). Multivariate regression analysis revealed that the low L1 SMD group had higher risks of all-cause death (hazard ratio 1.80; 95% confidence interval 1.05-3.11, P = 0.034) and cardiac death (hazard ratio 3.74; 95% confidence interval 1.43-9.79, P = 0.007). CONCLUSIONS: In initial-dialysis patients, there is high agreement between the L1 and L3 measures for SMI and SMD. Low SMD measured at L1, but not low SMI, is an independent predictor of both all-cause death and cardiac death.

The systemic inflammation indexes predict all-cause mortality in peritoneal dialysis patients.

BACKGROUND: Chronic inflammation is a common complication in peritoneal dialysis (PD) patients. The aim of this study is to investigate the capacity of aggregate index of systemic inflammation (AISI), systemic immune-inflammation index (SII), and systemic inflammation response index (SIRI) to predict all-cause mortality in PD patients. METHODS: This was a single-center retrospective study. The optimal cutoff values were identified by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was calculated to evaluate the predictive ability of these indexes. The Kaplan-Meier curves and log-rank test were performed to estimate cumulative survival rate. Cox proportional hazards regression analyses were conducted to determine the independent prognostic power of inflammation indexes. RESULTS: A total of 369 incident PD patients were involved. During a median follow-up period of 32.83 months, 65 patients (24.2%) died. The ROC analysis indicated the largest value of AUC was obtained for SII (AUC = 0.644, 95% CI = 0.573-0.715, p < .001), followed in order by AISI (AUC = 0.617, 95% CI = 0.541-0.693, p = .003), and SIRI (AUC = 0.612, 95% CI = 0.535-0.688, p = .004). The Kaplan-Meier survival curves revealed significantly lower survival rate with higher AISI (p = .001), higher SSI (p = .001), and higher SIRI (p = .003). Even after adjustment for the confounding factors, higher AISI [hazard ratio (HR)=2.508, 95% confidence intervals (CI)=1.505-4.179, p < .001), SII (HR = 3.477, 95% CI = 1.785-6.775, p < .001), and SIRI (HR = 1.711, 95% CI = 1.012-2.895, p = .045) remained as independent predictors of all-cause mortality. CONCLUSIONS: The higher AISI, SII, and SIRI were independent indicators of all-cause mortality in PD patients. Furthermore, they could provide comparable predictive value and assist clinicians to ameliorate PD management.

Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in neurons in the central nervous system. It has been documented that HCN channels regulate the intrinsic excitability of pyramidal cells in the medial prefrontal cortex (mPFC) of rodents. Here, we report that HCN channels limited GABAergic transmission onto pyramidal cells in rat mPFC. The pharmacological blockade of HCN channels resulted in a significant increase in the frequency of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs) in mPFC pyramidal cells, whereas potentiation of HCN channels reversely decreases the frequency of mIPSCs. Furthermore, such facilitation effect on mIPSC frequency required presynaptic Ca2+ influx. Immunofluorescence staining showed that HCN channels expressed in presynaptic GABAergic terminals, as well as in both soma and neurite of parvalbumin-expressing (PV-expressing) basket cells in mPFC. The present results indicate that HCN channels in GABAergic interneurons, most likely PV-expressing basket cells, constrain inhibitory control over layer 5-6 pyramidal cells by restricting presynaptic Ca2+ entry.

The effect of hepcidin in rats with renal ischemia/reperfusion injury.

OBJECTIVE: This study sought to investigate the effectiveness of hepcidin in renal ischemia/reperfusion injury by using a rat model of renal IRI. METHODS: In our study, male Sprague-Dawley rats were divided into a hepcidin-treated group and a control group before establishing the animal models. According to the difference of the modelling methods (renal pedicle occlusion for 45 minutes or not) and renal reperfusion time, the rats were then respectively divided into four subgroups: sham, IRI 4 h, IRI 12 h, and IRI 24 h. After the establishment of the IRI model, the rats were killed to determine renal function, histology, iron metabolism indexes in plasma and tissues, and the expression level of hepcidin and ferroportin-1. RESULTS: The results indicated that the levels of serum creatinine, blood urea nitrogen and serum iron, the renal iron content, and the kidney injury score were significantly decreased in the hepcidin group (P<0.05). The serum hepcidin and the splenic iron content were significantly increased while the duodenal iron content was significantly decreased in the hepcidin group (P<0.05). Hepcidin expression in the liver and ferroportin-1 expression in the kidneys were significantly decreased in the hepcidin group (P<0.05). CONCLUSION: Hepcidin has a reno-protective effect in renal IRI by possibly promoting iron intake in the spleen, inhibiting iron absorption and exportation in the duodenum, alleviating the degree of serum iron, and reducing renal iron accumulation in the renal IRI.

A Synthetic Human Antibody Antagonizes IL-18Rß Signaling Through an Allosteric Mechanism.

The interleukin-18 subfamily belongs to the interleukin-1 family and plays an important role in modulating innate and adaptive immune responses. Dysregulation of IL-18 has been implicated in or correlated with numerous diseases, including inflammatory diseases, autoimmune disorders, and cancer. Thus, blockade of IL-18 signaling may offer therapeutic benefits in many pathological settings. Here, we report the development of synthetic human antibodies that target human IL-18Rß and block IL-18-mediated IFN-γ secretion by inhibiting NF-κB and MAPK dependent pathways. The crystal structure of a potent antagonist antibody in complex with IL-18Rß revealed inhibition through an unexpected allosteric mechanism. Our findings offer a novel means for therapeutic intervention in the IL-18 pathway and may provide a new strategy for targeting cytokine receptors.

MicroRNA-30/Cx43 axis contributes to podocyte injury by regulating ER stress in diabetic nephropathy.

BACKGROUND: The microRNA-30 family plays a critical role in the pathogenesis of podocyte injury. Cx43 plays an essential role in intercellular communication, which is essential for coordinated kidney function. This study was conducted to explore the function of microRNA-30s/Cx43 in podocyte injury in diabetic nephropathy (DN), both in vivo and in vitro. METHODS: SD rats were given streptozotocin (STZ) injections to induce DN. Podocytes were incubated in the medium in the presence or absence of high glucose (HG). The effects of the microRNA-30/Cx43 axis on DN and its underlying mechanisms were investigated by TUNEL assay, PAS, immunohistochemical staining, immunofluorescence staining, Western blot, RT-qPCR, RNA interference, and luciferase reporter assay. Podocytes were transfected with microRNA-30 family mimics, microRNA-30 family inhibitors, Cx43 siRNA, and negative controls to detect the effect of the microRNA-30/Cx43 axis. MicroRNA-30 family mimic AAVs, and microRNA-30 family inhibitor AAVs applied to regulate microRNA-30 family expression in the kidneys of the STZ-induced DN model rats to reveal the underlying mechanisms of the microRNA-30/Cx43 axis in DN. RESULTS: MicroRNA-30 family member expression was downregulated in HG-treated podocytes and the glomeruli of STZ-induced DN rats. Luciferase reporter assays confirmed Cx43 is a directed target of microRNA-30s. The overexpression of microRNA-30 family members attenuated the HG-induced podocyte injury and protected against podocyte apoptosis and endoplasmic reticulum stress (ERS) both in vivo and in vitro. Also, silencing Cx43 expression eased podocyte apoptosis, injury, and ERS induced by a HG+microRNA-30 family inhibitor. Double-immunofluorescence staining assays proved the co-localization of caspase12 and Cx43. CONCLUSIONS: The overexpression of microRNA-30 family members prevents HG-induced podocyte injury and attenuates ERS by modulating Cx43 expression. The microRNA-30/Cx43/ERS axis might be a potential therapeutic target to treat DN.

Protective effect of dabrafenib on renal ischemia-reperfusion injury in vivo and in vitro.

AIMS: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which can lead to poor outcome and increased risk of mortality. Dabrafenib (DAB) is an approved cancer treatment. Little is known about the effect of DAB in prevention or treatment of renal IRI. METHODS: For in vivo experiments, C57BL/6 mice were divided into four groups: sham (no IRI, no DAB), IRI, DAB, and DAB + IRI. IRI was induced by clamping of bilateral renal pedicles for 30 min. For in vitro experiments, HK-2 cells were used to establish the hypoxia/reoxygenation (H/R) injury model, with four groups: control (no H/R, no DAB), H/R, DAB, and DAB + H/R. Renal function and renal histological changes were recorded. Expression of NGAL and KIM-1 proteins and mRNAs were determined by western blotting and qRT-PCR; secretion of inflammatory cytokines (IL-6 and TNF- α) was determined by qRT-PCR; Cell death was determined using the TUNEL assay, measurement of cleaved caspase-3, and flow cytometry. Necroptosis-related proteins were determined by western blotting. RESULTS: In mice, DAB pretreatment improved renal function and also reduced histological injury, inflammation, cell death, and expression of necroptosis-associated proteins. In HK-2 cells, DAB significantly decreased the levels of NGAL and KIM-1, inflammatory cytokines, cell death, and necroptosis-related proteins. CONCLUSION: Our in vitro and in vivo experiments indicated that DAB appears to alleviate renal IRI by suppressing cell death and inhibiting inflammatory responses. DAB has potential use for the clinical prevention and treatment of AKI-induced IRI.

Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats.

The number and subtype composition of N-methyl-d-aspartate receptor (NMDAR) at synapses determines their functional properties and role in learning and memory. Genetically increased or decreased amount of GluN2B affects hippocampus-dependent memory in the adult brain. But in some experimental conditions (e.g., memory elicited by a single conditioning trial (1 CS-US)), GluN2B is not a necessary factor, which indicates that the precise role of GluN2B in memory formation requires further exploration. Here, we examined the role of GluN2B in the consolidation of fear memory using two training paradigms. We found that GluN2B was only required for the consolidation of memory elicited by five conditioning trials (5 CS-US), not by 1 CS-US. Strikingly, the expression of membrane GluN2B in CA1was training-strength-dependently increased after conditioning, and that the amount of membrane GluN2B determined its involvement in memory consolidation. Additionally, we demonstrated the increases in the activities of cAMP, ERK, and CREB in the CA1 after conditioning, as well as the enhanced intrinsic excitability and synaptic efficacy in CA1 neurons. Up-regulation of membrane GluN2B contributed to these enhancements. These studies uncover a novel mechanism for the involvement of GluN2B in memory consolidation by its accumulation at the cell surface in response to behavioral training.

A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences.

Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis.

Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

BACKGROUND: Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations. METHODS: Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence. RESULTS: We identified twelve amino acid deletions that can retain the fluorescence of GFP(UV). Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence. CONCLUSION: Our results suggested that a "size-minimized" GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.

Signaling mechanism underlying α2A -adrenergic suppression of excitatory synaptic transmission in the medial prefrontal cortex of rats.

Stimulation of α2A -adrenoceptors (ARs) in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. A previous study in our laboratory showed that α2A -AR stimulation suppresses excitatory synaptic transmission in layer V-VI pyramidal cells of the rat medial PFC (mPFC). However, the intracellular mechanism underlying the α2A -AR suppression remains unclear. In the present study, we recorded evoked excitatory postsynaptic current (eEPSC) in layer V-VI pyramidal cells of the mPFC, using whole-cell patch-clamp recording. We found that the α2A -AR agonist guanfacine significantly suppresses eEPSC in mPFC pyramidal cells. The α2A -AR inhibition is mediated by the Gi-cAMP-PKA-PP1-CaMKII-AMPAR signaling pathway, as such inhibition no longer exists when each step of this pathway is blocked with NF023, Rp-cAMP, PKI5-24 or H89, tautomycin, and KN-62 or KN-93, respectively.

Delay-dependent impairment of spatial working memory with inhibition of NR2B-containing NMDA receptors in hippocampal CA1 region of rats.

Hippocampal N-methyl-D-aspartate receptor (NMDAR) is required for spatial working memory. Although evidence from genetic manipulation mice suggests an important role of hippocampal NMDAR NR2B subunits (NR2B-NMDARs) in spatial working memory, it remains unclear whether or not the requirement of hippocampal NR2B-NMDARs for spatial working memory depends on the time of spatial information maintained. Here, we investigate the contribution of hippocampal NR2B-NMDARs to spatial working memory on delayed alternation task in T-maze (DAT task) and delayed matched-to-place task in water maze (DMP task). Our data show that infusions of the NR2B-NMDAR selective antagonists, Ro25-6981 or ifenprodil, directly into the CA1 region, impair spatial working memory in DAT task with 30-s delay (not 5-s delay), but severely impair error-correction capability in both 5-s and 30-s delay task. Furthermore, intra-CA1 inhibition of NR2B-NMDARs impairs spatial working memory in DMP task with 10-min delay (not 30-s delay). Our results suggest that hippocampal NR2B-NMDARs are required for spatial working memory in long-delay task, whereas spare for spatial working memory in short-delay task. We conclude that the requirement of NR2B-NMDARs for spatial working memory is delay-dependent in the CA1 region.