Establishment and validation of the cut-off values of estimated glomerular filtration rate and urinary albumin-to-creatinine ratio for diabetic kidney disease: A multi-center, prospective cohort study.

Objective: We aimed to study the cut-off values of estimated glomerular filtration rate (eGFR) and the urinary albumin creatinine ratio (UACR) in the normal range for diabetic kidney disease (DKD). Methods: In this study, we conducted a retrospective, observational cohort study included 374 type 2 diabetic patients who had baseline eGFR ≥60 mL/min/1.73 m2 and UACR <30 mg/g with up to 6 years of follow-up. The results were further validated in a multi-center, prospective cohort study. Results: In the development cohort, baseline eGFR (AUC: 0.90, cut-off value: 84.8 mL/min/1.73 m2, sensitivity: 0.80, specificity: 0.85) or UACR (AUC: 0.74, cut-off value: 15.5mg/g, sensitivity: 0.69, specificity: 0.63) was the most effective single predictor for DKD. Moreover, compared with eGFR or UACR alone, the prediction model consisted of all of the independent risk factors did not improve the predictive performance (P >0.05). The discrimination of eGFR at the cut-off value of 84.80 mL/min/1.73 m2 or UACR at 15.5mg/g with the largest Youden's index was further confirmed in the validation cohort. The decrease rate of eGFR was faster in patients with UACR ≥15.5mg/g (P <0.05). Furthermore, the decrease rate of eGFR or increase rate of UACR and the incidence and severity of cardiovascular disease (CVD) were higher in patients with eGFR ≤84.8 mL/min/1.73 m2 or UACR ≥15.5mg/g (P <0.05). Conclusions: In conclusion, eGFR ≤84.8mL/min/1.73 m2 or UACR ≥15.5mg/g in the normal range may be an effective cut-off value for DKD and may increase the incidence and severity for CVD in type 2 diabetic patients.

Beta-Hydroxybutyric Acid Inhibits Renal Tubular Reabsorption via the AKT/DAB2/Megalin Signalling Pathway.

Aim: Patients with diabetic ketosis often exhibit albuminuria. We previously found that acute hyperglycaemia can cause nephrotoxic injury. Here, we explored whether an excessive ketone body level causes kidney injury and the potential underlying mechanism. Methods: Fifty-six type 2 diabetes without ketosis (NDK group), 81 type 2 diabetes with ketosis (DK group), and 38 healthy controls (NC group) were enrolled. Clinical data were collected before and after controlling diabetic ketosis. Beta-hydroxybutyric acid (BOHB), an AKT activator, an AKT inhibitor, or plasmids encoding DAB2 were transformed into human renal proximal tubule epithelial cells (HK-2 cells). Results: The urinary albumin-to-creatinine ratio (ACR), transferrin (TF), immunoglobulin G (IgG), Beta2-microglobulin (ß2-MG), retinol-binding protein (RBP), N-acetyl-beta-glucosaminidase (NAG), and Beta-galactosidase (GAL) were higher in the DK than NC and NDK groups. The proportion of patients with an increased urinary level of TF, IgG, ß2-MG, RBP, NAG, or GAL was higher in the DK group too. After controlling ketosis, urinary microalbumin, TF, IgG, ß2-MG, and RBP decreased significantly. In HK-2 cells, albumin endocytosis and megalin expression decreased with increasing BOHB concentration. Compared with BOHB treatment, BOHB with AKT activator significantly increased the DAB2, megalin levels and albumin endocytosis; the AKT inhibitor treatment exhibited the opposite effects. Compared with BOHB treatment, megalin expression and albumin endocytosis were significantly increased after BOHB with DAB2 overexpression treatment. Conclusions: Patients with diabetic ketosis may suffer from glomerular and tubular injuries that recover after ketosis control. High concentrations of BOHB downregulate megalin expression by inhibiting the AKT/DAB2/megalin signalling pathway and albumin endocytosis in proximal renal tubules.

Hyperinsulinemia impairs the metabolic switch to ketone body utilization in proximal renal tubular epithelial cells under energy crisis via the inhibition of the SIRT3/SMCT1 pathway.

Objective: To investigate the effects and mechanism of hyperinsulinemia on the metabolic switch to ß-hydroxybutyrate (BHB) absorption and utilization under a starvation or hypoxic environment in proximal tubular epithelial cells. Methods: A high-fat diet-induced hyperinsulinemia model in ZDF rats was used to test the expression of key enzymes/proteins of ketone body metabolism in the kidney. Notably, 12-week-old renal tubule SMCT1 specific knockout mice (SMCT1 flox/floxCre+) and control mice (SMCT1 flox/floxCre-) were used to confirm the roles of SMCT1 in kidney protection under starvation. The changes of key enzymes/proteins of energy metabolism, mitochondrial function, and albumin endocytosis in HK2 cells under low glucose/hypoxic environments with or without 50 ng/mL insulin were studied. Silent information regulation 2 homolog 3 (SIRT3) was overexpressed to evaluate the effect of hyperinsulinemia on the metabolic switch to BHB absorption and utilization through the SIRT3/SMCT1 pathway in HK2 cells. Results: In ZDF rats, the expression of HMGCS2 increased, the SMCT1 expression decreased, while SCOT remained unchanged. In renal tubule SMCT1 gene-specific knockout mice, starvation for 48 h induced an increase in the levels of urine retinol-binding protein, N-acetyl-ß-glucosaminidase, and transferrin, which reflected tubular damages. In HK2 cells under an environment of starvation and hypoxia, the levels of key enzymes related to fatty acid oxidation and ketone body metabolism were increased, whereas glucose glycolysis did not change. The addition of 2 mmol/l BHB improved ATP production, mitochondrial biosynthesis, and endocytic albumin function, while cell apoptosis was reduced in HK2 cells. The addition of 50 ng/ml insulin resulted in the decreased expression of SMCT1 along with an impaired mitochondrial function, decreased ATP production, and increased apoptosis. The overexpression of SIRT3 or SMCT1 reversed these alterations induced by a high level of insulin both in low-glucose and hypoxic environments. Conclusions: The increased absorption and utilization of BHB is part of the metabolic flexibility of renal tubular epithelial cells under starvation and hypoxic environments, which exhibits a protective effect on renal tubular epithelial cells by improving the mitochondrial function and cell survival. Moreover, hyperinsulinemia inhibits the absorption of BHB through the inhibition of the SIRT3/SMCT1 pathway.

Sex-specific mediating effect of gestational weight gain between pre-pregnancy body mass index and gestational diabetes mellitus.

BACKGROUND: Inappropriate weight gain may increase the risk of gestational diabetes mellitus (GDM). However, the relationship between pre-pregnancy body mass index (BMI), weight gain, and GDM has not been precisely quantified. This study aimed to explore whether gestational weight gain played a mediating role between pre-pregnancy BMI and GDM and whether the mediating effect was sex specific. METHODS: This study established a population-based observational cohort to assess weight gain in pregnant women. Mediation analyses were performed to quantify whether weight gain mediated the association between pre-pregnancy BMI and GDM. RESULTS: A total of 67,777 pregnant women were included in the final analysis, among whom 6751 (10.0%) were diagnosed with GDM. We verified that both pre-pregnancy BMI and weight gain were associated with GDM, and that BMI negatively contributed to weight gain. We also found that weight gain had a significant mediating effect on the relationship between pre-pregnancy BMI and GDM (Za × Zb confidence intervals [CIs] 0.00234-0.00618). Furthermore, the effect was sex-specific, in that it was only significant in overweight women carrying female fetuses (Za × Zb CIs 0.00422-0.01977), but not male fetuses (Za × Zb CIs -0.00085 to 0.01236). CONCLUSIONS: Weight gain during pregnancy had a fetal sex-specific mediating effect between pre-pregnancy BMI and GDM.

Pathological Hyperinsulinemia and Hyperglycemia in the Impaired Glucose Tolerance Stage Mediate Endothelial Dysfunction Through miR-21, PTEN/AKT/eNOS, and MARK/ET-1 Pathways.

Background: Impaired glucose tolerance (IGT) is an important prediabetic stage characterized by elevated concentrations of glucose and insulin in the blood. The pathological hyperglycemia and hyperinsulinemia in IGT may regulate the expression of microRNA-21 (miR-21) and affect the downstream insulin signaling pathways, leading to endothelial cell dysfunction and early renal damage. Methods: The individual and combined effects of insulin and glucose were investigated using human glomerular endothelial cells (HGECs). The expression levels of miR-21, and PTEN/AKT/eNOS and MAPK/ET-1 pathway proteins in the treated cells were measured. The levels of nitric oxide (NO) and endothelin-1 (ET-1) secreted by the cells were also measured. The role of miR-21 in mediating the regulatory effects of insulin and glucose was assessed by overexpression/inhibition of this miRNA using mimics/inhibitor. Results: High (>16.7 mmol/L) concentration of glucose upregulated the expression of miR-21, leading to the activation and inhibition of the PTEN/AKT/eNOS and MAPK/ET-1 pathways, and upregulation of NO and downregulation of ET-1 secretion, respectively. High (>25 ng/mL) concentration of insulin downregulated the expression of miR-21, and lead to the activation of the MAPK/ET-1 and inhibition of the PTEN/AKT/eNOS pathway, thereby upregulating the expression of ET-1 and downregulating the secretion of NO. MiR-21 was observed to play a key role by directly controlling the activation of the insulin signaling pathways when the cells were cotreated with different concentrations of insulin and glucose. The expression of miR-21 was found to be dependent on the relative concentration of insulin and glucose. Under simulated conditions of the IGT stage (8.3 mmol/L glucose + 50 ng/mL insulin), the inhibitory effect of high insulin concentration on miR-21 expression in the cells attenuated the activation by high glucose concentration, resulting in the downregulation of miR-21, upregulation of ET-1 and downregulation of NO secretion. Conclusion: Taken together, these results indicate that high insulin and glucose concentrations regulate the secretory function of glomerular endothelial cells in opposite ways by regulating the expression of miRNA-21. Pathological concentrations of insulin and glucose in the IGT stage may lead to a decrease in miR-21 expression, thereby disordering the secretion of vasoactive factors, resulting in renal tubule ischemia.

Hyperinsulinemia contributes to impaired-glucose-tolerance-induced renal injury via mir-7977/SIRT3 signaling.

BACKGROUND: Increasing evidence indicates that impaired glucose tolerance (IGT) is independently associated with chronic kidney disease, but the characteristics and underlying mechanisms remain largely unknown. METHODS: Here, the cross-sectional study was performed to study the characteristics of IGT-induced renal injury (IGT-RI). Furthermore, urine microRNA profile was evaluated and microRNAs involved in tubular injury were determined by in-vitro experiments. RESULTS: It was found that 12.1% of IGT patients had microalbuminuria, which we termed "IGT-RI." Overall, 100% of patients with IGT-RI exhibited reabsorption dysfunction and 58.3% had structural damage in the renal tubules. Two-hour postprandial insulin, retinol-binding protein, and N-acetyl-ß-glucosaminidase were significantly associated with microalbuminuria and they were independent risk factors for IGT-RI. The expression of mir-7977 was altered in IGT-RI patients and may be involved in cellular response to oxidative stress. In proximal tubule epithelial cells in vitro, a high level of insulin increased the expression of mir-7977 and decreased that of sirtuin 3 (SIRT3), leading to oxidative stress. Overexpression of mir-7977 further decreased SIRT3 expression, whereas inhibition of mir-7977 had the opposite effect. Furthermore, mir-7977 can bind to the 3'-untranslated region of SIRT3 mRNA and inhibit its expression. Moreover, inhibition of SIRT3 reduced the expression of cubilin and the endocytosis of albumin. CONCLUSIONS: In conclusion, IGT-RI mainly manifests as tubular injury, especially reabsorption dysfunction. Compensatory hyperinsulinemia may be involved. A high level of insulin can activate mir-7977/SIRT3 signaling, resulting in tubular injury by inducing oxidative stress as well as reabsorption dysfunction by inhibiting the expression of cubilin, ultimately contributing to IGT-RI.

Preventive Role of Salsalate in Diabetes Is Associated With Reducing Intestinal Inflammation Through Improvement of Gut Dysbiosis in ZDF Rats.

A safe and effective approach is needed to prevent and reduce the incidence of diabetes worldwide. The hypoglycemic efficacy of salicylic acid (salsalate, SAL), which has anti-inflammatory properties, has been empirically demonstrated in studies conducted at the Joslin Diabetes Center and elsewhere. Here, we investigated the potential role of SAL in preventing the onset of diabetes in Zucker diabetic fatty (ZDF) rats and attempted to elucidate its underlying mechanisms. ZDF and Zucker lean (ZL) rats were administered a high-fat diet with or without SAL intervention, and their relative rates of diabetes were compared. Our results showed that all rats in the placebo group developed diabetes, whereas only 10% of the SAL-treated rats presented with impaired glucose tolerance (IGT). None of the latter progressed to diabetes. Relative to the untreated rats, SAL lowered plasma glucagon and insulin while improving insulin sensitivity and ß-cell function. SAL may protect against hyperglycemia by increasing the microbial diversity, ameliorating gut dysbiosis, restoring intestinal epithelial cell connections, inhibiting endotoxin influx into the blood, and attenuating inflammation. Together, these findings suggest that SAL may be a candidate prophylactic therapy against diabetes. The protective role of SAL may be attributed to its ability to reduce intestinal inflammation and improve gut dysbiosis.

Establishment and Validation of a Risk Prediction Model for Early Diabetic Kidney Disease Based on a Systematic Review and Meta-Analysis of 20 Cohorts.

BACKGROUND: Identifying patients at high risk of diabetic kidney disease (DKD) helps improve clinical outcome. PURPOSE: To establish a model for predicting DKD. DATA SOURCES: The derivation cohort was from a meta-analysis. The validation cohort was from a Chinese cohort. STUDY SELECTION: Cohort studies that reported risk factors of DKD with their corresponding risk ratios (RRs) in patients with type 2 diabetes were selected. All patients had estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 and urinary albumin-to-creatinine ratio (UACR) <30 mg/g at baseline. DATA EXTRACTION: Risk factors and their corresponding RRs were extracted. Only risk factors with statistical significance were included in our DKD risk prediction model. DATA SYNTHESIS: Twenty cohorts including 41,271 patients with type 2 diabetes were included in our meta-analysis. Age, BMI, smoking, diabetic retinopathy, hemoglobin A1c, systolic blood pressure, HDL cholesterol, triglycerides, UACR, and eGFR were statistically significant. All these risk factors were included in the model except eGFR because of the significant heterogeneity among studies. All risk factors were scored according to their weightings, and the highest score was 37.0. The model was validated in an external cohort with a median follow-up of 2.9 years. A cutoff value of 16 was selected with a sensitivity of 0.847 and a specificity of 0.677. LIMITATIONS: There was huge heterogeneity among studies involving eGFR. More evidence is needed to power it as a risk factor of DKD. CONCLUSIONS: The DKD risk prediction model consisting of nine risk factors established in this study is a simple tool for detecting patients at high risk of DKD.

Acute Hyperglycemia May Induce Renal Tubular Injury Through Mitophagy Inhibition.

Aim: Acute hyperglycemia is closely related to kidney injury. Oxidative stress activation and notable mitochondria damages were found under acute hyperglycemia treatment in our previous work. In the present study, we explored the dose-effect relationship and the pivotal role of mitophagy in acute hyperglycemia induced tubular injuries. Methods: Forty non-diabetic SD rats were randomly divided and treated with different concentrations of hyperglycemia respectively during the 6-h clamp experiment. Renal morphological and functional alterations were detected. Rat renal tubular epithelial cells were treated with different concentrations of glucose for 6 h. Markers and the regulation pathway of mitophagy were analyzed. Results: Significant tubular injuries but not glomeruli were observed under both light and electron microscope after acute hyperglycemia treatment, which manifested as enlargement of tubular epithelial cells, disarrangement of epithelial cell labyrinths and swelling of mitochondria. Urinary microalbumin, ß2-MG, CysC, NAG, GAL, and NGAL were increased significantly with the increase of blood glucose (P < 0.05). ROS was activated, mitochondrial membrane potential and LC3-II/LC3-I ratio were decreased but P62 and BNIP3L/Nix were increased in hyperglycemia groups (P < 0.05), which were reversed by AMPK activation or mTOR inhibition. Conclusion: Acute hyperglycemia causes obvious tubular morphological and functional injuries in a dose-dependent manner. Acute hyperglycemia could inhibit mitophagy through AMPK/mTOR pathway, which would aggravate mitochondria damage and renal tubular impairment.

Evaluation of urinary biomarkers for prediction of diabetic kidney disease: a propensity score matching analysis.

BACKGROUND: The aim of this study was to evaluate the diagnostic value of six urinary biomarkers for prediction of diabetic kidney disease (DKD). METHODS: The cross-sectional study recruited 1053 hospitalized patients with type 2 diabetes mellitus (T2DM), who were categorized into the diabetes mellitus (DM) with normoalbuminuria (NA) group (n = 753) and DKD group (n = 300) according to 24-h urinary albumin excretion rate (24-h UAE). Data on the levels of six studied urinary biomarkers [transferrin (TF), immunoglobulin G (IgG), retinol-binding protein (RBP), ß-galactosidase (GAL), N-acetyl-beta-glucosaminidase (NAG), and ß2-microglobulin (ß2MG)] were obtained. The propensity score matching (PSM) method was applied to eliminate the influences of confounding variables. RESULTS: Patients with DKD had higher levels of all six urinary biomarkers. All indicators demonstrated significantly increased risk of DKD, except for GAL and ß2MG. Single RBP yielded the greatest area under the curve (AUC) value of 0.920 compared with the other five markers, followed by TF (0.867) and IgG (0.867). However, GAL, NAG, and ß2MG were shown to have a weak prognostic ability. The diagnostic values of the different combinations were not superior to the single RBP. CONCLUSIONS: RBP, TF, and IgG could be used as reliable or good predictors of DKD. The combined use of these biomarkers did not improve DKD detection.

Renal impairment markers in type 2 diabetes patients with different types of hyperuricemia.

AIMS/INTRODUCTION: Hyperuricemia (HUA) occurs because of decreased excretion of uric acid, increased synthesis of uric acid or a combination of both mechanisms. The proportions of these three types of HUA in type 2 diabetes patients are not known. In the mean time, we assume that different types of HUA might manifest with different renal damage, even in patients with normal renal filtration function. MATERIALS AND METHODS: We included 435 inpatients with type 2 diabetes at the Metabolic Disease Hospital of Tianjin Medical University from 2015 to 2016. Based on the clearance of uric acid, 90 patients with HUA were divided into three types: synthesis-increased HUA, excretion-decreased HUA and mixed type of HUA. RESULTS: Patients with the mixed type of HUA had the severest kidney injury manifested by a high level of 24 h urinary microalbumin, urinary immunoglobulin G, transferrin, α-galactosidase and ß2-microglobulin compared with the normal uric acid group. Urinary immunoglobulin G, transferrin and α-galactosidase were also increased in patients with synthesis-increased HUA compared with the normal uric acid group. Patients with excretion-decreased HUA did not have an increased level of renal impairment markers; however, these patients had an increased body mass index, which might cause dysfunction of kidney excretion. CONCLUSIONS: Excretion-decreased HUA is a more common type of HUA in type 2 diabetes patients that might be caused by dysfunction of tubular excretion instead of structural damage. The mixed type of HUA patients had the severest kidney glomerular and tubular damage compared with the normal uric acid group. Clinically, different types of hyperuricemia should be given individualized treatment according to their own characteristics.