Volume 07 · Issue 11 · 2015

It has been believed that the most important pathophysiological changes in the progression of diabetic nephropathy (DN) to end-stage renal disease (ESRD) are irreversible glomerular fibrosis and scarring^[1]。 With further research, renal tubulopathy, especially fibrosis of renal tubules and renal interstitium, also plays an important role in the progression of DN^[2,3]。 Although there are many pathological types of DN, the degree of fibrosis is always an important marker of DN progression, from early diffuse thickening of the glomerular basement membrane to late glomerular sclerosis or tubular atrophy^[1,2]。

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease (ESRD), which occurs in about 30% of diabetic patients. At present, for the clinical treatment of DN, including controlling blood sugar, blood lipids, and blood pressure, the only drugs verified by evidence-based medicine to delay the progression of DN are angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB). However, even long-term use of sufficient amounts of ACEI or ARB in DN patients cannot completely prevent their progression to ESRD. Multiple clinical studies have shown that ACEI or ARB can reduce the risk of progression of DN to ESRD by 20% to 53%^[1]。 Therefore, new therapeutic targets need to be explored, and this depends on new exploration of the pathogenesis of DN.

To investigate the characteristics of the 24 h ambulatory blood pressure in type 2 diabetic patients with chronic kidney disease(CKD).

A total of 510 type 2 diabetic patients hospitalized from September 2009 to December 2013 were enrolled in this study. The patients were divided into six groups according to K/DOQI staging of CKD: non-CKD(94 cases), stage 1 CKD(134 cases), stage 2 CKD(122 cases), stage 3 CKD(80 cases), stage 4 CKD(36 cases) and stage 5 CKD(44 cases). The incidence of hypertension in those groups were calculated. The data were compared with one-way analysis of variance among groups. Multiple stepwise regression analysis was applied to evaluate the effect of every indicators on estimated glomerular filtration rate.

The incidence of hypertension increased significantly in type 2 diabetic patients with CKD, with a incidence rate of 95.4% in stage 5 patients(χ²=54.533, P<0.05). With the progression of CKD the prevalence of isolated systolic hypertension increased accordingly, with a proportion of 56.8% in stage 5 patients(χ²=29.456,P<0.05). While there was no significance in the prevalence of isolated diastolic hypertension and systolic-diastolic hypertension among the groups(χ²=8.477,10.505,P>0.05). The predictors of atherosclerosis, pulse pressure and ambulatory arterial stiffness index, increased significantly along with the progression of CKD(F=32.829, 10.134,P<0.05). The decrease of nighttime systolic and diastolic blood pressure declined along with CKD progression (F=17.827, 10.519,P<0.05). Multiple stepwise regression analysis indicated that estimated glomerular filtration rate was negatively correlated with systolic blood pressure and pulse pressure(β=-0.201,-0.237, bothP<0.01),and positively correlated with body mass index, percentage of night reduction rate of diastolic pressure and smoothness index of systolic blood pressure (β=0.134,0.151,0.145, allP<0.05).

The incidence of hypertension increased significantly in type 2 diabetic patients with CKD, with increasing of systolic blood pressure and pulse pressure and obvious abnormal circadian rhythm. So monitoring on blood pressure rhythm of these patients should be strengthened.

To investigate altered expression of two endothelin receptor subtypes, e.g. endothelin A- and B-receptors (ETAR and ETBR), in local renal tissue of patients with type 2 diabetic nephropathy (DN) with progression of diabetic nephropathy.

Twenty eight inpatients with type 2 diabetes mellitus(T2DM) who had biopsy-confirmed DN from October 2011 to February 2013 were divided into 3 groups by pathologic classification: DN Ⅰ grade (group S1,n=8), DN Ⅱ grade (group S2,n=10) and DN Ⅲ grade (group S3,n=10). The normal renal tissue apart from kidney tumor was collected from patients with kidney tumor as control group (group C, n=5). Double-labeling immunofluorescence was used to detect the distribution of ETAR and ETBR receptors. Semi-quantitative analysis of the expression in renal tissue was performed with immunohistochemistry. The rate of glomerulosclerosis was calculated, too. Data were anaylzed using ANOVA, correlation andq test.

(1) Mean age of patients with DN (including 13 male and 15 female) was (51±10) years old. No statistical difference in age, blood pressure, serum creatinine and albumin was observed among groups. Significant difference was observed in 24-hour proteinuria, which increased with the progression of DN. (2) The majority of ETAR and ETBR was expressed in the proximal tubular and few in glomrular. (3) The expression of ETAR was lower than that of ETBR in control group. The expression of ETAR progressively increased with DN aggravation. Statistical difference was observed among the groups (F=194.78, P<0.05). (4)The ratio of ETAR/ETBR in group C was 0.370±0.021, group S1 0.830± 0.188, group S2 1.230±0.093, group S3 2.110±0.142, respectively; There were statistical significance among groups (F=249.21, P<0.05). (5) The expression of ETAR and the ratio of ETAR/ETBR had a significant positive correlation with the course of disease, urine protein and the rate of glomerular sclerosis (r=0.522-0.833, allP<0.05) in patients with DN.

The increase of ETAR and ETBR in local renal tissue and inverted ratio of ETAR over ETBR were positively correlated to severity and progression of DN.

To explore the effects and mechanism of 1,25-dihydroxyvitamin D₃ on the diabetic nephropathy with different levels of vitamin D receptor (VDR) expression in rats.

The models of diabetic nephropathy SD rats (n=36) were established by intraperitoneal injection of streptozotoein, then the rat models were divided into 3 groups: VDR over-expression group (kidney injection of VDR virus,n=12), VDR normal expression group (kidney injection of viral vector,n=12) and VDR low expression group (kidney injection of VDR interfered virus,n=12). Then the rats in the three groups were given calcitriol (0.03 μg·kg ^-1·d^-1) or peanut oil for 37 days, six rats with one treatment in each group. So the rats were divided into 6 groups: VDR over-expression+VD group, VDR over-expression+peanut oil group, VDR low expression+VD group, VDR low expression+peanut oil group, VDR normal expression+VD group, VDR normal expression+peanut oil group. The real time-polymerase chain reaction (RT-PCR) and immunohistochemistry staining were used to detect the mRNA and protein levels of VDR, transforming growth factor(TGF)-β1, phosphatase and tensin homolog deleted on chromosome ten (PTEN), cologen-1(COL-1) in kidney. And the HE and Masson staining were used to detect changes of structure and the level of fibrosis in kidney. Data were compared by usingt-test and one-way analysis of variance.

The expressions of VDR and PTEN mRNA and protein in VDR over-expression+VD group were significantly higher than those in VDR over-expression+peanut oil group (t=-19.67, -5.18, -25.57, -10.06, allP<0.01). The expressions of TGF-β1 and COL-1 mRNA and protein in VDR over-expression+VD group significantly decreased than those in VDR over-expression+peanut oil group(t=15.62, 8.20, 28.28, 5.81, allP<0.01). The expressions of VDR and PTEN mRNA and protein in VDR low-expression+VD group were significantly higher than those in VDR low-expression+peanut oil group (t=-53.67, -7.00, -19.81, -8.06, allP<0.01). The expressions of TGF-β1 and COL-1 mRNA and protein in VDR low-expression+VD group decreased significantly than those in VDR low-expression+peanut oil group(t=12.00, 8.50, 21.22, 5.42, allP<0.01). The expressions of VDR and PTEN mRNA and protein in VDR normal expression+VD group were significantly higher than those in VDR normal expression+peanut oil group (t=-20.39, -5.46, -25.92, -6.38, allP<0.01). The expression of TGF-β1 and COL-1 mRNA and protein in VDR normal expression+VD group significantly decreased than those in VDR normal expression+peanut oil group (t=17.80, 10.61, 47.36, 8.23, allP<0.01). The pathological structure of the kidney in kidney disorders was associated with the low levels of VDR. These fibrosis changes could be improved by calcitriol interventions.

The 1,25-dihydroxyvitamin D₃ has some anti-fibrosis effects on diabetic nephropathy by the pathway of VDR in kidney.

To investigate the effect and mechanism of glucagon like peptide-1(GLP-1) on endothelial cell endoplasmic reticulum stress (ER stress) and apoptosis induced by constant or fluctuating high glucose.

Cultured human umbilical vein endothelial cells (HUVECs) were divided into 6 groups: normal glucose control (cultured with 5.5 mmol/L glucose), constant high glucose (cultured with 30 mmol/L glucose), fluctuating high glucose (cultured with high glucose and normal glucose, changed every 12 hours), normal glucose control+10 nmol/L GLP-1, constant high glucose+10 nmol/L GLP-1, fluctuating high glucose+10 nmol/L GLP-1. The later 3 groups were pretreated with 10 nmol/L GLP-1 for 30 minutes, then cultured with corresponding glucose. After treatment for 48 h, the apoptosis rate of HUVECs was measured by flow cytometer, the glucose regulated proteins 78(GRP78) expression level was measured by Western blotting methods. HUVECS were treated by 30 mmol/L high glucose plus GLP-1 or not, then GRP78 expression level at different times (0, 3, 6, 12, 24, 48 h) was measured by using Western blotting methods. Differences between two groups were compared by LSD-t test, differences among more than two groups were compared by one-way analysis of variance.

Assuming apoptosis rate of normal control group was 1, the apoptosis rate in fluctuating high glucose group was (1.2±0.05), which was significantly higher than that of control group(F=45.086, P<0.05); After GLP-1 was added, the apoptosis rate of cells in fluctuating high glucose group decreased to 0.87±0.01, which was significantly lower than that in fluctuating high glucose group (t=17.4,P<0.05). After treatment of 30 mmol/L high glucose for 3 hours, GRP78 expression in HUVECS increased to (1.62±0.48), which was significantly higher than that of in control group (treated with high glucose for 0 h, GRP78 expression assumed as 1,t=2.584,P<0.05); after 6 hours, GRP78 expression peaked to 2.82±0.59, and was significantly higher than that in control group(t=6.123,P<0.05); 12 hours later, GRP78 expression began to decline and it decreased to 0.98±0.59 after treated with high glucose for 48 hours, no significant difference was found between it and that in control group(t=0.378,P>0.05). The GRP78 expression in constant high glucose group was 0.96±0.10, while it was 0.77±0.04 in fluctuating high glucose group(t=3.134,P<0.05).

Both constant and fluctuating high glucose can induce ER stress and apoptosis in HUVECS, the latter has stronger effect by further inhibition of GRP78 expression, GLP-1 can alleviate the ER stress and apoptosis by inducing the expression of GRP78.

To study the effect of intermittent hyperglycemia on rat insulinoma cell line Ins-1 apoptosis and the expression of phosphatase and tensin homologue-deleted chromosome ten gene (PTEN) in those cells.

Ins-1 cells were divided into three groups: control group (CG, cultured in 11.1 mmol/L glucose), sustained high glucose group (SHG, cultured in 33.3 mmol/L glucose) and intermittent high glucose group (IHG, cultured in 33.3 or 11.1 mmol/L glucose each for 12 h) respectively. After three days of incubation, cell viability was determined by Owen's reagent(MTS), apoptosis rate was detected by flow cytometry after staining with Annexin V/PI, insulin secretion was measured but radioimmunoassay, intracellular reactive oxygen species(ROS) was detected by DCFH-DA fluorescence probe, the morphology of cells of the three groups were observed under inverted phase contrast microscope. The data was compared with one-way ANOVA analysis.

Compared with CG group, the cellular apoptosis rate, intracellular ROS levels, calcium levels, and the expressions of PTEN protein in cells of SHG group were all significantly increased (t=8.310, 10.261, 17.890, 5.123, allP<0.05), while cell viability and insulin secretion decreased significantly(t=-19.630, -2.053, bothP<0.05). However, compared to both of the CG group and the SHG group, the cellular apoptosis rate, intracellular ROS levels, intracellular calcium levels, and the expression of PTEN protein in cells of IHG group increased significantly(t=6.20-18.227, all P<0.05), while cell viability and insulin secretion decreased significantly(t=-27.800, -2.79, -8.167, -1.503, allP<0.05). Compared to CG group and the SHG group, the pleomorphism of the cells, the reduction in cell number and the disarrangement of cells in the IHG group were all the most significant.

Oxidative stress may play an important role in the apoptosis of Ins-1 cells under high glucose environment and it may be related to the high expressions of PTEN. The adverse effect of intermittent high glucose on the Ins-1 cell-line may be greater than that of sustained high glucose.

To observe the effects of islet amyloid polypeptide (IAPP) on K_ATP channel in rat islet cell tumor cell line Ins-1 stimulated by high concentration of glucose.

Ins-1 cells, as the research object, were randomly divided into 16.7 mmol/L glucose control group and 0.1, 1, 10 μmol/L amylin pretreatment group. Whole cell patch clamp technique was employed to study the influences of short exposure to IAPP on electrophysiological characteristics of K _ATP channel upon glucose stimulation. Meanwhile, insulin secretion was measured by enzyme-linked immunoassay. Group data were compared by usingt test with theP value set at 0.05.

The insulin secretion and half-maximal activation voltage (V_0.5) of K_ATP in cells incubated with 16.7 mmol/L glucose were (5.57±0.93) μg/L and (9.4±1.4) mV, respectively; and those were (4.95±0.49)μg/L and (13.0±2.4) mV in cells pretreated with 0.1 μmol/L amylin; and there was no significant differences between the two groups( t=1.022, -2.244, bothP>0.05). While the insulin secretion in cells pretreated with 1 μmol/L and 10 μmol/L amylin significantly decreased ((3.44±0.32)μg/L and (2.23±0.55) μg/L, respectively) and the V_0.5 increased significantly((28.2±2.7) mV and (33.3±2.1) mV, respectively) when compared with those in cells treated with 16.7 mmol/L glucose (t=3.751, 5.354, -5.053, -10.707, allP<0.05).

Short-term exposure to high concentration of amylin inhibites the closure of ATP-sensitive potassium channels upon glucose stimulation, thus may ultimately lead to the reduction of insulin secretion in Ins-1 cells.

To explore the effect of 1,25-dihydroxy vitamin D₃ (VD) on the expression of C-Jun N-terminal kinase/C-Jun (JNK/C-Jun) inflammatory pathway in the liver of type 2 diabetes mellitus (T2DM) rats and the possible mechanisms.

Sprague Dawley(SD) rats were divided into normal control group(NC), T2DM group(DM) and calcitriol-treated group(VD) via random number table. Calcitriol was given to VD group by gavage for 8 weeks, while the DM and NC group received peanut oil. After sacrifice, body weight was measured and fasting blood glucose(FBG), alanine transaminase(ALT), asparate transaminase(AST), total cholesterol(TC), triacylglycerol(TG) in serum were tested. Liver histopathology was examined by hematoxylin-eosin (HE) staining. The mRNA levels of JNK, C-Jun and its downstream cytokines tumor necrosis factor(TNF-α) and interleukin(IL-1β) were measured by real-time fluorescence quantitative PCR. The protein expression was analyzed by immunohistochemical staining and Western blotting.Thet test was performed for pair-wise comparison and wilcoxon rank sum test was used for ranked data.

The level of FBG, ALT, AST, TC and TG in DM group increased and body weight decreased compared with those in NC group((25.7±2.3)vs(5.8±0.9) mmol/L, (137±12)vs(53±6)U/L,(162±13)vs(65±7)U/L, (1.4±0.2)vs(1.21±0.08)mmol/L, (1.32±0.15)vs(0.73±0.10)mmol/L,(362±12)vs(452±16) g;t=29.573,26.142,14.395, 10.632,11.746,9.539, respectively, allP<0.05). Compared with DM group, ALT,AST and TG decreased significantly in VD group((112±10)vs(137±12) U/L, (129±11)vs (162±13) U/L, (1.06±0.14)vs (1.32±0.15) mmol/L;t=5.382,6.043,5.268, allP<0.05). HE staining showed that liver cells were in alignment and normal in NC group, whereas appeared swelling and fatty degeneration with inflammatory cells infiltration in DM group. VD treatment could alleviate the pathologic changes. The mRNA and protein levels of JNK, C-Jun, TNF-α and IL-1β in DM group increased compared with those in NC group, VD treatment down-regulated this inflammatory factors compared with DM group(χ²=19.958, 20.710, 20.969, 21.255, respectively, allP<0.05). JNK was positive correlated significantly with C-Jun, TNF-α, IL-1β (r=0.857,0.821,0.836,P<0.05).

1,25(OH)₂D₃ may protect diabetes-induced liver complications by down-regulating the inflammatory pathway of JNK/C-Jun.

Adult occult autoimmune diabetes mellitus (LADA) suffers slow immune damage to pancreatic islet β cells, and progressively reduces insulin secretion, which ultimately requires insulin therapy. Dumping syndrome is one of the causes of functional hypoglycemia. We recently treated a patient with LADA complicated with dumping syndrome, which is presented below.

Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes. In recent years, as the prevalence of diabetes has increased year by year, the prevalence of DN has also increased significantly. In some parts of China, up to one-third of diabetic patients have kidney complications^[1]。 DN is one of the main causes of end-stage renal disease, and it is also a high risk factor for cardiovascular events such as coronary heart disease and stroke. Recent studies have shown that in addition to glucose and lipid metabolism disorders and hemodynamic abnormalities, chronic inflammation, oxidative stress, fibrosis and other pathological processes are involved in the occurrence and development of DN^[2]。

The prevention and treatment of diabetic peripheral neuropathy (DPN) has always been a difficult point in clinical practice. The occurrence of DPN is related to hyperglycemia, dyslipidemia, insulin resistance, oxidative stress caused by chronic inflammation, vascular ischemia and hypoxia, etc. The treatment of DPN mainly includes: (1) strengthening blood sugar control and risk factor management; (2) treatment for pathogenesis; (3) Pain relief. Pain is the main symptom of DPN, and about 1/3 of DPN patients may have neuralgia^[1]and the incidence of neuralgia in patients with type 2 diabetes mellitus (T2DM) is higher than in patients with type 1 diabetes mellitus (T1DM). The mechanism of neuralgia is still not fully understood. Possible mechanisms include peripheral mechanism and central mechanism theory^[2]。 Since numbness, tingling, burning pain and other symptoms of DPN seriously affect patients' life, often cause insomnia, and even cause pessimism and disappointment because of unbearable disturbance, resulting in suicidal tendency. Therefore, symptomatic pain relief is an important link in DPN treatment^[3]。

The prevention and treatment of diabetic complications is the key to the treatment of diabetes, and it is also the decisive factor of the quality of life and longevity of diabetic patients. Microvascular disease is its characteristic complication, mainly including diabetic nephropathy, diabetic retinopathy and diabetic peripheral neuropathy, which is the main cause of high disability and mortality rate of diabetic patients. The complexity of its pathogenesis makes multi-target therapy possible. Glucagon-like peptide 1 (GLP-1) is an incretin secreted by intestinal L cells. After specifically binding to GLP-1 receptor (GLP-1R) on pancreatic β cells, it functions through adenosine cyclic phosphate (cAMP) as a second messenger signaling pathway to induce voltage-gated Ca²⁺The channel is opened to promote intracellular insulin synthesis and secretion, and achieve glucose-dependent blood glucose regulation. Natural GLP-1 has a very short half-life and can be rapidly degraded by dipeptidyl peptidase IV (DPP-4)^[1]。 At present, there are two main classes of drugs for the treatment of type 2 diabetes based on GLP-1: DPP-4 inhibitors and GLP-1R agonists (GLP-1RA)^[2]。 Exenatide is a commonly used GLP-1R agonist in clinical practice, which has 53% homology with GLP-1, and can resist its degradation by DPP-4^[3]。 GLP-1R belongs to G protein-coupled receptor family, which is widely distributed and expressed in nervous system, eye and kidney tissues^[4,5]and can mediate the different biological roles of GLP-1 in different tissues^[3]Therefore, it can be speculated that GLP-1 may play an important role in the development of diabetic microvascular complications, and has potential therapeutic value. Currently, large prospective trials clinically based on the effects of incretin drugs on related complications in patients with type 2 diabetes are ongoing, but the results have not been published. Therefore, this paper is still mainly limited to a review of the related scientific research achievements obtained in animal experiments and cell culture.

The development of insulin has experienced 3 milestones. The first generation of insulin is animal insulin, the second generation is recombinant human insulin, and the third generation is insulin analogs. The latter solves the problem that human insulin preparations cannot well simulate the physiological insulin secretion pattern. According to the rate of onset of action, insulin analogs can be divided into: fast-acting insulin analogs (including aspart, lispro and insulin glulain) and long-acting insulin analogs (including insulin glargine and insulin detemir). The onset time of subcutaneous injection of conventional human insulin (RHI) is 30 to 45 minutes, but more than 75% of patients cannot inject insulin 30 minutes before meals. Therefore, rapid-acting insulin analogs with rapid onset of action are particularly important for postprandial blood glucose management. Insulin glulain is the latest member of the family of fast-acting human insulin analogs, which was launched in China at the end of 2012. This article reviews the pharmacological properties, clinical efficacy and tolerability of insulin glulain as follows.

From April 15 to 18, 2015, the 8th International Symposium on Pregnancy Complicated by Diabetes, Hypertension and Metabolic Syndrome (hereinafter referred to as DIP) was held in Berlin, Germany. The meeting focused on the current status of diagnosis and treatment of gestational hyperglycemia in various countries, reviewed the historical origin of gestational diabetes mellitus (GDM), the changes in diagnostic criteria and the huge controversy that still exists, and reaffirmed the importance of hyperglycemia and adverse pregnancy outcomes (HAPO) research, that is, pregnant women who do not meet the diagnostic criteria for diabetes will also have a significant increase in the risk of adverse pregnancy outcomes with the increase of gestational blood glucose^[1]。 The conference also looks ahead to the ongoing HAPO follow-up study, which will explore the long-term effects of varying degrees of hyperglycemia during pregnancy on the health of pregnant women and offspring. In addition, the meeting summarized the application of insulin analogs in blood glucose management during pregnancy in recent years, and believed that the fast-acting insulin analogue insulin aspart is superior to human insulin in controlling postprandial blood glucose and reducing the risk of hypoglycemia, and the long-acting insulin analogue insulin detemir is comparable to human insulin in safety and efficacy, and is superior to human insulin in some aspects. This meeting focused on the comprehensive discussion of hot issues related to hyperglycemia in pregnancy, which triggered our thinking on the current situation and future of diabetes management in pregnancy in China.