Multiplex Bead Array Assay of a Panel of Circulating Cytokines and Growth Factors in Patients with Albuminuric and Non-AlbuminuricDiabetic Kidney Disease.

A panel of cytokines and growth factors, mediating low-grade inflammation and fibrosis, was assessed in patients with type 2 diabetes (T2D) and different patterns of chronic kidney disease (CKD). Patients with long-term T2D (N = 130) were classified into four groups: no signs of CKD; estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 without albuminuria; albuminuria and eGFR ≥60 mL/min/1.73 m2; albuminuria and eGFR <60 mL/min/1.73 m2. Thirty healthy subjects were acted as control. Twenty-seven cytokines and growth factors were assessed in serum by multiplex bead array assay. Serum hs-CRP, urinary nephrin, podocine, and WFDC2 were measured by ELISA. Patients with T2D showed elevated IL-1Ra, IL-6, IL-17A, G-CSF, IP-10, MIP-1α, and bFGF levels; concentrations of IL-4, IL-12, IL-15, INF-γ, and VEGF were decreased. IL-6, IL-17A, G-CSF, MIP-1α, and bFGF correlated negatively with eGFR; IL-10 and VEGF demonstrated negative associations with WFDC2; no relationships with podocyte markers were found. Adjusted IL-17A and MIP-1α were predictors of non-albuminuric CKD, IL-13 predicted albuminuria with preserved renal function, meanwhile, IL-6 and hsCRP were predictors of albuminuria with eGFR decline. Therefore, albuminuric and non-albuminuric CKD in T2D patients are associated with different pro-inflammatory shifts in the panel of circulating cytokines.

SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes.

Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.

Circulating Wnt1-inducible signaling pathway protein-1 (WISP-1/CCN4) is a novel biomarker of adiposity in subjects with type 2 diabetes.

BACKGROUND: Wnt1-inducible signaling pathway protein 1, or cellular communication network factor 4 (CCN4), a member of CCN family of secreted, extracellular matrix associated signaling proteins, recently was validated as a novel adipose tissue derived cytokine. OBJECTIVE: To assess the relationships between circulating CCN4, adipose tissue distribution and function, and chronic low-grade inflammation in subjects with type 2 diabetes. METHODS: We observed 156 patients with type 2 diabetes and 24 healthy controls. Serum levels of CCN4, hsCRP and alpha1-acid glycoprotein (alpha1-AGP) were measured by ELISA. Serum concentrations of leptin, resistin, visfatin, adipsin, adiponectin, IL-6, IL-8, IL-18 and TNF-alpha were determined by multiplex analysis. Fat mass and distribution was assessed by DEXA. Mean diameter of adipocytes was estimated in samples of subcutaneous adipose tissue. RESULTS: Patients with diabetes had higher levels of circulating CCN4, leptin, resistin, adipsin, visfatin, hsCRP, alpha1-AGP, and IL-6 (all p < 0.02). The CCN4 concentration correlated positively with percentage of fat mass in central abdominal area, as well as with leptin, resistin and visfatin levels; negative correlation was found between CCN4 and mean adipocyte diameter. In multiple regression analysis fat mass in central abdominal area was independent predictor for CCN4 concentration. CONCLUSION: In subjects with type 2 diabetes serum levels of CCN4 are associated with central abdominal fat mass and adipose tissue dysfunction.

Linagliptin alleviates fatty liver disease in diabetic db/db mice.

AIM: To study the effects of linagliptin on the structural signs of non-alcoholic fatty liver disease (NAFLD) in db/db mice. METHODS: Male diabetic db/db mice (BKS.Cg-Dock7m+/+Leprdb/J) aged 10 wk received the dipeptidyl peptidase 4 (DPP4) inhibitor linagliptin (10 mg/kg) or saline as a placebo once per day by gavage for 8 wk. Intact db/db mice served as controls. Structural changes in the liver were analyzed from light and electron microscopic images of sections from intact, placebo-treated and linagliptin-treated animals. We estimated the changes in hepatocytes, sinusoidal cells, liver microvasculature and lymphatic roots. Hepatic staining for lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) was assessed by immunohistochemistry. RESULTS: In 18-wk-old diabetic mice, liver steatosis (predominantly microvesicular and mediovesicular steatosis) was accompanied by dilation of the roots of the lymphatic system, interlobular blood vessels and bile canaliculi. Compared to saline-treated mice, linagliptin-treated mice exhibited a reduction in the mean numeral densities of hepatocytes with lipid droplets (92.4% ± 1.7% vs 64.9% ± 5.8% per field of view, P = 0.0002) and a lower proportion of hepatocytes with a high density of lipid droplets (20.7% ± 3.6% vs 50.4% ± 3.1%, P = 0.0007). We observed heterogeneous hepatocytes and relatively preserved cell structures in the linagliptin group. Dilation of blood and lymphatic vessels, as well as ultrastructural changes in the hepatocyte endoplasmic reticulum and mitochondria, were alleviated by linagliptin treatment. In intact and placebo-treated mice, immunohistochemical staining for LYVE-1 was observed in the endothelial cells of interlobular lymphatic vessels and on the membranes of some endothelial sinusoidal cells. We observed an enlarged LYVE-1 reaction area in linagliptin-treated mice compared to intact and placebo-treated mice. The improvement in the structural parameters of the liver in linagliptin-treated mice was independent to changes in the plasma glucose levels. CONCLUSION: The DPP4 inhibitor linagliptin alleviates liver steatosis and structural changes in the hepatic microvasculature and lymphatic roots in a model of NAFLD in diabetic db/db mice.

Association of IL2, TNFA, IL4 and IL10 Promoter Gene Polymorphisms with the Rate of Progression of the HIV Infection.

The rate of transition from one stage to another during the course of HIV infection is characterized by changes in the cytokine network balance. The alterations in the cytokine network balance during HIV infection depend on the individual profile of cytokine production predetermined by the functioning of the genes encoding the immunomodulators. The purpose of this research is to study the distribution in the frequency of allelic variants of the promoter regions of the genes encoding pro-inflammatory (T-330G IL2 and G-308A TNFA) and anti-inflammatory (C-590T IL4 and C-597A IL10) cytokines among healthy individuals of European origin and in HIV-infected patients with various rates of HIV progression (fast and slow). Four polymorphic loci of the promoter regions were analyzed in 127 HIV-infected patients and 52 healthy individuals using the polymerase chain reaction - restriction light fragment polymorphism (PCR-RLFP) methodology. We have obtained data indicating an increased allelic content of genotypes T/T IL2 (OR = 1.67), G/A TNFA (OR = 4.21), T/T IL4 (OR = 3.43), C/A IL10 (OR = 1.34) in HIV-infected patients as compared to healthy individuals. The correlation between the genotypes and allelic combinations of the investigated cytokines, and the rate of the infection progression in AIDS has been investigated. The association of T/G IL2, G/A TNFA, T/T IL4, A/A IL10 allelic variants of the immunomodulator genes with a fast rate of HIV infection has been established.

Polymorphism of Immune Response Genes as a Factor for Predisposition to Development of Diseases.

The association of particular allelic variants of genes coding proteins, such as transporter associated with processing (TAP) and heat shock protein (HSP), involved in endocellular processing of endogenous antigens, with synovium pathology accompanied with urogenital infection was observed. The particular alleles of TAP1 and TAP2 genes appeared to be the factor of predisposition or resistance for Chlamidia trachomatis but not for Mycoplasma hominis. At the same time, the relative risk for C. trachomatis and M. hominis infection development was increased among persons with HSP70-2*A/B phenotype. Some TAP2 gene allelic variants were determined as the factor of an increased relative risk to develop synovium pathology, especially accompanied with urogenital infection. The preliminary data concerning gene polymorphism of some proinflammatory cytokines in human immunodeficiency virus (HIV) infected patients are presented. We observed an increased frequency of C/T allele of IL-4 gene among HIV patients. None of patients showed A/A homozygous variant of IL-10 and TNF-alpha genes. Our obtained results may be used as informative criteria for prognosis of resistance or predisposition to different diseases.