| BACKGROUND:As we all know, high blood sugar is the initiating factor leading to DKD, so Explore the effects of high glucose on the kidney and its mechanism of early prevention and treatment DKD inevitable part of the study. On the impact of hyperglycemia on kidney and related mechanisms have been numerous animal studies, but high glucose on human kidney cells with specific hazards and specific the mechanism remains to be further clarified, so that research into this field research focus. Especially high-sugar through a variety of cytokines, a variety of cell signaling pathways, such as intrinsic to the kidney mesangial cells, podocytes, tubular epithelial cells, endothelial cells and the impact of research, more and more deeper. High glucose on the kidney connective tissue growth factor (Connective tissue growth factor, CTGF) expression and the corresponding mechanism of the present study is one of the focuses.Mesangial cells has important physiological functions:glomerular capillary plexus is the main stand, with a contraction, secretion, membrane through its Department of vasoactive substances on the cell membrane receptors, according to the contraction of the body needs to adjust to changes in kidney filtration membrane filtration area; mesangial cells also have a strong phagocytic ability of the glomerular filtration clearance of certain macromolecules play an important role. As we all know, high blood sugar is the occurrence of the initiating factors and DKD core elements, and mesangial cells' physiological characteristics determine that it is main target cell of a number of pathogenic factors in diabetes. However, human glomerular mesangial cells in primary culture there has been a lot of difficulties, such as:the low success rate of cultured cells, a shorter survival time, passage or passages too difficult issues, which hinder the experimental study of mesangial cells, and greatly influenced the progress of researching in glomerular disease.CTGF is a cysteine-rich secreted protein, has a strong pro-fibrotic effects. The main mechanism through autocrine, paracrine, etc. to induce a variety of cell differentiation, proliferation, involved in mediating the migration of these cells, adhesion, and promote extracellular matrix synthesis and deposition, leading to the corresponding target organ fibrosis. In addition, CTGF also induce angiogenesis, wound repair in tissue cells and induction of apoptosis and a series of biological functions. CTGF in a variety of human tissues is widespread, as an important cytokine involved in the synthesis and deposition of extracellular matrix, which is an important mechanism for tissue fibrosis. Study found that the kidney is the body's many tissues and organs in most organs with one of CTGF, a variety of intrinsic renal cells can express CTGF, and in some factors such as oxidative stress, hypoxia, under the effect of high sugar with high expression. Experiments have been confirmed, CTGF mediated by advanced glycation end product of this approach for kidney mesangial cells to synthesize collagen and fibronectin, which participated in the progress of renal fibrosis. Recent studies not only in animal models of kidney DKD found high expression of CTGF, and kidney tissue in patients with DKD had a similar discovery, so that CTGF as an important factor in promoting fibrosis DKD occurrence and development of play important role.Recent studies by our group shows that in the early diabetic kidney disease began to appear high expression of CTGF in renal tissue and blood, urine, increasing the concentration of CTGF, and angiotensinâ…¡receptor antagonist can reduce the blood level of CTGF reduce kidney damage in diabetic rats. There is also a scholar to detect patients with type 1 diabetes, elevated levels of CTGF, this change even before the onset of microalbuminuria has occurred, and its levels in patients with proteinuria level and renal function (endogenous creatinine clearance rate) has a high correlation. Tip of CTGF and DKD, development is closely related to the factors in the future may become an early diagnosis and treatment of DKD and an important indicator of prognosis. However, these blood and urine levels of CTGF in the specific sources and their pathophysiological significance of increased expression is not yet clear. Have been reported in the literature, CTGF can be induced in vitro by a mechanism of renal mesangial cells and Sertoli cell hyperplasia, hypertrophy []. DKD mesangial cell hypertrophy is an early kidney is one of the main pathological features []. This study showed that high glucose can induce cultured human mesangial cells, increased expression of CTGF protein, and thus that CTGF as an important cytokine involved in mesangial cell hypertrophy of the pathological process.OBJECTIVE:1. To develop the human mesangial cells(HMC) which can be reproducible and repeatedly passaged.2. To explore the expression of connective tissue growth factor (CTGF) and its receptor low density lipoprotein receptor related protein (LRP), and the possible signaling pathway for the modulation in cultured HMC exposed in high glucose.METHODS:1 In vitro cultured HMC:adult kidney tumor surgery relative to take away from the tumor of the kidney tissue, induction of fetal cystic kidney, three-layer mesh filtration through a separation, select the bottom sieve renal tissue by collagenase digestion develop normal glomerular mesangial cells, with systematic observation and immunofluorescence methods such as identification after subculture.2. High glucose experiments:in vitro cultured HMC for the study, using double-antibody sandwich enzyme-linked immunosorbent assay (ABC-ELISA) determination of the normal glucose group (NG group), high glucose (HG group) and mannitol group (MG group) medium CTGF, pERK and LPR concentration changes were observed in high glucose on CTGF protein expression in mesangial cells, mitogen-activated protein kinase (MAPKS) signaling pathway and the expression of CTGF receptor LRP.RESULTS:1 The morphological, immunofluorescence and other methods confirmed the identification of cultured cells are mesangial cells, adult kidney 2 weeks after the primary culture before passage, fetal mesangial cells in primary culture 1 week can be passaged, and continuous train to the 7th generation or more.2. Lower basal levels of CTGF protein were observed in cultured HMC, the higher levels of CTGF protein were detected by high glucose medium groups on the 1st day, got to the peak on the 3rd day(P<0.05), then began to lower on the 4th day (P>0.05). The levels of CTGF in normal glucose and mannitol glucose groups did not change notably. High glucose substratum induced the phosphorylation of ERK at the first 1 hour, and reached the peak at 8 hours; maintained the activity at 24 hours, then began to lower to the basal level in 48 hours. However, no pERK was detected in normal glucose and mannitol glucose groups. Interestingly, interception of phosphorylation of ERK with PD98059 (a specific ERK activation inhibitor) counteracted the high glucose induced expression of CTGF protein. But, high glucose did not effect the expression of LRP protein at each point.CONCLUSION:1 Using 3-layer filter isolated from human glomerular mesangial cells method is simple and efficient, the cultured primary mesangial cell line of human glomerular mesangial cells, biological characteristics, and further confirmed that embryonic kidney tissue than adult kidney primary cell culture easy success.2 These results demonstrate that high glucose influences the expression of CTGF through the pERK-dependent signaling pathway in cultured HMC, while high glucose does not effect the expression of LRP in HMC.
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