Experimental Study Of MK And CTGF In The Kidney Tissue Of Type2Diabetic Nephropathy Rats

Objective Diabetic nephropathy (DN) is a life-threatening condition associated with diabetes mellitus(DM) and is the leading cause of end stage renal failure in many countries. DN is characterized by sequential functional progression from hyperfiltration to microalbuminuria to macroalbuminuria to renal failure. This progression is accompanied by various mechanisms, including polyols, the activation of PKC way, protein nonenzymatic glycosylation, synthesis of macromolecular end products, and cytokine. Although DN was not considered as an inflammatory disease, there is an increasing body of evidence that inflammation is involved in the pathogenesis of this disease. However so far, only a few cytokine including connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) have been identified as key players in this inflammation. CTGF has been identified as a prosclerotic cytokine acting downstream of TGF-β and shown to be involved in the process of extracellular matrix accumulation. This cytokine has been shown to play a critical role in the development of glomerulosclerosis and tubulointerstitial injury of DN. The growth factor midkine (MK), a heparin-binding growth factor, has been implicated in neuronal survival and differentiation, cancer development, and inflammation-related diseases. Recent studies indicate that MK also plays a critical role in the pathogenesis of DN. The progression of DN in MK-deficient (mdk-/-) mice was postponed than in mdk+/+ones. However, there was no report on the relationship between MK and the other cytokines in DN. In this study we aim to intervene prophase DN with MK antisense oligodeoxynucleortides(AS-ODNs) and then to determine the effect of MK AS-ODNs on the expression of MK and CTGF in kidney of rats with DN, in order to reveal the role of MK AS-ODNs in the treatment of prophase DN.Methods Experiments were performed on8-week-old male Sprague-Dawlry rats of pathogen-free weighing200-250g. After1wk’ adaptability breeding, rats were randomly divided into normal control group (n=10), DN group (n=10) and MK AS-ODNs group (n=10). DN group and MK AS-ODNs group were treated with high-glucose and high-fat diet for4weeks and then intraperitonesl STZ (30mg/kg body weight) after unilateral nephritic excision. Normal control group were treated with general feed for4weeks and then intraparitonesl citric acid buffer after incision surgery. During the first week after injection of STZ, the blood glucose levels were measured three times using a glucometer, and rats with blood glucose levels of more than16.7mmo1/L were regarded as diabetes-induced rats. After confirmation of diabetes, water intake, urine output, body weights, and24-h urinary albumin excretion were measured at0wk,3wk,6wk. At the end of6th week, rats in MK AS-ODNs group were administrated with MK AS-ODNs (1mg/kg body weight), while normal control group and DN group were administrated with saline. After4days of intervention, the kidneys were rapidly dissected and stored for subsequent pathological observation, immunohistochemical and fluorescence quantitative PCR analysis.Results1. After confirmation of diabetes mellitus, the rats of DN group and MK AS-ODNs group all presented the symptom such as polydipsia, polyuria, polyphagia, weight loss, hair withered and yellow, lacklustre and slow-moving. Compared with normal group,24-h urinary albumin excretion at6wk was significantly higher in DN group and MK AS-ODNs group rats (P<0.01) and the renal pathology changing was clear, so the model of early T2DN was made successfully.2. Immunohistochemistry staining and RT-PCR results showed that there was rarely expression of MK and CTGF in control kidneys. The expression of MK and CTGF was significantly elevated in DN kidneys compared with control kidneys (P<0.05). Treatment with MK AS-ODNs significantly attenuated MK and CTGF mRNA expression (P<0.01).3. Spearman correlation analysis indicated that the expression of MK was positively correlated with the expression of CTGF (r=0.498, P<0.05).Conclusion1. Early T2DN model was successfully induced through triple method of the unilateral nephrectomy, fed with high-sugar and high-lipid fodder and intraperitoneal injection with small dose of STZ.2. Compared with normal rats, the expression of MK and CTGF was significantly elevated in the renal tissue of prophase T2DN rats.3. Treatment with MK AS-ODNs reduced the expression levels of MK and CTGF in the renal tissue of early T2DN rats.4. There were positive correlations between the expression of MK and CTGF.