| Objective:To discover the early biomarkers of the diabetic nephropathy(DN) and analyze the relationship between the markers and the path physiological mechanism of diabetic nephropathy, and find the possible treatment target of prevention of traditional Chinese medicine. As recent epidemiological data indicate that the prevalence of patients with diabetic mellitus (DM) more than 20 years old is 9.7% in our country. That means China has the largest population of 92.4 million patients with DM. Among the DM patients,20 to 30 percent of them will undergo DN, and especially DN could contribute 20 to 40 percent of end stage renal disease. DN has been a heavy burden on the development of our country and is becoming a more and more important social-economical problem which needs the government and public to focus on. As we have known, the earlier diagnosis and prevention have been applied, the better prognosis of DN will be. That is also true to the discovery of the early biomarker for the diagnosis of DN is very important. The most common diagnostic biomarker of DN, albuminuria, has its limitations both in specificity and sensitivity. To find new and effective markers, will contribute to improvement of the diagnosis and treatment of DN and deepening our understanding on the DN pathophysiology, and help find new therapeutic targets. Based on our recently study results and the international research progression, we had chosen three proteins, i.e. liver-type fatty acid protein(L-FABP), transforming growth factors -β1(TGFβ1), and glutathione peroxidase 3(GPx3) as our observing subjects. We investigated the urinary L-FABP/plasma L-FABP levels, urinary TGFβ1 levels, and GPx3 levels in 3 groups (DM with normal albuminuria/DN with microalbuminuria/DN with macroalbuminuria). And we observed the effect of a Chinese medicine formula Tangshen Formula on the urinary levels of these three proteins. Furthermore, we observed the expression levels of these three proteins in the DN models which were established by injection with STZ and execution of unilateral nephrectomy in rats. At last, we hoped to find the clues about early diagnosis biomarkers and therapeutic targets of DN.Methods:1. The clinical study about the relationship between L-FABP, GPx3,TGFβ1 and the progression of DN:The recruited patients with Type 2 DM were from four hospitals in two cities (Beijing and Tangshan). They were divided into 3 groups group 1(DM with normal albuminuria (n=30)), group 2 (DM with microalbuminuria (n=30)), and group 3 (DM with macroalbuminuria (n=30)). The general clinical information, urinary albuminuria/24h urine protein parameters, blood and urine routine parameters, blood biochemistry parameters were collected. We assay urinary L-FABP/plasma L-FABP, urinary TGFβ1, and urinary GPx3 by enzyme linked immunosorbent assay(ELISA). And analysis was performed on the relationship between the urine/plasma levels of these three proteins and the DN stages and clinical parameters..2. Effect of Tangshen Formula on urine L-FABP/GPx3/TGFβ:the subjects were recruited from 4 hospitals in two cities Beijing and Tangshan:30 cases diagnosed Type 2 DN with microalbuminuria, and 30 cases diagnosed Type 2 DN with macroalbuminuria, the two populations were randomly divided into two groups respectively:Tangshen formula prevention group, and placebo control group. All the groups were followed for 24 weeks. At the 0 week,12th week and 24th week, the parameters of the general clinical information, blood routine, blood biochemistry(blood sugar, lipids, liver function, renal function), urine microalbuminuria/24h urine protein was collected;3. Expression of L-FABP, GPx3 and TGFβin DM rats induced by STZ injection and unilateral nephrectomy:DN models were established by Wistar Rats which were injected with streptozocin (STZ) and executed by right unilateral nephrectomy. After models established, the parameters of rats'weight, blood sugar, and 24h urine protein were collected at 0 week,4th week,8th week,12th week and 20th week. At the 20 week, the rats were sacrificed under anesthesia, and the rat's body was perfused by physiological salt. The left kidney was collected for histopathological and molecular biological analysis. We assay the expression levels of L-FABP, TGFβ1, and GPx3 in rat kidney by Western Blot, a semi-quantitative method4. The construction of rat L-FABP recombinant DNA:we designed the forward primer with a Bam HⅠrestriction enzyme cutting site and reverse with an EcoRⅤrestriction enzyme cutting site. The L-FABP fragment was amplified by PCR, and the product was purified and recycled. The L-FABP fragment and the expression plasmid vector pc DNA3.1 A/V5-His were digested by BamHⅠand EcoRⅤenzyme, respectively; the digested product was linked by T4 DNA ligase to form a recombinant DNA. The recombinant DNA was transformed into competent E.Coli. and positive clones were selected for sequencing, The clone whose sequence was perfectly accurate was cultured with large quantity, and the recombinant DNA was extracted and purified. Furthermore, the recombinant DNA was transfected into HEK293 cell line by calcium phosphate methods. After 72h in transfected HEK293 cell culture, the total RNA were extracted, and the transcription levels of L-FABP were assayed by RT-PCR.Results:1. the clinical study about the relationship between L-FABP/GPx3/TGFβand T2DN:The general data (sex, age, weight, height,BMI,BP and HbA1C) and blood glucose and lipid were compared between patients of different groups, and the results showed no statistical difference in patients with normal albuminuria or microalbuminuria or macroalbuminuria (p>0.05). Patients with macroalbuminuria had obviously higher levels of serum creatinine (Scr) and BUN, compared to those with normal or microalbuminuria(p<0.01).As the development from normal albuminuria to macroalbuminuria, the level of urine L-FABP (uL-FABP) was significantly elevated;and the uL-FABP level in the patients with normal albuminuria was markedly higher than that in the normal control group. There was a linear correlation between In uL-FABP and blood glucose, BUN, total cholesterol and LDL-C(p<0.05). Although plasma L-FABP (pL-FABP) elevated as DN progressed, no statistical difference was found in this elevation (p>0.05) The level of urine GPx3 (uGPX3) was obviously decreased in patients with micraoalbuminuria, but the levels between patients with normal albuminuria and macroalbuminuria were found no differences. It was proved to be correlation between uGPX3 and plasma triglycerin. The level of urine (uTGFβ) in each group was found no difference.2. Effect of Tangshen Formula on urine L-FABP/GPx3/TGFβ:The effect of Tangshen Formula was observed comarped with the placebo control group in patients with miroalbuminuria and macroalbuminuria. Between the two group in both stages, the general data, blood routine and blood biochemical parameters were found no statistical difference. Therefore, these groups were compatible. After follow-up for 24 weeks, uL-FABP was found a decrease in patients treated by Tangshen Formula with microalbuminuria or macroalbuminuria but no effect on p-LFABP was observed. There was no effect of Tangshen Formula on urine GPx3. Urine TGFβwas decreased in patients treated by Tangshen Formula with microalbuminuria rather than those with macroalbuminuria. 3. Expression of L-FABP, GPx3 and TGFβin DM rats induced by STZ injection and unilateral nephrectomy:Parameters in the model group and the sham operation group were compared. In the model group, the level of blood glucose was significantly elevated at 0 week; body weight started to decrease at the 4th week; the level of urinary albumin was markedly elevated and the difference was significant (p<0.05).The Scr level, extracellular matrix area/glomerular area percentage and intertubular fibrosis index were obviously higher than those in the sham operation group (p<0.01). No expression of L-FABP in the renal tissue was detected by Western Blot in both groups. The expression of TGFβin the renal tissue was greatly elevated in the model group (p<0.05). A trend of elevation was also detected in the expression of GPx3 but no statistical difference was found (p>0.05)4. Construciton of rat L-FABP recombinant DNA:Related primers was designed with the additional cutting site of BamH I or EcoR V and PCR was used to amplify the target L-FABP sequence by the rat liver cDNA sucessfully. The L-FABP target sequence and vector was cut by both of BamH I and EcoR V restriction endonuclease, respectively.And the digested product was linked by the T4DNA ligase. The compound was then transfected into competent E.Coli and positive clones of E.Coli were screened. The recombined DNA was purified and sequenced, which indicated 100% of base accuracy. The success rate of transfecting recombined DNA into HEK293 cell line by calcium phosphate methods reached a transfecting ratio of 70%.RT-PCR revealed that the expression of L-FABP mRNA was greatly up-regulated in the recombined DNA group compared wtih control group of pcDNA3.1 A vector.Conclusion:Urine L-FABP might be an effective biomarker for early diagnosis of diabetic nephropathy and monitoring the progression of diabetic nephropathy. It was sensitive in response of Traditional Chinese Medicine treatment and it could be used as an evaluation of therapeutic effect of Traditional Chinese Medicine. The recombined rat DNA of L-FABP provided a new source for further exploration of pathophysiological mechanism of DN and TCM targets.
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