| Background and Objectives:With the rising incidence of diabetes, diabetic nephropathy (DN), the main pathological change of which is renal interstitial fibrosis, is one of the main primary diseases to result in the end-stage renal disease (ESRD). Tubular epithelial myofibroblast transdifferentiation (TEMT), along with extracellular matrix accumulating, play important roles in renal interstitial fibrosis.Integrin-linked kinase (ILK) is a serine/threonine protein kinase, combinated with the intracellular region of integrin, regulates cell adhesion, migration, extracellular matrix accumulation, and is closely related with the pathogenesis of kidney diseases. Studies have shown that ILK, which participates in ECM accumulation, is an important downstream effector molecule of TGF-β1/Smad signaling pathway, and the committed step of TGF-β1/Smad induced TEMT.Matrix metalloproteinases-9(MMP-9) can specifically degrade the main ingredient-collagen IV in tubular epithelial basement membrane. Tissue inhibitor of metalloproteinase-1(TIMP-1) can specifically combine with MMP-9and keep their activity in a state of homeostasis. Recently scholars have been focused on the ratio of MMP-9/TIMP-1and considered it as a potential index for evaluating MMP-9effect in the process of renal fibrosis. In the condition of TEMT, the normal MMP-9/TIMP-1ratio is disturbed. Excessive MMP-9degradates type IV collagen in tubular basement membrane, trough which the transdifferentiated tubular epithelial cells pass and reach the renal interstitium, promotes further development of interstitial fibrosis.Rhein is a precursor component isolated from rhubarb anthraquinone derivatives, and a main renoprotective ingredient of rhubarb. Cumulative evidence suggests that some Chinese herbal medicines, including rhubarb, have a beneficial role in slowing the progression of CKD. Our previous studies suggested that rhein inhibited the pathogenesis of TEMT, however, whether this effect was related with regulation of ILK and MMP-9/TIMP-1ratio, is still unclear.This research studied from three aspects:chronic kidney disease patients, diabetic nephropathy rats and high glucose environment cultured human proximal tubular epithelial cells; meanwhile inhibited ILK expression with small interfering RNA (siRNA), and intervened the above process with rhein. The aims of this research were:observe TEMT in DN tubulointerstitial lesions, and the changes of ILK expression and MMP-9/TIMP-1ratio; explore the regulating effects of ILK on MMP-9/TIMP-1ratio in TEMT; investigate the therapeutic effect of rhein on DN and its impact on the above-mentioned process, elucidate the specific pathogenesis and the possible new target of DN prevention.Methods:1. Renal biopsy specimens of diabetic nephropathy patients (n=6) were collected. HE, PASM, Masson staining were used to observe renal pathological changes. Immunohistochemistry was used to detect E-cadherin, a-SMA expression in renal biopsy specimens and compared with the control group (n=6); the aim was to observe whether there is TEMT process in diabetic nephropathy patients. 2. Healthy male Wistar rats of8weeks old were randomly divided into normal group (n=12), diabetic nephropathy group (n=12), rhein intervention group (n=12) and valsartan intervention group (n=12). Six rats of each group were killed at the end of the8th and16th week. The renal interstitial injuries and the relative area of renal interstitial collagen were measured by HE and Masson staining. The protein expression of E-cadherin, a-SMA, TGF-β1, ILK, MMP-9, TIMP-1and AP-1in renal tubular epithelial cells were measured by immunohistochemistry.Human proximal tubular epithelial cells (HK-2) were cultured and divided into the following groups:normal group; high glucose group; ILK-siRNA group; negative siRNA group; high glucose+low concentration rhein group; high glucose+medium concentration rhein group; high glucose+high concentration rhein group; and hypertonic group. After cultured for48hours, total cell RNA and total cell protein were extracted. The mRNA and protein expression of E-cadherin, a-SMA, TGF-β1, ILK, MMP-9, TDVIP-1was detected by Real-time RT PCR, Western blot and cytoimmunohistochemistry.Results:1. HE, Masson and PASM staining showed that there were tubular and interstitial lesions in the kidneys of DN patients. Comparing to the patients of the control group, the E-cadherin expression in the cell membrane and cytoplasm decreased, and a-SMA expression increased in the renal tubular cells of diabetic nephropathy patients, with significant difference (P<0.05). 2. Comparing to the normal group, the relative area of renal interstitial collagen of diabetic nephropathy rats increased. The expression of E-cadherin in renal tubular epithelial cells decreased and the expression of a-SMA significantly increased at the level of P<0.05. Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed increases in the relative area of renal collagen. The expression of E-cadherin increased, while the expression of α-SMA significantly decreased. There is no significant difference between rhein intervention group and valsartan intervention group at the P>0.05level.Comparing to the normal group, the expressions of MMP-9and TIMP-1in renal tubular epithelial cells of diabetic nephopathy rats changed while the ratio of MMP-9/TIMP-1was disturbed. Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in MMP-9/TIMP-1ratio. There is no significant difference between rhein intervention group and valsartan intervention group at the P>0.05level.Comparing to the normal group, the expressions of TGF-β1, ILK, AP-1in renal tubular epithelial cells of diabetic nephopathy rats increased significantly with the disease progression (P<0.05). Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in expression of TGF-β1, ILK, AP-1(P<0.05). There is no significant differnce between rhein intervention group and valsartan intervention group at the P>0.05level.3. The mRNA and protein expression of cultured HK-2cells were evaluated by Real-time RT PCR, Western blot and cytoimmunohistpchemistry respectively. The results indicated: Comparing to the normal group, the expression of E-cadherin decreased; the a-SMA and ILK expression significantly increased in the high glucose group (P<0.05). Inhibition of ILK by ILK-siRNA resulted in the increased expression of E-cadherin, decreased expression of a-SMA and ILK at the P<0.05level. In the rhein intervention groups, the HK-2cells showed a similar change with the ILK-siRNA group and there was no significant difference between these groups (P>0.05).Comparing to the normal group, the MMP-9/TIMP-1ratio was disturbed in the high glucose group (P<0.05). Inhibition of ILK by ILK-siRNA resulted in a normal return of MMP-9/TIMP-1ratio. Similar change was observed in the rhein intervention groups and there was no significant difference between them (P>0.05).Conclusions:1. TEMT may exist in the process of human diabetic nephropathy.2. E-cadherin expression decreased and a-SMA expression increased in high glucose environment cultured HK-2cells and the tubular epithelial cells of DN rats, indicating that TEMT exists in the process of tubulointerstitial lesions.3. In the pathological state of DN, ILK expression of renal tubular cells increased; ILK participated and mediated the process of TEMT; ILK inhibition could counteract with TEMT, and regulate the disturbed ratio of MMP-9/TIMP-1, thus inhibit TEMT. 4. Rhein could down-regulate ILK expression in HK-2cells under high glucose environment and in renal tubular epithelial cells of DN rats, improve the status of MMP-9/TIMP-1ratio imbalance, and attenuate the progress of TEMT. The inhibition effect of rhein towards the ILK overexpression in DN and its regulating role to MMP-9/TIMP-1ratio may be one of its renoprotective mechanisms. |
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