| Background:Diabetic nephropathy (DN) is one of the most common microvascularcomplications of diabetes mellitus. It is now the leading cause of end-stage renaldisease (ESRD) in developed countries. Proteinuria and glomerular hypertrophy aretwo cardinal features of DN. Studies show that multiple mechanisms including theactivation of renin-angiotensin system (RAS) are involved in the development ofproteinuria. AngiotensinⅡ (AngⅡ) is the most important effector in RAS and mostof its effects are demonstrated to be mediated by AngⅡ type1(AT1) receptor.Numerous clinical and experimental studies have demonstrated the role of AT1receptor blocker (ARB) in ameliorating kidney injury and proteinuria in DN.However, the mechanism that ARB attenuates proteinuria in DN remains to beinvestigated.Lipoxygenases (LO) are a family of enzymes that insert molecular oxygen intopolyunsaturated fatty acids. They are classified as5-,8-,12-and15-LO according tothe carbon atom of arachidonic acid at which the oxygen is inserted.12-LOactivation can produce12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], theproduct from arachidonic acid. It is now clear that12-LO and12(S)-HETEcontribute to the development and progression of DN by enhancing oxidative stressand inflammation. Studies show that12-LO in glomerular cells are activated afterAngⅡ stimulation, and ARB could downregulate12-LO activity in the glomeruli ofobese Zucker rats and reduce proteinuria levels. However, it remains unclear howproteinuria is decreased by inhibiting the AngⅡ-12-LO pathway in DN.Podocyte constitutes the last glomerular filtration barrier. It is now clear that reduced podocyte numbers and dysfunction of slit diaphragm proteins are importantcauses of proteinuria in DN. Studies show that the role of AngⅡ in proteinuria isrelated to podocyte epithelial-mesenchymal transition (EMT) and decreasedexpression of slit diaphragm proteins nephrin and P-cadherin. The12-LO product12(S)-HETE was also demonstrated to decrease P-cadherin expression in glomeruli.Therefore, we hypothesized that AngⅡ blocked by ARB could lead to12-LOinhibition, preventing podocyte EMT and upregulating nephrin and P-cadherinexpression, thus ameliorating proteinuria in the end.Insulin resistance (IR) is a key feature of type2diabetes, and is demonstrated tobe involved in proteinuria of DN by numerous mechanisms. Studies show that12-LO is involved in albuminuria of type2DN, but not type1. It is well known thatthe most significant difference between type1and type2diabetes is IR, thus wehypothesized that12-LO and IR interaction may be related to the pathogenesis ofproteinuria in type2DN.Methods:1. Podocytes were treated with AngⅡ (10-7M) for24h, and cell supernatantswere harvested for12(S)-HETE detection.2. Rats were randomly assigned to receive either vehicle (ethanolamine) or AngⅡ (infusion rate400ng Kg-1min-1) through osmotic mini-pumps.14days later,glomeruli were isolated for12(S)-HETE, nephrin and P-cadherin detection.3. Rats were randomly assigned to receive either vehicle (ethanolamine) or12(S)-HETE (infusion rate1mg Kg-1d-1) through osmotic mini-pumps.7days later,glomeruli were isolated for AngⅡ, nephrin and P-cadherin detection.4. Type2diabetes was induced by high fat diet combined with low dozestreptozocin (STZ) injection. After the induction of diabetes, rats were randomlydivided into2groups: type2diabetes (DN), type2diabetes+ARB treatment(losartan,5mg Kg-1d-1, by gavage). Rats fed regular chow were used as control.6weeks later, rats were sacrificed.24h urine and glomeruli were collected. Renalslices were stained with periodic acid schiff (PAS).12(S)-HETE, nephrin, P-cadherin, podocyte epithelial marker proteins and mesenchymal marker proteinswere detected.5. Wild type and12-LO gene knock-out mice were divided into four groups:wide type (WT),12-LO gene knock-out (LOKO), wide type+STZ (WT+STZ),12-LO gene knock-out+STZ (LOKO+STZ). Type1diabetes was induced by singleinjection of STZ intraperitoneally. Blood glucose levels of mice were monitored and24h urine were collected.6. Type2diabetes was induced by high fat diet combined with low doze STZinjection. After the induction of diabetes, rats were randomly divided into2groups:type2diabetes (DN), type2diabetes+CDC treatment (cinnamyl-3,4-dihydroxy-α-cynanocinnamate,12-LO inhibitor)(8mg Kg-1d-1,3times/week, subcutaneously inthe hind leg). Rats fed regular chow were used as control.8weeks later, rats weresacrificed. Blood and24h urine were collected. Fasting insulin levels were detected.Glomeruli were isolated with a sieving method and classified into largeglomeruli (125μm) and small glomeruli (75μm) according to sieve meshes. Thelevels of AngⅡ,12(S)-HETE and urinary albumin were tested by ELISA. Fastinginsulin levels were determined by radioimmunoassay. Protein expression wasdetected by RT-PCR and Western blot analysis. Renal slices were stained with PAS.Results:1. Ang Ⅱ increased12(S)-HETE levels in podocytes and glomerulisignificantly compared with the relative control (P<0.01). Subcutaneous injection of12(S)-HETE with osmotic mini-pumps increased Ang Ⅱ levels in glomeruli(P<0.01).2. Subcutaneous injection of AngⅡ and12(S)-HETE with osmotic minipumpsdecreased nephrin protein expression in large glomeruli (P<0.05), but increasednephrin protein expression in small glomeruli (P<0.05). P-cadherin proteinexpression was decreased significantly in both large glomeruli and small glomeruliafter AngⅡ and12(S)-HETE treatment (P<0.05).3. Blood glucose, kidney weight/body weight ratio and24h urinary albumin levels were increased significantly in DN group compared with the relative control(P<0.01). PAS stainings showed glomerular hypertrophy and extracellular matrixaccumulation in DN group. Losartan had no effect on blood glucose levels, butdecreased kidney weight/body weight ratio and24h urinary albumin levels in type2diabetic rats significantly (P<0.05). Losartan treatment also ameliorated kidneyinjury induced by type2diabetes.4. Compared with the levels of control,12(S)-HETE levels were increasedsignificantly in the glomeruli of type2DN rats (P<0.01). Losartan treatmentdecreased12(S)-HETE levels in type2diabetic glomeruli (P<0.05).5. Compared with control, nephrin protein expression was decreased in largeglomeruli and increased in small glomeruli in DN group (P<0.01). P-cadherinprotein expression was decreased in both large glomeruli and small glomeruli in DNgroup (P<0.01). Losartan treatment increased nephrin protein expression in largeglomeruli and P-cadherin protein expression in both large and small glomeruli(P<0.05).6. Compared with control, the expression of podocyte epithelial marker proteinszonula occludens-1(ZO-1) and podocin was decreased (P<0.05) in type2diabeticglomeruli, while the expression of mesenchymal marker proteins, such as collagenⅠ,fibronectin (FN), fibroblast specific protein (FSP-1) and desmin was increased intype2diabetic glomeruli (P<0.05). Losartan treatment increased the expression ofP-cadherin, ZO-1and podocin (P<0.05), but decreased the expression of collagenⅠ,FN, FSP-1and desmin (P<0.05).7. Urinary albumin levels were increased significantly in LOKO+STZ groupcompared with WT and LOKO group (P<0.01). There were no difference in urinaryalbumin levels between LOKO+STZ group and WT+STZ group. Urinary albuminlevels in type2DN group were increased significantly compared with control(P<0.01), and the increase was attenuated by CDC treatment (P<0.05).8. After the induction of type1diabetes, blood glucose levels in LOKO+STZgroup were lower than those in WT+STZ group in1month. CDC had no significant effect on blood glucose levels after the induction of type2diabetes.9. Fasting insulin levels were increased and insulin sensitivity index weredecreased significantly in DN group compared with relative control (P<0.01). CDCtreatment decreased fasting insulin levels and increased insulin sensitivity indexsignificantly (P<0.05) in type2diabetic rats.Conclusions:1. The interaction of AngⅡ and12-LO in type2diabetic glomeruli promotespodocyte EMT and decrease the expression of slit diaphragm proteins nephrin andP-cadherin.2. ARB can block the interaction of AngⅡ and12-LO in type2DN, andprevent podocyte EMT and upregulate nephrin and P-cadherin protein expression,thereby ameliorating proteinuria.3. The progression of proteinuria in type2DN is related to IR that induced by12-LO activation. |
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