The Expression And Role Of TRB3in Renal Tubular Cells Of Diabetic Kidneys

Background and ObjectiveDiabetic nephropathy (DN) is a serious and common complication of diabetes mellitus (DM), leading to end-stage renal failure in up to30%of patients with diabetes, so it is important to better understand the underlying mechanisms in order to develop new and improved therapeutic strategies. The glomerulus has been at the center of attention as the primary site of injury in DN. Although there is no question that there are changes seen in the glomerulus, it is also well known that tubulointerstitial changes are a prominent component of the disease.TRB3, a mammalian homolog of Drosophila tribbles, also named NIPK, SINK, SKIP3, belongs to a group of proteins named pseudokinase. It contains a consensus serine/threonine kinase catalytic core structure, but do not possess kinase activity due to strong deviations from the kinase active site consensus sequence. TRB3has been found upregulated in many organs in diabetes milieu such as liver, heart, and pancreatic islets. It possesses broad biological activity such as increases insulin resistance and regulates cell proliferation and involves in several signal pathways as an important regulatory protein and works at least through Akt, CDC25/String and MAPK. Recently Cunard and his colleagues reported that TRB3expression is increased in the kidneys of both streptozotocin-induced (type1) and db/db (type2) mice and regulated by endoplasmic reticulum (ER) stress, however, the investigation of its effect in DN is far from enough. A recent study demonstrated that silencing of TRB3expression could ameliorate fibrosis in an animal model of diabetic cardiomyopathy. In addition, another study revealed TRB3mediates overexpression of type I collagen in rat cardiac fibroblasts. Based on these findings, we explored the expression of TRB3in diabetic kidneys and the role it played on extracellular matrix (ECM) accumulation in vivo and in vitro. Although the mechanisms underlying tubulointerstitial injury remain unclear, apoptosis is an attractive one because it is believed to contribute to the gradual loss of renal mass and function in DN. Studies now available have provided abundant evidence for tubular cell apoptosis in streptozotocin-induced diabetic mice and rats, as well as in diabetic patients. Besides, study of the role of TRB3on apoptosis is now a research hotspot. Thus, we hypothesized that TRB3might participate in the apoptotic process of DN.Based on the backgrounds mentioned above, we tested our strategy in streptozotocin-induced diabetic rats by gene silencing technique to explore the expression and effect of TRB3in diabetic kidneys. Meanwhile, we cultured renal tubular epithelial cell (NRK-52E) in vitro to further explore the underlying mechanisms.Methods1.80male Wistar rats (8-week old) weighing200-220g were randomly divided into diabetic group (50rats) and control group (30rats) after an adaptation period of one week. After overnight fast, diabetic group rats received an intraperitoneal injection of60mg/kg STZ prepared in0.1M citrate buffer (pH4.5), and control group rats received an equivalent volume of citrate buffer only. Blood glucose and body weight were monitored every week till animal sacrifice. Rats were sacrificed8,12,16and20weeks after model establishment. Physical, biochemical and morphological parameters were measured. Blood glucose, urinary albumin excretion (UAE), serum creatinine (Scr) and were detected by laboratory mathods; TRB3expression was measured by quantitative RT-PCR, western blot and immunohistochemistry (IHC). PAS staining were performed to assess the glomerular sclerotic injury. Rats in DM+vehicle group and DM+TRB3-siRNA group received vehicle or TRB3-siRNA treatment after12weeks of diabetes.4weeks later, the expression of Collagen Ⅰ and fibronectin (FN) in kidney tissues were measured by IHC and western blot. NRK-52E cells were cultured in supplemented DMEM. High glucose and albumin were used as stimulation separately, and Collagen I and FN secretion were detected with ELISA. After silencing the expression of TRB3, Collagen I and FN were detected again.2.40male Wistar rats (8-week old) weighing200-220g were randomly divided into control group, DM group, DM+vehicle group and DM+TRB3-siRNA group. After overnight fast, diabetic group rats received an intraperitoneal injection of60mg/kg STZ prepared in0.1M citrate buffer (pH4.5), and control group rats received an equivalent volume of citrate buffer only. Rats in DM+vehicle group and DM+TRB3-siRNA group received vehicle or TRB3-siRNA treatment after16weeks of diabetes.4weeks later, Kidneys were removed to detect TRB3expression and apoptosis. NRK-52E cells were stimulated with albumin then TRB3expression and apoptosis were investigated. After silencing the expression of TRB3, apoptosis was detected again. In addition, Akt signaling pathway was explored to understand its role in this apoptotic process.Results1. TRB3, up-regulated in kidneys of rats with typel diabetes, mediates extracellular matrix (ECM) accumulation in vivo and in vitro.1.1Physical, biochemical and histological changes in ratsBlood glucose level in the group with diabetes was significantly elevated compared with that in the normal control group. On the other hand, body weight in the group with diabetes was significantly lower than that in the normal control group. UAE detection indicated an obvious increase of albumin excretion in group with diabetes than that in the normal control group. Scr also showed a markedly increase in group with diabetes early from8w. Histologically, kidneys from rats with diabetes contained many abnormal glomeruli with increased deposits of ECM protein in the mesangium as revealed by PAS staining. The ratio of abnormal glomeruli was obviously increased after diabetes induction.1.2TRB3mRNA and protein expression was up-regulated in the kidneys of rats with typel diabetesIHC staining showed that positive cells were barely observed in kidneys of control rats, but obtained in renal tubules especially proximal tubules of rats with diabetes. Quantitative real-time PCR showed renal expression of TRB3mRNA was not significantly changed throughout the duration of study in rats without diabetes, but course-dependently increased in rats with diabetes. Western blotting demonstrated that the protein of TRB3was present at low abundance in normal rat kidneys, but its expression was up-regulated early from the8th week in kidneys of rats with diabetes.1.3Elevated expression of ColⅠ and FN in the kidneys of rats with diabetes was partially reversed by TRB3silencingWhen compared with vehicle treatment, TRB3-siRNA treatment significantly down-regulated mRNA expression of renal TRB3. To explore the effect of TRB3gene silencing on albuminuria, UAE was determined before transfection and before sacrifice separately. The results demonstrated TRB3silencing did not work on albuminuria. To assess the role of TRB3on ECM accumulation, the expression of ColⅠ and FN was determined by IHC staining first. ColⅠ and FN were increased in DM group compared with control, but decreased after TRB3-siRNA transfection, while transfection with vehicle did not affect their expression. The results were then verified by western blotting. Similarly, the expression of these two ECM proteins was also significantly increased in kidneys of rats with diabetes compared with control. However, their expression in kidneys of diabetic rats transfected with TRB3-siRNA was distinctly decreased compared with that transfected with vehicle1.4TRB3expression was up-regulated in NRK-52E cells induced by albumin-overloadTo investigate whether TRB3was induced by albumin-overload, cells were exposed to various concentrations of fatty acid free BSA in serum-free medium for72h. Quantitative real-time PCR showed an increase in TRB3mRNA expression in a concentration-dependent manner. Then cells were incubated with BSA (10mg/ml) for various durations. Quantitative real-time PCR showed the expression of TRB3mRNA was increased over time. Western blotting for various durations of10mg/ml BSA treatment elucidated an increase of TRB3in a duration-dependent way in protein level. 1.5Elevated secretion of ColI and FN induced by albumin-overload was partially reversed by TRB3silencingTo clarify the role of TRB3on ECM accumulation, TRB3-siRNA or vehicle was transfected into NRK-52E cells. Then the expression of TRB3mRNA was detected to decrease to31%of control after transfection. Cells transfected with vehicle or TRB3-siRNA or without transfection were treated for72h in basic medium with BSA (10mg/ml) and then supernatant was collected for ELISA assay. The concentrations of ColI and FN in the medium supplemented with BSA significantly increased by105%and141%respectively compared with the BSA-free control. However, by exposure to BSA for72h, ColI and FN concentration in media in which cells were transfected with TRB3-siRNA was obviously decreased compared with that transfected with vehicle.2. TRB3mediates apoptosis of renal tubular cells in vivo and in vitro2.1TRB3mRNA and protein expression was up-regulated in the kidneys of rats with typel diabetesImmunohistochemical staining showed that positive cells were barely observed in kidneys of control rats, but obtained in renal tubules especially proximal tubules of rats with diabetes. Quantitative real-time PCR and western blotting demonstrated that TRB3was up-regulated early from the8th week in kidneys of rats with diabetes.2.2Apoptosis is increased in diabetic kidneys, but partially recovered after TRB3silencingIn situ TUNEL assay showed positive cells mainly existed in the tubules and the number of positive nuclei in diabetic kidneys was obviously increased compared with that in control ones. But transfection of TRB3-siRNA indeed decreased the number of TUNEL-positive cells compared with DM+vehicle group. In addition, Caspase3activity was distinctly elevated in DM group and did not changed after vehicle treatment, but transfection of TRB3-siRNA significantly decreased renal caspase3activity in diabetic kidneys.2.3TRB3expression is upregulated in NRK-52E cells induced by albumin-overload, but not regulated by high glucose Hyperglycemia is the main feature of diabetes, so we wondered whether TRB3was regulated by high glucose. Unexpectedly, neither quantitative real-time PCR nor western blotting showed statistical change in TRB3expression. Massive proteinuria is a hallmark of progressive DN, so we investigated whether TRB3was regulated by albumin-overload. As a result, cells exposed to BSA exhibit overexpression of TRB3in a dose-and duration-dependent way.2.4Albumin-overload induces apoptosis in NRK-52E cells, but inhibition of TRB3expression protects against apoptosisthe percentage of apoptotic cells is significantly increased after BSA treatment, but after TRB3-siRNA transfection, the percentage of apoptotic cells and caspase3activity was significantly decreased compared with that after transfection of vehicle, suggesting that TRB3mediates apoptosis of NRK-52E cells induced by albumin-overload.2.5Failure to phosphorylate Akt is implicated in apoptosis induced by albumin-overload, but partially reversed by TRB3silencingThe value of p-Akt/Akt decreased after BSA exposure. While inhibition of Akt phosphorylation increased apoptosis in cells induced by BSA, illustrating that Akt pathway indeed participated in this apoptotic process. To further explore the relationship between TRB3and Akt, cells transfected with TRB3-siRNA or vehicle were exposed to BSA and Akt phosphorylation level was detected by western blotting. As a result, the value of p-Akt/Akt of cells transfected with TRB3-siRNA was significantly elevated compared with that of cells transfected with vehicle, indicating that the inhibitory phosphorylation of Akt was partially restored after TRB3silencing and further demonstrating that TRB3worked through Akt pathway in this process.Conclusion1. TRB3is upregulated in renal tubular cells of diabetic kidneys.2. TRB3mediates ECM accumulation in diabetic kidneys.3. TRB3mediates apoptosis of renal tubular cells in diabetic kidneys.4. TRB3gene silencing ameliorate ECM accumulation and apoptosis in DN.