Protein kinase C and protein kinase A are involved in the protection of recombinant human glucagon-like peptide-1 on glomeruli and tubules in diabetic rats

Abstract

Aims/introduction: Blockade or reversal the progression of diabetic nephropathy is a clinical challenge. The aim of the present study was to examine whether recombinant human glucagon-like peptide-1 (rhGLP-1) has an effect on alleviating urinary protein and urinary albumin levels in diabetic rats.

Materials and methods: Streptozotocin-induced diabetes rats were treated with rhGLP-1 insulin and saline. Using immunostaining, hematoxylin-eosin, electron microscopy and periodic acid-Schiff staining to study the pathology of diabetic nephropathy, and we carried out quantitative reverse transcription polymerase chain reaction, western blot and immunohistochemistry to identify the differentially expressed proteins. The mechanism was studied through advanced glycation end-products-induced tubular epithelial cells.

Results: rhGLP-1 inhibits protein kinase C (PKC)-β, but increases protein kinase A (PKA), which reduces oxidative stress in glomeruli and in cultured glomerular microvascular endothelial cells. In tubules, rhGLP-1 increased the expression of two key proteins related to re-absorption - megalin and cubilin - which was accompanied by downregulation of PKC-β and upregulation of PKA. On human proximal tubular epithelial cells, rhGLP-1 enhanced the absorption of albumin, and this was blocked by a PKC activator or PKA inhibitor.

Conclusions: These findings suggest that rhGLP-1 can reverse diabetic nephropathy by protecting both glomeruli and tubules by inhibiting PKC and activating PKA.

Keywords: Diabetic nephropathy; Protein kinase; Recombinant human glucagon-like peptide-1.

Figure 1

The efficacy of recombinant human glucagon‐like peptide‐1 (GLP‐1) in diabetic nephropathy. (a) Blood glucose, (b) urinary protein and (c) urinary albumin were measured at different time‐points after treatment for up to 3 months. (d) Serum C‐peptide levels were measured by enzyme‐linked immunosorbent assay at the end of the experiment. (e) Insulin level in islets were detected by immunohistochemical staining in pancreatic tissue. Scale bar, 20 μm. Data are presented as mean ± standard error the mean (*P < 0.05, **P < 0.01 and ***P < 0.001; one‐way anova test). G, diabetic rats treated with recombinant human glucagon‐like peptide‐1 (n = 8; 5 male, 3 female); I, diabetic rats treated with insulin (n = 8; 3 male, 5 female); N, normal rats (n = 8l; 4 male, 4 female); V, diabetic rats treated with saline (n = 8; 4 male, 4 female).

Figure 2

Recombinant human glucagon‐like peptide‐1 protects the glomerular filtration barrier in diabetic rats. After treatment for 12 weeks, the animals in all groups were euthanized and the kidneys were harvested for structural evaluation of the glomerular filtration barrier. (a) Glomerular morphology was carried out by periodic acid–Schiff staining. Glomerulosclerosis scores were semiquantified in the normal group (N), vehicle group (V), recombinant human glucagon‐like peptide‐1 group (G), and insulin group (I). ***P < 0.001 (vs V). (b) Transmission electron microscopy for the structure of the glomerular basement membrane (GBM) and foot processes of podocytes. Scale bars, 2 μm. The GBM thicknesses were measured with a ruler in Adobe PDF files at >50 sites, and the results are presented as mean ± standard error of the mean. (c) Real‐time polymerase chain reaction, (d) western blot and (e) immunofluorescence imaging was used to measure the expression of podocin in the glomeruli. (f) Western blot, (g) real‐time polymerase chain reaction and (h) immunofluorescence imaging was used to measure the expression of nephrin in the glomeruli. Data are presented as mean ± standard error of the mean, n = 8 (*P < 0.05, **P < 0.01 and ***P < 0.001; one‐way anova test).

Figure 3

The effect of recombinant human glucagon‐like peptide‐1 treatment on the expression of protein kinase C (PKC) ‐β. (a–b) Eight weeks after treatment, animals were euthanized and the PKC‐β expression in kidneys was evaluated by immunohistochemistry and reverse transcription polymerase chain reaction. (c,d) The expression level of PKC‐β was also measured in isolated glomerular tissue and tubular tissue by western blotting (n = 8 each group, one‐way anova test). In addition, (e) PKC activity was evaluated by measuring the amount of phosphorylated PKC in isolated glomerular tissue (left) and tubular tissue (right). Data are presented as means ± standard error of the mean (*P < 0.05, **P < 0.01 and ***P < 0.001, NS, not significant, by one‐way anova test). G, recombinant human glucagon‐like peptide‐1 group; I, insulin group; N, normal group; V, vehicle group.

Figure 4

Recombinant human glucagon‐like peptide‐1 inhibits oxidative stress and diacylglycerol (DAG) in the glomeruli of diabetic nephropathy rats. (a) The expression of nitric oxide synthase (iNOS) and superoxide dismutase 1 (SOD1) was measured in kidneys by immunohistochemistry. Scale bar, 20 μm. In addition, the expression levels of iNOS and SOD1 were also measured in isolated glomeruli by (b) reverse transcription polymerase chain reaction and (c) western blotting. (d) DAG contents were measured by enzyme‐linked immunosorbent assay in the isolated glomerular tissue. (e) Immunofluorescence images of collagen IV in the diabetic nephropathy rat's kidney. G, recombinant human glucagon‐like peptide‐group; I, insulin group; N, normal group; V, vehicle group.

Figure 5

Recombinant human glucagon‐like peptide‐1 inhibits oxidative stress in rat glomerular endothelial cells (RGECs). (a) The effects of protein kinase C (PKC) and protein kinase A (PKA) on the expression of nitric oxide synthase (iNOS) were analyzed in by western blotting. RGEC was incubated with advanced glycation end‐products (200 μg/mL) to mimic diabetic injury, and the activator, inhibitor of PKC or PKA, was used to analyze the effect of the two pathways. **P < 0.01, ***P < 0.001. (b,c) Production of reactive oxygen species and nitric oxide was measured in cultured RGECs under different conditions. Data are presented as mean ± standard error of the mean (***P < 0.001 vs normal; #P < 0.05, ###P < 0.001 vs advanced glycation end‐products group; one‐way anova test; NS, not significant). A, advanced glycation end‐products; G, recombinant human glucagon‐like peptide‐1; I, insulin; iNOS, nitric oxide synthase; N, normal; PKAa, protein kinase A activator; PKCa, protein kinase C activator; PKAi, protein kinase A inhibitor; PKCi, protein kinase C inhibitor; SOD1, superoxide dismutase 1.

Figure 6

Recombinant human glucagon‐like peptide‐1 promotes the absorption of albumin in advanced glycation end‐product (AGE) ‐induced tubular epithelial cells through protein kinase C (PKC) ‐β and protein kinase A (PKA). (a) HK‐2 cells were cultured with AGEs to mimic diabetic nephropathy. The effects of PKC and PKA on the expression of megalin and cubilin were detected by western blotting (*P < 0.05, **P < 0.01, ***P < 0.001). (b) PKC and PKA regulate the absorption of fluorescein‐labeled albumin by HK‐2 cells observed under microscopy. (c) Quantitative fluorescence intensity was measured with a fluorescence plate reader and cell numbers were normalized to total protein. Data are presented as means ± standard error of the mean (*P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant vs advanced glycation end‐products group; one‐way anova test). A, advanced glycation end‐products; G, recombinant human glucagon‐like peptide‐1; GLP‐1, glucagon‐like peptide‐1; I, insulin; N, normal; PKAa, protein kinase A activator; PKAi, protein kinase A inhibitor; PKCa, protein kinase C activator; PKCi, protein kinase C inhibitor.