Glomerular expression of p27Kip1 diabetic db/db mouse:: Role of hyperglycemia

Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G(1)-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27(Kip1). The present study was performed to investigate the renal expression of p27(Kip1) in db/db mice, a model of diabetes mellitus type II. Glomerular p27(Kip1) protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27(Kip1) positive glomerular cells showed a significant increase of these cells in db/db mice compared with nondiabetic db/+ animals. Although tubular cells revealed a positive staining for p27(Kip1) protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27(Kip1) protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27(Kip1) expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27(Kip1) expression in mesangial cells. Transfection of cells with p27(Kip1) antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db, mesangial cells. Furthermore, p27(Kip1) expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27(Kip1) expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27(Kip1) may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy.