Response of mesangial cells to low-density lipoprotein and angiotensin II in diabetic (OLETF) rats.
BACKGROUND
Progression of diabetic nephropathy is closely associated with morphological changes in glomeruli, such as thickening of the glomerular basement membrane, mesangial expansion, and glomerulosclerosis. To elucidate early glomerular events, we compared the mitogenic activity and extracellular matrix production in mesangial cells (MC) isolated from diabetic rats prior to the manifestation of nephropathy and those showing overt nephropathy. This study may help to clarify the mechanisms underlying diabetic nephropathy and provide clues about early therapeutic interventions for preventing or slowing this process.


METHODS
Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a chronic model for human type 2 diabetes mellitus, and age-matched control (LETO) rats were used. Glomerular cell numbers, expression of immediate early genes (c-Fos and c-Myc) and proliferating cell nuclear antigen (PCNA), and low-density lipoprotein (LDL) deposition were determined in renal tissue sections from rats aged 15 to 75 weeks. Mesangial cells (MCs) from OLETF rats at two different stages of the disease, that is, young (12- to 14-week-old) OLETF rats (y-OLETF) prior to the manifestation of nephropathy and old (48- to 50-week-old) OLETF rats (o-OLETF) showing nephropathy, were isolated and cultured. After stimulation with native (n-) or oxidized (ox-) LDL or angiotensin II (Ang II), DNA synthesis and extracellular matrix (ECM) production were examined. Cellular expression of LDL/scavenger receptors was analyzed using fluorescence-labeled LDL and binding to 125I-labeled-LDL.


RESULTS
The number of cells per glomerular cross section was significantly higher in OLETF rats than in LETO rats between 25 and 65 weeks of age. In OLETF glomeruli, c-Fos, c-Myc, and PCNA were transiently expressed in the early phase. Glomerular LDL deposition increased with the age of OLETF rats. Addition of a low dose of n-LDL (10 microg/mL) to the culture medium significantly stimulated DNA synthesis of y-OLETF MCs, as compared with o-OLETF MCs and LETO MCs (P < 0.05). A high dose of n-LDL (100 microg/mL) caused cytotoxic effects in all cells. Exposure to ox-LDL minimally affected DNA synthesis of OLETF or LETO MCs. LDL receptors and scavenger receptors were predominant in y-OLETF and o-OLETF, respectively. After stimulation with n-LDL and ox-LDL, expression of type I and type III collagen, along with transforming growth factor-beta (TGF-beta), was higher in o-OLETF MCs that in y-OLETF MCs or LETO MCs. Exposure to Ang II markedly induced DNA synthesis and ECM mRNA expression in y-OLETF MCs and o-OLETF MCs, respectively.


CONCLUSIONS
These findings indicate that the cell proliferation process precedes the evolution of diabetic glomerulopathy. The responses of OLETF MCs to n-LDL/ox-LDL and Ang II differed depending on the stage of diabetes. In the early phase, MCs were prone to proliferate, whereas in the late stage, MCs, which expressed higher levels of TGF-beta, tended to synthesize ECM. A functional switch in MCs may contribute to the development of glomerulosclerosis in diabetic nephropathy.
Okada
Mitsuru
M
Department of Pediatrics, Kinki University School of Medicine, Osaka-Sayama, Japan.
Takemura
Tsukasa
T
Yanagida
Hidehiko
H
Yoshioka
Kazuo
K
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002452:Cell Count; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D008078:Cholesterol, LDL / Q000493:pharmacokinetics*; D024042:Collagen Type I / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D003924:Diabetes Mellitus, Type 2 / Q000473:pathology / Q000503:physiopathology; D003928:Diabetic Nephropathies / Q000473:pathology* / Q000503:physiopathology; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000187:drug effects / Q000473:pathology*; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL / Q000493:pharmacokinetics; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020334:Rats, Inbred OLETF; D011973:Receptors, LDL / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000235:genetics; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D008078:Cholesterol, LDL; D024042:Collagen Type I; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D018809:Proliferating Cell Nuclear Antigen; D016760:Proto-Oncogene Proteins c-fos; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011973:Receptors, LDL; D016212:Transforming Growth Factor beta; D014662:Vasoconstrictor Agents; C099252:oxidized low density lipoprotein; D000804:Angiotensin II; D004247:DNA; D020778:Matrix Metalloproteinase 2