title: Expression and cellular distribution of TLR4, MyD88, and NF-κB in diabetic renal tubulointerstitial fibrosis, in vitro and in vivo.
abstract: OBJECTIVE
  Inflammation and extracellular matrix hyperplasia are crucial in the pathogenesis of tubulointerstitial fibrosis (TIF) involved in diabetic nephropathy (DN). Macrophage accumulation plays a major role, but whether immune factors contribute to DN pathogenesis is not well understood. This study aimed to investigate TLR4-MyD88-NF-κB-dependent pathway's involvement in TIF pathogenesis.
  
  
  METHODS
  STZ-induced diabetic rats and rat renal tubular epithelial NRK-52E cells cultured under high glucose conditions were used as in vivo and in vitro models, respectively. Real-time RT-PCR, western blot, immunohistochemistry and immunofluorescence were performed to examine the mRNA and protein levels of TLR4, MyD88, NF-κB, MCP-1, and α-SMA.
  
  
  RESULTS
  Compared with 5.5 mmol/L glucose, treatment of NRK-52E cells with 25 and 50 mmol/L d-glucose resulted in significantly increased TLR4 and MyD88 mRNA and protein levels (P<0.05). TLR4 and MyD88 were detected in the cytoplasm of most NRK-52E cells cultured under high glucose. Pronounced damage in the renal tubulointerstitium was observed in diabetic rats (scores: 3.82 ± 0.65 vs. 0.38 ± 0.08, P<0.01). Compared with the normal controls, a sharp upregulation of TLR4, MyD88, NF-κB p65, MCP-1, and α-SMA mRNA and protein levels was observed in diabetic rat kidneys (P<0.05). In diabetic animals, TLR4 and MyD88 were strongly expressed in the cytoplasm, while NF-κB p65 was widely expressed in cytoplasm and nuclei of renal tubular epithelial cells.
  
  
  CONCLUSIONS
  The inflammatory reaction and epithelial-mesenchymal transformation observed in renal tubulointerstitium may be the result of overactivation of the TLR4-MyD88-NF-κB-dependent innate immunity under high glucose, and may be involved in DN occurrence and progression.
authors.affiliation: Department of endocrinology, General hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Province, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of endocrinology, General hospital of Tianjin Medical University, Tianjin 300052, China.
authors.forename: Feng'ao
authors.initials: F
authors.lastname: Li
authors.affiliation: Department of endocrinology, General hospital of Tianjin Medical University, Tianjin 300052, China.
authors.forename: Mingcai
authors.initials: M
authors.lastname: Qiu
authors.affiliation: Cardiovascular and Cerebrovascular Disease Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Province, China. Electronic address: helanjie@medmail.com.cn.
authors.forename: Lanjie
authors.initials: L
authors.lastname: He
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D003928:Diabetic Nephropathies / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D005355:Fibrosis; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007249:Inflammation / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D007684:Kidney Tubules / Q000378:metabolism* / Q000473:pathology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009395:Nephritis, Interstitial / Q000150:complications / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013549:Sweetening Agents / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C507781:Myd88 protein, rat; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D012333:RNA, Messenger; D013549:Sweetening Agents; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D005947:Glucose
keywords: Diabetic nephropathy; MyD88; NF-κB; Renal tubulointerstitial fibrosis; TLR4