J Korean Med Sci.  2009 Oct;24(5):837-843. 10.3346/jkms.2009.24.5.837.

Regulation of Type IV Collagen alpha Chains of Glomerular Epithelial Cells in Diabetic Conditions

• Ha TS
• Hong EJ
• Ahn EM
• Ahn HY

• KMID: 1782003
• DOI: http://doi.org/10.3346/jkms.2009.24.5.837

Abstract

An early feature of diabetic nephropathy is the alteration of the glomerular basement membrane (GBM), which may result in microalbuminuria, subsequent macroproteinuria, and eventual chronic renal failure. Although type IV collagen is the main component of thickened GBM in diabetic nephropathy, cellular metabolism of each alpha chains of type IV collagen has not been well studied. To investigate the regulation of alpha(IV) chains in diabetic conditions, we examined whether glucose and advanced glycosylation endproduct (AGE) regulate the metabolism of each alpha(IV) chains in the diabetic tissue and glomerular epithelial cells (GEpC). Glomerular collagen alpha3(IV) and alpha5(IV) chains protein were higher and more intense in immunofluorescence staining according to diabetic durations compared to controls. In vitro, mainly high glucose and partly AGE usually increased total collagen protein of GEpC by [3H]-proline incorporation assay and each alpha(IV) chain proteins including alpha1(IV), alpha3(IV), and alpha5(IV) in time-dependent and subchain-specific manners. However, the changes of each alpha(IV) chains mRNA expression was not well correlated to the those of each chain proteins. The present findings suggest that the metabolism of individual alpha(IV) chains of GBM is differentially regulated in diabetic conditions and those changes might be induced not only by transcriptional level but also by post-translational modifications.

Keyword

Glycosylation End Products, Advanced; Diabetic Nephropathies; Collagen Type IV; Glucose; Podocytes

MeSH Terms

Animals
Cells, Cultured
Collagen Type IV/genetics/*metabolism/physiology
Diabetic Nephropathies/*metabolism
Epithelial Cells/*metabolism
Glomerular Basement Membrane/metabolism
Glucose/metabolism
Glycosylation End Products, Advanced/metabolism
Male
Podocytes/*metabolism
RNA, Messenger/metabolism
Rats
Rats, Sprague-Dawley

Figure

• 

  Fig. 1 Immunofluorescence staining of α3(IV) and α5(IV) in rat tissues. (A) Double-staining of α5(IV) with P-cadherin and α3(IV) showed colocalization in podocyte foot processes around capillary loops in rat glomeruli at 48 hr of diabetes (magnification ×1,000). (B) Immunofluorescence microscopy of rat kidney tissue stained with anti-α3(IV) and α5(IV) antibodies at each experimental time (magnification ×400). The further diabetic nephropathy advanced, the more intense stainings of α3(IV) and α5(IV) chains along the glomerular capillaries were observed compared with those of age-matched controls.

• 

  Fig. 2 [3H]-Proline incorporation by high glucose and AGE. High glucose increased [3H]-proline incorporation of GEpC significantly (*P<0.05).

• 

  Fig. 3 Effects of glucose and AGE on the α(IV) chains protein in cultured GEpC assayed by Western blotting. (A) Sequential changes of α3(IV) proteins, (B) Sequential changes of α5(IV) proteins ; Both α3(IV) and α5(IV) chains were not changed initially, then, increased at 48 hr by high glucose, (C) High glucose (B30 and A30) increased the amounts of α1(IV) proteins significantly compared with group B5, (D) High glucose (B30 and A30) increased the amounts of α5(IV) proteins significantly compared with group B5. Data on the densitometric analysis of α1(IV) and α5(IV)/β-actin ratio are expressed as mean±SD. Control (100%); the value of B5. *P<0.05 versus control.

• 

  Fig. 4 Regulation of α(IV) chains mRNA in cultured GEpC assayed by RT-PCR. Sequential changes of α1(IV) mRNA (A), α3(IV) mRNA (B), α5(IV) mRNA (C). (D) High glucose and AGE (A30) increased the amounts of α1(IV) mRNA significantly at 48 hr incubation compared with group B5. Change of α3(IV) mRNA (E) and α5(IV) mRNA (F) at 48 hr incubation. Data on the densitometric analysis of each α(IV)/GAPDH ratio are expressed as mean±SD. Control (100%); the value of B5. *P<0.05.

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