In-situ hybridization studies of protein kinase C α and β I isoforms in human diabetic nephropathy

Abstract

Background. Hyperglycemia is the most important contributor to the development of diabetic nephropathy (DN). The activation of protein kinase C (PKC) caused by hyperglycemia is implicated in the pathogenesis of DN. PKC, which comprises a family of enzymes, plays a role in many signal transduction pathways and is involved in the regulation of cell growth and differentiation. However, the precise role of PKC in the progression of human DN is not fully understood. Methods. To evaluate the pathological role of PKC in DN, we examined the mRNA expression levels of PKC α and PKC I isoforms in normal renal tissues and in renal tissues affected by DN. Tissues from open renal biopsies were obtained from 20 type 2 diabetic patients with DN. The expression levels of PKC α and PKC I mRNA in kidneys with DN and in normal human kidney (NHK) were evaluated by in-situ hybridization, using digoxigenin-labeled oligonucleotide probes. Results. Cells positive for PKC α and I mRNA were mainly observed in glomeruli, and some mRNA was also detected in the tubulointerstitium. The percentages of glomerular cells positive for PKC α and I mRNA were higher in kidneys with DN than in NHK. In the glomeruli of kidneys with DN, the percentage of cells positive for PKC I mRNA, but not the percentage of cells positive for PKC α mRNA, tended to be decreased with the degree of mesangial expansion. Conclusions. Our results suggest that the expression of mRNA for PKC α and/or I may be associated with the progression of DN.

High expression of PKC-MAPK pathway mRNAs correlates with glomerular lesions in human diabetic nephropathy

“…CPCK isoforms, especially PKCa and b1, have been reported to be involved in the progression of DN [4,7,32]. We previously reported high expression of PKCa and b1 mRNA in renal tissue samples from patients with DN, and our results suggested an important role for PKCa and b in the development and progression of DN [5]. It is well known that activated PKC triggers the MAPK cascade [33].…”

“…Diacylglycerol (DAG)-induced activation of protein kinase C (PKC) contributes significantly to the pathogenesis of DN [1][2][3][4]. We previously reported that PKCa and/or b1 might play an important role in the development and progression of glomerulosclerosis in DN [5]. PKC is known to activate mitogen-activated protein kinase (MAPK) through activating MAPK kinase (MAPKK).…”

Glomerular expression of platelet-derived growth factor (PDGF)-A, -B chain and PDGF receptor-?, -? in human diabetic nephropathy

“…To evaluate the specificity of the signals, two types of control experiments were carried out, as described previously. [22][23][24][25] (1) Pretreatment with RNase was performed after proteinase K digestion, and after sections were prehybridized and hybridized, and (2) a competitive study was performed by adding a 100-fold excess amount of homologous or unrelated, unlabeled oligonucleotides to the hybridization buffer together with the antisense probe.…”

“…In situ hybridization was performed using the modified technique developed in our laboratory. [22][23][24][25] Briefly, fresh renal biopsy tissues were embedded in optimol cutting temperature (OCT) compound and stored at Ϫ70°C until use. Specimens were cut into 4-µm-thick sections and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), and then deproteinized by HCl and digested with proteinase K (Sigma Chemical, St. Louis, MO, USA).…”

Dietary Perilla Seed Oil Supplement Increases Plasma Omega-3 Polyunsaturated Fatty Acids and Ameliorates Immunoglobulin A Nephropathy in High Immunoglobulin A Strain of ddY Mice

“…At the same time, the total number of glomerular resident cells in each glomerulus was also counted. To quantify TGF-β 1 mRNA expression, the percentage of positive cells to all glomerular resident cells was calculated as described in the literature [18,19,20]. Counting the TGF-β 1 -positive cells of at least 10 randomly selected nonsclerotic glomeruli was repeated three times by 3 independent investigators who were unaware of the others’ results.…”