Mechanisms Involved In 12/15-lipoxygenase-induced Renal Glomerular Mesangial Cellular Hypertrophy Associated With High Glucose

Diabetic nephropathy is one of the major complications of diabetes, which could cause end stage renal dysfunction. Glomerular hypertrophy is a key feature of Diabetic Nephropathy (DN) in the early stage, usually glomerular mesangial cell (MC) and endocellar cell which highly associated with renal hypertrophy and substitute glomerular fibrosis. Lipoxygenases (LO) are a family of nonheme iron-containing enzymes that insert molecular oxygen into polyunsaturated fatty acids such as arachidonic and linoleic acids. They are classified as 5-, 8-, 12-, and 15-LO on the basis of the carbon atom of arachidonic acid at which oxygen is inserted.12-LO activation can lead to the formation of oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]. Human and rabbit 15-LO as well as the leukocyte-type 12-LO have high homology and are classified as 12/15-LO because they can form both 12(S)-HETE and 15(S)-HETE from arachidonic acid. Recently, there has been heightened interest in 12/15-LO pathway because of key data implicating it in the pathogenesis of atherosclerosis, restenosis, and hypertension. It has been demonstrated that there are high expression of 12-LO, as well as activate formation such as 12(S)-HETE in the glomerular mesangial cells and vessel smooth muscle cells. The activate 12(S)-HETE is also associated with abnormal proliferation and inflammation at endothelial and glomerular cells. They could induce cellular hypertrophy, extracellular matrix components such as collagen, fibronectin overexpression and this effect was amplified under diabetic condition. In the studies of signaling transduction, we noticed that oxidized product of 12-LO could active extracellular kinase (ERK) or p38MAPK signaling pathway, as well as mediated activation of some growth factors and cytokine. In addition, LO enhance cellular nitrogen wasting which promote hypertension, artery sclerosis and renal disease.In this study, we noticed that 12(S)-HETE induce MC hypertrophy; MC from wild type (WT) and 12-LO knockout (LOKO) mice were cultured under normal or high glucose and cellular hypertrophy index showed that glucose associated mesangial cellular hypertrophy was lower in LOKO MC than in WT. Furthermore, in type 1 diabetes of WT and LOKO mice, we observed LOKO diabetic mice expressed low levels of albuminuria and proteinuria; the indices of renal hypertrophy and glomerular size was less in LOKO than in WT mice. Taken together, these results from in vitro and in vivo demonstrated that 12-LO metabolic pathway is highly associated with diabetic renal hypertrophy and pathological progress; repression of 12-LO in the course of DN is in favor of control cellular hypertrophy and nephropathy proceeding. It's well known that the process of cell proliferation, hypertrophy and apoptosis is finally decided at cell cycle control. Cell cycle is defined as the interval from the last cell division till this division. It is unilateral and phase character, usually stops at any phase, and continues transit to the next phase when the growth condition recovered. The cell cycle is controlled by positive and negative regulation proteins. The positive proteins include cyclin and cyclin dependent kinase (CDK); while the negative proteins include cyclin dependent kinase inhibitors (CDKI). They combined with CDK and inhibited their activity, affect DNA copy to restrain cell proliferation. They are divided as INK4 and CIP/KIP family according to the combined cyclin-CDK complex. The CIP/KIP family members such as p21, p27 are able to restrain most cyclin-CDK complex activity, control early or late stage of G1 and S phase, are major studies objects. Some people observed diabetic relative cell hypertrophy is associated with G1/S cell cycle arrest, and p21 as well as p27 protein overexpression is the major reason. Therefore we presume 12-LO may participate CDKI regulation.We observed in the study that 12(S)-HETE infusion significant enhanced p21 and p27 protein expression; Furthermore, HG induced overexpression of p27kip1 and p21cip1 were attenuated in MC and renal glomeruli from LOKO mice and reciprocally increased in an MMC cell line stably overexpressing 12/15-LO. From these results we conclude 12-LO induce p21 and p27 elevation to promote diabetic glomerular hypertrophy.P21 is the founding member of the Cip/Kip family. The expression of p21 is controlled mostly at the transcriptional level. P53 protein is known as the key transcription factor of p21, the expression, subcellular distribution and binding ability to DNA of p53, would influence p21 transcriptional activity. Studies demonstrate phosphorylation on serine 15 of p53 reduce protein disassemble and increase protein expression, therefore result in p21 gene regulation. In the present study, we observed 12(S)-HETE enhanced p21 mRNA level in MC, correspondingly 12-LO knockout significantly inhibit high glucose associated p21 increment suggest 12(S)-HETE could transcriptionally regulate p21 gene expression. We also noted 12(S)-HETE induce p53 protein expression and its phosphorylation on serine 15 from nuclear protein, indicated 12(S)-HETE related p21 regulation is p53 dependent. In order to further certify the mechanism, we build new luciferase reporter plasmids containing mouse p21cip1 promoter region (-4542～+133bp,WT) and deletion of p53 binding cites including Mutant 1 (-2815～-2796) and Mutant 2 (-1923～-1904); According to the measurement of p21cip1 promoter transcript activity which construct containing p53 binding cites (wild type) and their mutant sites, we mentioned 12(S)-HETE enhanced activity of its WT promoter, while enhanced activity was abolished in mutant constructs, suggest that 12(S)-HETE induced p21cip1 transcriptional activity was depend on p53 transcript manner.In contrast to p21cip1, the mechanisms of regulation in p27kip1 expression are mainly at the level of post-transcription regulated by changes in protein translation and degradation through the ubiquitin proteolytic pathway; in another, it is also posttranslational modified by phosphorylation or subcellular distribution. We observed in this study that in spit of increase p27 protein expression, 12(S)-HETE treatment significantly induce p27 congregation in the nuclear. Immunofluerence results show that p27 focus in the nucleus at quiescent cell; 15%FBS induce cell proliferation and p27 was translocated from nuclear to cytoplasma, infusion of 12(S)-HETE lead to p27 reenter into the nuclear again. Taken together, 12(S)-HETE can modulate p27 activity through mediate its translocation from cytoplasm to nuclear in spite of the affection to its protein abundance.It's widely accepted that p38MAPK signaling pathway was involved in G1 cell cycle arrest. We previously demonstrated that LO production 12(S)-HETE primarily activates p38MAPK, lead to hypertrophy and matrix protein production, while specific p38 MAPK inhibitor blocked these effects completely in MC. In this study, we noted that 12(S)-HETE induced over expression of p21 and p27 were blocked by SB-202190 other than U-0126, a specific inhibitor of ERK1/2 pathway, further indicated that p38MAPK activation was associated with 12(S)-HETE-induced MC hypertrophy. We have demonstrated 12(S)-HETE phosphorylate and activate Smad pathway, as well as induce TGF-βgene expression recently; in another, TGF-βcould also activate MAPK signaling system including ERK, p38 and JNK in spite of classical Smad system,so 12(S)-HETE related TGF-βincrement may be the main reason of Smad and p38MAPK activation by 12(S)-HETE. Our results demonstrated pretreatment with both TGF-βneutralized antibody and receptor inhibitor attenuated Smad phosphyrylation, but failed to impact to p38MAPK activation related of 12(S)-HETE treatment, indicated 12/15-LO induced p38MAPK activation is independent of TGF-βand its pathway. Thereby, 12/15-LO activation induces hypertrophy in MC by regulating p21Cip1 and p27Kip1 expression and this procession is mediated through p38MAPK and TGF-βpathway.In summery, our study firstly demonstrates mechanisms of oxidized products of insaturated fatty acid in the progress of diabetic glomerular hypertrophy and observes lipoxygenase pathway crosstalk with TGF-b to activate each other, accelerate diabetic pathological progress. This study affords a new therapeutic target and research direction for postpone the progress of diabetic nephropathy and end stage of renal disease.