Regulation Of UCH-L1 Expression Of Podocytes In Diseased Glomeruli And In Vitro

To our knowledge, the degradation of ubiquitin-conjugated cellular proteins by the proteasome system plays a key role in many cellular processes, including cell cycle control, DNA repair, regulation of transcription, growth differentiation, receptor function, immune and inflammatory responses and oncoprotein degradation. This system is also regulated by a reversible process involving many deubiquitination enzymes. Among these deubiquitination enzyme families, the best characterized are the ubiquitin COOH-terminal hydrolases (UCHs) and the ubiquitin-specific processing proteins(UBPs). UCH-L1 is an important member of UCH family, whose expression normally exhibits a prominent specificity of tissues including the brain, the testis and the kidney. Besides regulating targeted proteins through ubiquitin-proteasome pathway, a large amount of researches indicate that UCH-L1 also associates with some diseases, such as neurodegenerative diseases and cancers.In recent years, more and more attentions have been paid to the expression of UCH-L1 in kidney. Using immunohistochemistry, Shirato et al. observed UCH-L1 expression both in some parietal epithelial cells of normal rat and in crescentic cells of Masugi glomerulonephritis model. Diomedi et al. also demonstrated UCH-L1 was expressed in human tubular epithelial cells and parietal epithelial cells in normal and pathological renal tissues. But whether UCH-L1 is expressed in the glomeruli is still indefinite.In order to confirm the hypothesis of UCH-L1 expression in the glomeruli, we firstly detected its expression in paraffin-embedded tisssues of kidney needle biopsy from 98 cases by immunohistochemistry and discovered that UCH-L1 was expressed not only in parietal epithelial cells of Bowman's capsules and tubular epithelial cells, but also at the peripheral area of the capillary tufts of glomeruli. In addition, compared to normal kidney tissues, the expression level of UCH-L1 was markedly higher in acute proliferative glomerulonephritis (APGN), lupus nephritis (LN), membranous glomerulonephritis (MGN) and IgA nephropathy while little changed in minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS) and minor abnormalities.The distribution of UCH-L1 immunostaining by immunohistochemistry suggested that podocytes should be the cells which expressed UCH-L1 in some diseased glomeruli. Double immunofluorescence labling and immunoelectron microscopy were performed next to prove this hypothesis. The experimental result displayed UCH-L1 was expressed in the cytoplasms and processes of podocytes in some glomerulonephritises.The statistical data of the 98 cases detected indicated the degree of UCH-L1 expression in capillary tufts was somewhat variant among different pathological types of glomerulonephritises. In order to discover the possible stimulating factors that elevated UCH-L1 expression in podocytes of some nephritises, we treated the rat normal podocytes in vitro with some cytokins involved in inflammation reaction such as TGF-β1, TNF-αand IL-1, as well as immunocomplex and complements which participated in the pathogenesis of glomerulonephritis. Western blot revealed the expression of UCH-L1 was weak in normal rat podocytes as a basal level and little increased in other groups exposed to cytokins, while immunecomplex was a main factor which induced over-expression of UCH-L1 in podocytes. Complements alone may be a reason for UCH-L1 expression in podocytes at the early stage of damage responses.UCH-L1 over-expression has been reported mainly in cancers, including non-small-cell lung carcinoma, colorectal carcinoma and renal cell carcinoma, and further been confirmed to be closely related to abnormal regulation of cancer cells' proliferation. UCH-L1 is also expressed in the germ cells and regulates cells' multiplication, differentiation and apoptosis. The renal tubular epithelial cells with elevated expression of UCH-L1 perform more actively in the metabolism and proliferation. All the data suggest that there are some relationship between UCH-L1 expression and cell proliferation. So we used immunohistochemistry to detect PCNA in podocytes, and the result showed that podocyte number was nearly the same among different types of glomerulonephritises, while the percent number of PCNA positive cells in total podocytes was obviously higher in LN, APGN and IgA nephropathy than that in FSGS, MCD and normal control. PCNA was also increased with P27 decrease in rat podocytes under immune stimulation in vitro. This result further supports that immnocomplex induces the podocyte's regeneration as a repairing reaction to its damage and loss in renal diseases. Moreover UCH-L1 up-regulation in podocytes may probably associate with this response and be a symbol of podocytes' immune injury in glomerulonephritis.In summary, our experiment discovered that UCH-L1 was expressed not only in parietal epithelial cells of Bowman's capsules and tubular epithelial cells in kidney, but also in podocytes both in normal and pathological states. The expression level of UCH-L1 was markedly higher in APGN, LN, MGN and IgA nephropathy than that in FSGS, MCD, minor abnormalities and normal kidney tissue. In vitro experiment, we further confirmed that the immune injury was one main factor which induced up-expression of UCH-L1 in podocytes. Complements alone may join to be a partial reason for UCH-L1 expression of podocytes at the early stage of damage responses. The study also indicated that the podocyte's expression of UCH-L1 might closely relate to its regeneration as a repairing response. These data may offer a favorable experimental basis for further investigation of podocyte's role in nephrosis and mechanism of its regulation.