The Functions of Clusterin Expression in Renal Mesenchymal Stromal Cells: Regulation of Cell Proliferation and Macrophage Activation

The expression of clusterin (CLU) in mice increases resistance to renal ischemia-reperfusion injury and promotes renal tissue repair. However, the mechanisms underlying of the renal protection of CLU remain largely unknown. Mesenchymal stromal cells (MSCs), found in different compartments of the kidney, may contribute to kidney cell turnover and injury repair. This study investigated the in vitro functions of CLU in kidney mesenchymal stromal cells (KMSCs). KMSCs were isolated by digestion of kidney tissues with collagenase type 1 and growth in plastic culture plates. Cell surface markers, apoptosis and phagocytosis were determined by flow cytometry, and CLU protein by Western blot. Here, we showed that KMSCs isolated from both wild type (WT) and CLU knockout (KO) mice positively expressed CD133, Sca-1, CD44, and CD117, and negatively of CD34, CD45, CD163, CD41, CD276, CD138 and CD79a. There was no difference in trilineage differentiation to chondrocytes, adipocytes and osteocytes between WT and KO KMSCs. CLU protein was expressed in and secreted by WT KMSCs, and it was up-regulated in response to hypoxia. However, lack of CLU expression did not negatively affect hypoxia-induced apoptosis in cultured KMSCs. The WT KMSCs proliferated faster than KO KMSCs in cultures. Furthermore, the incubation of macrophages with CLU-containing culture medium from WT KMSCs increased the CD206 expression in the macrophages and their phagocytic capacity. In conclusion, our data for the first time demonstrate the functions of CLU in the promotion of KMSCs proliferation, and may be required for KMSCs-regulated macrophage M2 polarization and phagocytic activity.

Immunohistochemical expression of clusterin in colorectal carcinoma

Background and objective: Colorectal cancer is a heterogeneous malignancy characterized by a wide range of genetic and epigenetic alterations. Clusterin is a heterodimeric glycoprotein widely expressed in a variety of tissues and secreted in many body fluids. Increased clusterin expression has been reported in the normal colonic mucosa, benign polyps, and colorectal carcinoma. This study aimed to detect the frequency of the clusterin immunoexpression in colorectal carcinoma and determine its association with some clinicopathological parameters. Methods: Sixty formalin-fixed paraffin-embedded sections of colorectal adenocarcinoma were obtained and randomly selected from the histopathology laboratory at Rizgary Teaching Hospital and some private histopathology laboratories in Erbil city over two years between December 2016 and December 2018. All patients had been diagnosed to have primary colorectal adenocarcinoma and had undergone surgery. The clinicopathological characteristics of the tumors were revised, and the specimens were analyzed immunohistochemically using anticlusterin mouse monoclonal antibody. Results: Twenty eight cases (46.6%) were labeled as clusterin positive, while 32 cases (53.4%) were negative for clusterin expression. Clusterin expression was significantly associated with the tumor type (Non-mucinous) (P = 0.01) and tumor grade (well to moderately differentiated) (P = 0.03). At the same time, no significant association was found between clusterin immunoexpression and other clinicopathological characteristics like age, gender, tumor site, and tumor stage. Conclusion: Our study indicated that clusterin is overexpressed in some colorectal carcinomas and is significantly associated with histological type and grade. These results suggest that clusterin may play a role in colorectal carcinogenesis. Further studies are required to understand the possible mechanism of clusterin association with carcinogenesis and cancer progression. Keywords: Colorectal cancer; Clusterin; Immunohistochemistry.

The Functions of Clusterin Expression in Renal Mesenchymal Stromal Cells: Regulation of Cell Proliferation and Macrophage Activation

BackgroundThe expression of clusterin (CLU) in mice increases resistance to renal ischemia-reperfusion injury and promotes renal tissue repair. However, the mechanisms underlying of the renal protection of CLU remain largely unknown. Mesenchymal stromal cells (MSCs), found in different compartments of the kidney, may contribute to kidney cell turnover and injury repair. This study investigated the in vitro functions of CLU in kidney mesenchymal stromal cells (KMSCs). MethodsKMSCs were isolated by digestion of kidney tissues with collagenase type 1 and growth in plastic culture plates. Cell surface markers, apoptosis and phagocytosis were determined by flow cytometry, and CLU protein by Western blot. ResultsKMSCs isolated from both wild type (WT) and CLU knockout (KO) mice positively expressed CD133, Sca-1, CD44, and CD117, and negatively of CD34, CD45, CD163, CD41, CD276, CD138 and CD79a. There was no difference in trilineage differentiation to chondrocytes, adipocytes and osteocytes between WT and KO KMSCs. CLU protein was expressed in and secreted by WT KMSCs, and it was up-regulated in response to hypoxia, but the degrees of hypoxia-induced apoptosis in WT KMSCs were not significantly different from those in KO KMSCs. The WT KMSCs proliferated faster than KO KMSCs in cultures. Furthermore, the incubation of macrophages with CLU-containing culture medium from WT KMSCs increased the CD206 expression in the macrophages and their phagocytic capacity. ConclusionOur data for the first time demonstrate the functions of CLU in the promotion of KMSCs proliferation, and may be required for KMSCs-regulated macrophage M2 polarization and phagocytic activity.

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process

Parkinson’s Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn’s aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aβ aggregation in Alzheimer’s Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.

Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

Clusterin, a glycoprotein encoded by the CLU gene, is expressed in many tissues, including the kidney, and clusterin expression is upregulated in the glomeruli of patients with various forms of kidney disease. Here, we investigated the role of clusterin in diabetic nephropathy (DN). In this study, we found that glomerular clusterin expression was increased in both patients with DN and streptozotocin-induced diabetic mice and that it co-localised with the podocyte marker WT1, indicating clusterin is expressed in podocytes. In our in vitro analysis, we found no significant change in CLU mRNA expression in podocytes following stimulation with high glucose and angiotensin II; in contrast, CLU mRNA expression was significantly upregulated following methylglyoxal stimulation. Methylglyoxal treatment also significantly decreased the mRNA expression of the slit diaphragm markers ZO-1 and NEPH1 and significantly increased the mRNA expression of the oxidative stress marker HO-1. Lastly, we showed that pre-incubating podocytes with recombinant human clusterin protein increased podocyte survival, prevented slit diaphragm damage, and reduced oxidative stress‒induced apoptosis following methylglyoxal stimulation. Taken together, our results indicate that glomerular clusterin is upregulated in DN, and this increase in clusterin expression may protect against oxidative stress-induced apoptosis in podocytes, providing a possible new therapeutic target for DN and other kidney diseases.

Preliminary Study on Clusterin Protein (sCLU) Expression in PC-12 Cells Overexpressing Wild-Type and Mutated (Swedish) AβPP genes Affected by Non-Steroid Isoprenoids and Water-Soluble Cholesterol

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (AβPP) processing and regulates clusterin protein levels. AβPP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sAβPPα) presence in AβPP-wt cells and Aβ expression in AβPP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 μM) or simvastatin (50 μM) as well as cholesterol chelator methyl-β-cyclodextrin (0.2 mM) diminished cell viability (p < 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p < 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in AβPP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.