Huijuan Wu

UCH-L1 expression of podocytes in diseased glomeruli and in vitro.

Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1), an important member of de‐ubiquitination enzyme families involved in the ubiquitin–proteasome pathway, is expressed mainly in neural and reproductive systems as well as in some tumours. Recently, expression of UCH‐L1 has been discovered in parietal epithelial cells of Bowmans capsules and some tubular epithelia in the kidney. However, whether UCH‐L1 is expressed in the capillary tufts of the glomeruli has not yet become clear. In this study, we used immunohistochemistry, double immunofluorescence labelling, immunoelectron microscopy in kidney biopsy tissues and western blot in cultured rat podocytes to investigate the expression of UCH‐L1 and the regulation of this expression in podocytes. The results demonstrated that UCH‐L1 was expressed in podocytes and its expression was significantly higher in acute proliferative glomerulonephritis (APGN), lupus nephritis (LN), membranous glomerulonephritis (MGN) and IgA nephropathy than that in focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), minor abnormality and normal kidney tissues (p < 0.05). In in vitro experiments, western blot showed that UCH‐L1 expression significantly increased in the two groups of podocytes co‐cultured with mesangial cells exposed to ATS 50 µl/ml and ATS 50 µl/ml with normal human serum 30 µl/ml, respectively (p < 0.05), while the other groups treated with TGFβ1 1 ng/ml, TNFα 10 ng/ml or IL‐1 10 ng/ml had little rise of UCH‐L1 expression, with no statistical significance compared with normal control (p > 0.05). Further tests indicated that the percentage of PCNA‐positive podocytes in LN, APGN and IgA nephropathy was significantly higher than that in MCD, FSGS and normal control (p < 0.05). These data show for the first time that podocytes do express UCH‐L1 and that its expression can be increased in these immunocomplex‐mediated nephrites. The immune injury is a main cause for stimulating podocytes to express UCH‐L1. The expression of UCH‐L1 may be associated with the regeneration of podocytes as a repair response to immunocomplex‐mediated injury. Copyright

Overexpression of decorin induces apoptosis and cell growth arrest in cultured rat mesangial cells in vitro.

Background:  Decorin (DCN) is a small leucine‐rich proteoglycan that plays an important role in the regulation of intercellular contact, cell migration and proliferation. DCN suppresses cell growth and induces apoptosis in various tumour cells. The aim of this study was to investigate whether overexpression of DCN could induce apoptosis and cell growth arrest in mesangial cells (MsCs) in vitro.

Classification and Differential Diagnosis of Diabetic Nephropathy

Diabetic nephropathy (DN) is a major cause of end-stage renal disease throughout the world in both developed and developing countries. This review briefly introduces the characteristic pathological changes of DN and Tervaert pathological classification, which divides DN into four classifications according to glomerular lesions, along with a separate scoring system for tubular, interstitial, and vascular lesions. Given the heterogeneity of the renal lesions and the complex mechanism underlying diabetic nephropathy, Tervaert classification has both significance and controversies in the guidance of diagnosis and prognosis. Applications and evaluations using Tervaert classification and indications for renal biopsy are summarized in this review according to recent studies. Meanwhile, differential diagnosis with another nodular glomerulopathy and the situation that a typical DN superimposed with a nondiabetic renal disease (NDRD) are discussed and concluded in this review.

Regulation of intracellular decorin via proteasome degradation in rat mesangial cells

Decorin (DCN) is a member of small leucine‐rich proteoglycan family that neutralizes the bioactivity of transforming growth factor‐beta1 (TGF‐β1). It has been proven to be a promising anti‐fibrotic agent to treat glomerulonephritis. But the underlining mechanism for regulating and degrading intracellular DCN is still not fully understood. In this study, we investigated the roles of ubiquitination in the regulation of cytoplasmic DCN metabolism in rat mesangial cells (MC) by immunoprecipitation and Western blot. The results showed that a proportion of cytoplasmic DCN was ubiquitinated in normal MC and was enhanced in N‐glycosylation inhibitor (tunicamycin)‐treated MC. After being treated with the proteasome inhibitor MG132, ubiquitinated DCN accumulated and displayed a prolonged half‐life, accompanied by decreased TGF‐β1 expression and reduced collagen IV mRNA level in MC. This study demonstrated that the stability and function of cytoplasmic DCN can be regulated by ubiquitin‐proteasome system (UPS) in MC, which implies that regulating the ubiquitination and degradation of DCN might be a novel approach for modulating MC bioactivity. J. Cell. Biochem. 111: 1010–1019, 2010.

Transgene therapy for rat anti-Thy1.1 glomerulonephritis via mesangial cell vector with a polyethylenimine/decorin nanocomplex

Polyethylenimine (PEI), a cationic polymer, is one of the most efficient non-viral vectors for transgene therapy. Decorin (DCN), a leucine-rich proteoglycan secreted by glomerular mesangial cells (MC), is a promising anti-fibrotic agent for the treatment of glomerulonephritis. In this study, we used PEI–DCN nanocomplexes with different N/P ratios to transfect MC in vitro and deliver the MC vector with PEI–DCN expressing into rat anti-Thy1.1 nephritis kidney tissue via injection into the left renal artery in vivo. The PEI–plasmid DNA complex at N/P 20 had the highest level of transfection efficiency and the lowest level of cytotoxicity in cultured MC. Following injection, the ex vivo gene was transferred successfully into the glomeruli of the rat anti-Thy1.1 nephritis model by the MC vector with the PEI–DCN complex. The exogenous MC with DCN expression was located mainly in the mesangium and the glomerular capillary. Over-expression of DCN in diseased glomeruli could result in the inhibition of collagen IV deposition and MC proliferation. The pathological changes of rat nephritis were alleviated following injection of the vector. These findings demonstrate that the DCN gene delivered by the PEI–DNA nanocomplex with the MC vector is a promising therapeutic method for the treatment of glomerulonephritis.

Expression of USP2-69 in mesangial cells in vivo and in vitro

Ubiquitin‐specific protease 2 (USP2) is a member of a family of de‐ubiquitinating enzymes. It may play an important role in the regulation of cell growth and differentiation. It is known that expression of the isoform USP2‐69 kD is high in kidney tissue, but its role remains unclear. Mesangial cell proliferation is a prominent element of various types of glomerulonephritides. Therefore, whether USP2 plays a role in mesangial cell proliferation during glomerulonephritides is an interesting question to explore. The purpose of the present study was to evaluate USP2‐69 expression in needle biopsies of human kidneys and in cultured rat mesangial cells. On immunohistochemistry USP2‐69 was upregulated in some mesangial proliferative glomerulonephritides. The proportion of USP2‐69 positive area in the glomeruli was 3.90% in normal kidney, 4.96% in minimal change disease, and 4.39% in membranous glomerulonephritides, while it was 14.84% in IgA nephropathy (IgAN) (mesangial proliferative type), 16.18% in lupus nephritis (LN; diffuse proliferative type) and 15.54% in acute proliferative glomerulonephritides (APGN); the difference of the percentages between IgAN, LN (IV subtype) and APGN and normal kidney were statistically significant (P < 0.05). Additionally, the number of proliferating cell nuclear antigen (PCNA)‐positive nuclei in the glomeruli was statistically significantly higher in the various glomerulonephritides than in the normal kidney (P < 0.05). Immunohistochemistry showed that the distribution of the USP2+ area and PCNA+ nuclei overlapped in the glomeruli. Treatment with interleukin‐1β for 12 h and 24 h, or with anti‐thymocyte serum for 6 h and 12 h resulted in elevated USP2‐69 mRNA and protein expression in the rat mesangial cells. Also, PCNA expression increased and p27 expression decreased significantly in the treated mesangial cells. These findings suggest that USP2‐69 was upregulated in mesangial cells during mesangial proliferative glomerulonephritides in vivo and in vitro, which may relate to the proliferation of mesangial cells.

OTUB1 Overexpression in Mesangial Cells Is a Novel Regulator in the Pathogenesis of Glomerulonephritis through the Decrease of DCN Level

Background OTUB1 is a member of OTUs (Ovarian-tumor-domain-containing proteases), a deubiquitinating enzymes family (DUBs), which was shown as a proteasome-associated DUB to be involved in the proteins Ub-dependent degradation. It has been reported that OTUB1 was expressed in kidney tissue. But its concrete cellular location and function in the kidney remain unclear. Decorin (DCN) in mesangial cells (MC) is considered to be a potentially important factor for antagonizing glomerulonephritides, and its degradation is mediated by ubiquitination. The aim of this study is to investigate the role of OTUB1 expression in MC and its relationship with DCN during glomerulonephritis. Methodology/Principal Findings Using quantitative RT-PCR and Western blot, we demonstrated that OTUB1 mRNA and protein were constitutively expressed in cultured rat MC and found to be upregulated by the stimulation of IL-1β or ATS. OTUB1 overexpression was detected in the mesangial area of glomeruli in some immunocomplex mediated nephritides such as IgA nephropathy, acute diffuse proliferative glomerulonephritis and lupus nephritis by immunohistochemistry. The immunoprecipitation assay demonstrated that OTUB1 interacted with DCN. The overexpression of OTUB1 enhanced the ubiquitination and degradation of DCN in MC. Conclusion/Significance These data showed the inflammatory injury could up-regulate OTUB1 expression in MC, which might attribute the promoting effect of OTUB1 on glomerulonephritides to the decrease of DCN level.

Detection of UCH-L1 expression by pre-embedding immunoelectron microscopy with colloidal gold labeling in diseased glomeruli.

The purpose of this report was to study the use of pre-embedding immunoelectron microscopy technique with gold and horseradish peroxidase (HRP) labeling in detecting the expression of ubiquitin C-terminal hydrolase 1 (UCH-L1) of podocytes in glomerulonephritis. The specimens of human IgA nephropathy and lupus nephritis were fixed with paraformaldehyde and lysine–HCl buffer, labeled by colloidal gold or HRP, embedded with epoxy resin, and examined under the transmission electron microscope. The high density of gold particles or peroxidase reaction products (DAB) combined with UCH-L1 was obvious in cytoplasm and processes of podocytes. This modified technique of pre-embedding immunoelectron microscopy could perfectly preserve the ultrastructure of kidney and expose antigens which is valuable for clinical diagnostic work and experimental research.

Clinicopathological features of idiopathic membranous nephropathy combined with IgA nephropathy: a retrospective analysis of 9 cases

BackgroundThe concomitant presence of idiopathic membranous nephropathy and IgA nephropathy is rare. Here, we report 9 cases of phospholipase-A2-receptor (PLA2R) positive idiopathic membranous nephritis combined with IgA nephropathy, while reviewing publications regarding the pathological characteristics of this glomerolonephritis complication.Case presentationNine cases of renal biopsy tissues were retrospectively reviewed, including the clinicopathological features, the results of the immunofluorescence assays, and the electron microscopic examination. The patients mainly presented proteinuria and microscopic hematuria, and the serum anti-PLA2R was detected as positive in all of the patients. Histologically, a wide thickening of the glomerular basement membrane was observed in each of the 9 cases. Additionally, there existed mild hyperplasia in the mesangial cell and the matrix of the mesangial area. Immunofluorescence assays showed prominent glomerular granular staining on the glomerular capillary loops for IgG (++/+++), IgG4 (++/++++), and PLA2R (+/++). In addition, moderate IgA positive stains were focally or sparsely limited to the mesangial areas. Electron microscopy revealed subepithelial and mesangial electron-dense deposits.ConclusionsThe results from the case analyses indicated that idiopathic membranous nephropathy combined with IgA nephropathy possess the clinicopathological features found in both components. It is suggested that serum anti-PLA2R and tissue PLA2R are important biomarkers that can assist in the diagnosis of idiopathic membranous nephropathy associated with IgA nephropathy.

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion–Induced Kidney Growth

The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys.