Endothelial-derived complement factor D contributes to endothelial dysfunction in malignant nephrosclerosis via local complement activation.

Malignant nephrosclerosis is a thrombotic microangiopathy associated with abnormal local activation of the complement alternative pathway (AP). However, the mechanism underlying local AP activation is not fully understood. We hypothesized that complement factor D (CFD) secreted by endothelial cells triggers vascular dysfunction in malignant nephrosclerosis via local complement activation. We investigated the deposition of CFD in human kidney biopsy tissues and the function of endothelial-derived CFD in endothelial cell cultures. Immunofluorescence microscopy and laser microdissection-targeted mass spectrometry revealed significant deposition of CFD in the kidneys of patients with malignant nephrosclerosis. Conditionally immortalized human glomerular endothelial cells (CiGEnCs) continuously expressed and secreted CFD in vitro. CFD knockdown in CiGEnCs by small interfering RNA reduced local complement activation and attenuated the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), von Willebrand factor (VWF), and endothelin-1 (ET-1) induced by Ang II. The expression of CFD in CiGEnCs was significantly higher than that in other types of microvascular endothelial cells. Our findings suggest that (i) glomerular endothelial cells are an important source of local renal CFD, (ii) endothelial-derived CFD can activate the local complement system, and (iii) endothelial-derived CFD mediates endothelial dysfunction, which may play a role in the pathogenesis of malignant nephrosclerosis.

Precise diagnosis and typing of early-stage renal immunoglobulin-derived amyloidosis by label-free quantification of parallel reaction monitoring-based targeted proteomics.

BACKGROUND: Early diagnosis and typing are crucial for improving the prognosis of patients with renal amyloidosis. Currently, Untargeted proteomics based precise diagnosis and typing of amyloid deposits are crucial for guiding patient management. Although untargeted proteomics achieve ultra-high-throughput by selecting the most abundant eluting cationic peptide precursors in series for tandem MS events, it lacks in sensitivity and reproducibility, which may not be suitable for early-stage renal amyloidosis with minor damages. Here, we aimed to develop parallel reaction monitoring (PRM)-based targeted proteomics to achieve high sensitivity and specificity by determining absolute abundances and codetecting all transitions of highly repeatable peptides of preselected amyloid signature and typing proteins in identifying early-stage renal immunoglobulin-derived amyloidosis. METHODS AND RESULTS: In 10 discovery cohort cases, Congo red-stained FFPE slices were micro-dissected and analyzed by data-dependent acquisition-based untargeted proteomics for preselection of typing specific proteins and peptides. Further, a list of proteolytic peptides from amyloidogenic proteins and internal standard proteins were quantified by PRM-based targeted proteomics to validate performance for diagnosis and typing in 26 validation cohort cases. The diagnosis and typing effectiveness of PRM-based targeted proteomics in 10 early-stage renal amyloid cases was assessed via a comparison with untargeted proteomics. A peptide panel of amyloid signature proteins, immunoglobulin light chain and heave chain in PRM-based targeted proteomics showed significantly distinguishing ability and amyloid typing performance in patients. The diagnostic algorithm of targeted proteomics with a low amount of amyloid deposits in early-stage renal immunoglobulin-derived amyloidosis showed better performance than untargeted proteomics in amyloidosis typing. CONCLUSIONS: This study demonstrates that the utility of these prioritized peptides in PRM-based targeted proteomics ensure high sensitivity and reliability for identifying early-stage renal amyloidosis. Owing to the development and clinical application of this method, rapid acceleration of the early diagnosis, and typing of renal amyloidosis is expected.

Identification of polo-like kinase 1 as a therapeutic target in murine lupus.

INTRODUCTION: The signalling cascades that contribute to lupus pathogenesis are incompletely understood. We address this by using an unbiased activity-based kinome screen of murine lupus. METHODS: An unbiased activity-based kinome screen (ABKS) of 196 kinases was applied to two genetically different murine lupus strains. Systemic and renal lupus were evaluated following in vivo PLK1blockade. The upstream regulators and downstream targets of PLK1 were also interrogated. RESULTS: Multiple signalling cascades were noted to be more active in murine lupus spleens, including PLK1. In vivo administration of a PLK1-specific inhibitor ameliorated splenomegaly, anti-dsDNA antibody production, proteinuria, BUN and renal pathology in MRL.lpr mice (P < 0.05). Serum IL-6, IL-17 and kidney injury molecule 1 (KIM-1) were significantly decreased after PLK1 inhibition. PLK1 inhibition reduced germinal centre and marginal zone B cells in the spleen, but changes in T cells were not significant. In vitro, splenocytes were treated with anti-mouse CD40 Ab or F(ab')2 fragment anti-mouse IgM. After 24-h stimulation, IL-6 secretion was significantly reduced upon PLK1 blockade, whereas IL-10 production was significantly increased. The phosphorylation of mTOR was assessed in splenocyte subsets, which revealed a significant change in myeloid cells. PLK1 blockade reduced phosphorylation associated with mTOR signalling, while Aurora-A emerged as a potential upstream regulator of PLK1. CONCLUSION: The Aurora-A → PLK1 → mTOR signalling axis may be central in lupus pathogenesis, and emerges as a potential therapeutic target.

Association between thrombotic microangiopathy and activated alternative complement pathway in malignant nephrosclerosis.

BACKGROUND: Malignant nephrosclerosis, defined as renal microangiopathy in the setting of severe hypertension, remains a critical renal emergency leading to end-stage renal disease despite aggressive anti-hypertensive treatment. Recently, activation of the complement alternative pathway (AP) has been reported to play a prominent role in the pathogenesis of malignant nephrosclerosis. However, subsequent study failed to recapitulate the findings of genetic complement abnormalities in the disease. This study aimed to determine the presence of AP activation and genetic complement defects and establish their correlations to renal microangiopathy lesions, clinical features and prognosis in patients with malignant nephrosclerosis. METHODS: Fifty patients with malignant hypertension and concomitant thrombotic microangiopathy (TMA) proven by renal biopsy were investigated; 25 cases of kidney donors who received zero-hour allograft biopsies were used as normal controls. Various renal TMA lesions in patients with malignant nephrosclerosis were reviewed and evaluated using a semi-quantitative scoring system. Deposition of C5b-9, C3a, C5a, C4d and mannose-binding lectin was assessed by immunohistochemistry. Co-localization of C5b-9 and CD34 was detected by confocal microscopy. Complement factor B (FB), factor P (FP; properdin), factor D (FD), factor H (FH), C3a and C5a levels were quantified by enzyme-linked immonosorbent assay in plasma and urine samples of patients with malignant nephrosclerosis and controls. Genetic abnormalities of complement components were analysed by whole-exome sequencing. RESULTS: Renal biopsies of malignant nephrosclerosis showed identical histopathological and ultrastructural features to atypical haemolytic uraemic syndrome. C5b-9, C3a and C5a deposits were found along the walls of arteries/arterioles and glomerular capillaries and localized in the endothelial cells. Elevated plasma and urinary levels of FB, FP, FD, C3a and C5a as well as decreased FH levels were observed in patients with malignant nephrosclerosis compared with normal controls. The urinary levels of complement AP components, but not the plasma levels, were correlated with renal functions, prognosis and active TMA lesions except for arteriolar thrombi. Finally, mutations of the MCP, CFB, CFH and CFHR5 genes were identified in 8 of 20 patients with malignant nephrosclerosis. CONCLUSIONS: Aberrant complement AP dysregulation was demonstrated and associated with the activity, severity and renal outcomes of malignant nephrosclerosis. This observation warrants screening for complement defects in patients with malignant nephrosclerosis for the potential use of complement regulators and also highlights the need for further investigation of the precise role of AP in the pathogenesis of the disease.

Curcumin Attenuates Both Acute and Chronic Immune Nephritis.

Curcumin is known to have immunomodulatory potential in addition to anti-oxidant, anti-inflammatory and anti-carcinogenic effects. The aim of the present study is to investigate the therapeutic effects of curcumin on immune-mediated renal disease in an anti-glomerular basement membrane (GBM) model (representing acute kidney Injury, AKI) and murine lupus model (representing chronic kidney disease, CKD). In the AKI model, female anti-GBM 129/svj mice were administered with curcumin right before disease induction. In the CKD model, female MRL.lpr mice at the age of 8-10 weeks old were treated with curcumin or placebo via oral gavage daily for two months. After treatment, serum autoantibody levels, splenomegaly and spleen cellularity were reduced in murine lupus. Collectively, curcumin ameliorated kidney disease in the two mouse models with either acute or chronic nephritis, as marked by reduced proteinuria, blood urea nitrogen, glomerulonephritis, crescent formation, tubule-interstitial disease, and renal infiltration by lymphocytes. In addition, curcumin treatment reduced activation of the NFkB, MAPK, AKT and pBAD pathways either systemically, or within the inflamed kidneys. These findings suggest that natural food supplements could become an alternative approach to ameliorating immune-mediated kidney diseases.