Defective complement activation has been associated with various types of kidney disease. This led to the hypothesis that specific urine complement fragments may be associated with kidney disease ...etiologies, and disease progression may be reflected by changes in these complement fragments. We investigated the occurrence of complement fragments in urine, their association with kidney function and disease etiology in 16,027 subjects, using mass spectrometry based peptidomics data from the Human Urinary Proteome/Peptidome Database. Twenty-three different urinary peptides originating from complement proteins C3, C4 and factor B (CFB) could be identified. Most C3-derived peptides showed inverse association with estimated glomerular filtration rate (eGFR), while the majority of peptides derived from CFB demonstrated positive association with eGFR. Several peptides derived from the complement proteins C3, C4 and CFB were found significantly associated with specific kidney disease etiologies. These peptides may depict disease-specific complement activation and could serve as non-invasive biomarkers to support development of complement interventions through assessing complement activity for patients' stratification and monitoring of drug impact. Further investigation of these complement peptides may provide additional insight into disease pathophysiology and could possibly guide therapeutic decisions, especially when targeting complement factors.
Background. The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a group of heterogeneous diseases. This study was undertaken to investigate the outcome of Wegener's ...granulomatosis (WG), microscopic polyangiitis (MPA) and renal-limited vasculitis (RLV). Furthermore, we analysed the differences in patients with proteinase 3-ANCA (PR3-ANCA) and those with myeloperoxidase-ANCA (MPO-ANCA), which have not been assessed in a homogeneously treated group of patients with renal involvement. Methods. In this retrospective analysis, 80 patients with a new diagnosis of WG, MPA or RLV with biopsy-proven renal involvement were followed over a median of 46.7 months (range: 0.8–181.9 months). All patients had induction treatment with cyclophosphamide and oral corticosteroids. Results. At the end of follow-up, 23% were dependent on dialysis. Renal survival was significantly worse in patients with WG compared with patients with MPA or RLV (P = 0.04). A higher rate of end-stage renal disease (ESRD) was noticed in PR3-ANCA- vs MPO-ANCA-positive patients. A total of 21 patients (26%) died. Predictors of patient mortality were development of ESRD, older age and the maximum creatinine in the first month. Mortality was found to be higher in patients with WG and was significantly higher in PR3-ANCA-positive cases (P = 0.02). The relative risk of death was 9.32 times higher in PR3-ANCA- vs MPO-ANCA-positive patients. Conclusions. Our data underscore the pathogenetic potential of ANCA by demonstrating a more aggressive disease state and a poorer outcome in patients with PR3-ANCA.
We report here the case of a 17-year-old boy who suffered acute renal failure after consuming 3 L of energy drink (ED) in combination with 1 L of vodka amounting to 4600 mg of taurine and 780 mg of ...caffeine mixed with 380 g of alcohol. The consumption of this mixture is extremely popular in adolescents, because the joint effects of caffeine and taurine reduce the effect of alcohol. Although there have been case reports of deaths linked to the consumption of EDs with and without alcohol, awareness of the possible dangers is still low. The fact that athletes and major sports events are sponsored by ED manufacturers implies that they may even be healthy and performance-enhancing.
Nitric oxide down-regulates connective tissue growth factor in rat mesangial cells.
Nitric oxide (NO) exerts complex regulatory actions on mesangial cell (MC) biology, such as inhibition of ...proliferation, adhesion or contractility and induction of apoptosis. In our previous studies the NO-donor S-nitroso-glutathione (GSNO) was found to be a potent inhibitor of MC growth. This effect was mediated at least in part by inhibitory effects of GSNO on the transcription factor early growth response gene-1 (Egr-1)10. We therefore were interested in the regulation of gene expression in MC after treatment with NO.
To identify the genes that are regulated by NO in MC, gene expression was analyzed by representational difference analysis. Expression of connective tissue growth factor (CTGF) was studied by Northern and Western blot analyses.
Cultured rat MCs treated with GSNO for 8 hours were compared with unstimulated MCs and the CTGF mRNA was found to be down-regulated. The down-regulation was dose-dependent and transient, with a maximum inhibition seen after 6 hours. In parallel, down-regulation of CTGF protein by GSNO was observed by Western blot analysis. Other NO-donors such as S-nitroso-N-acetyl-D,L-penicillamine and spermine-NO showed similar effects. The induction of the inducible NO-synthase by TNF-α, IL-1β and LPS provoked a transient down-regulation of CTGF mRNA, an effect that could be partially overcome by pretreatment with the NOS-inhibitor Nω-nitro-L-arginine methyl ester. The observed NO-effect could be simulated by treatment with the stable cGMP analog 8br-cGMP, and was abolished by blocking the guanylyl cyclase with the inhibitor NS2028.
NO acts as a strong repressor of CTGF expression in cultured rat MC. Thus, in addition to its antiproliferative effects, NO potentially exerts antifibrotic activity by down-regulation of CTGF.
Mesangial cells are thought to be important mediators of glomerular inflammation and fibrosis. Studies have established a direct role for nitric oxide (NO) in the regulation of gene expression in ...mesangial cells. Representational difference analysis was used to investigate changes in gene expression elicited by the treatment of S-nitroso-L-glutathione in rat mesangial cells. Seven upregulated and 11 downregulated genes were identified. Four out of 11 downregulated genes (connective tissue growth factor, thrombospondin-1, collagen type I α1 and collagen type I α2) are known to be linked to inflammation and fibrosis. Results were verified across species in mesangial cells treated with a series of NO donors using Northern blot analysis, quantitative real-time PCR and protein analysis methods. Induction of endogenous NO production by cytokine stimulation also triggered regulation of the genes. One example gene, connective tissue growth factor, was studied at the promoter level. Promoter-reporter gene studies in mesangial cells demonstrated that NO acts at the transcriptional level to suppress gene expression. Our results reveal a complex role of NO in regulating gene expression in mesangial cells and suggest an antifibrotic potential for NO.
Abstract
BACKGROUND AND AIMS
Currently >20 000 native peptides in urine are known that are highly dynamic and able to display the status of different organs, especially the kidney. The ...characterization of urinary peptide profiles (UPP) enables the depiction of kidney disease severity, progression, fibrosis, and informs about the disease etiology. Advanced machine learning algorithms enable combining the changes in the very complex UPP associated with specific disease etiologies and reducing the dataspace to only few dimensions. Here, we show the application of a supervised machine learning pipeline for the visualization of different CKD etiologies based on high-dimensional peptidomics data, toward non-invasive disease classification.
METHOD
The Uniform Manifold Approximation and Projection (UMAP) algorithm was used as a novel nonlinear dimensionality-reduction technique to visualize and differentiate the UPP of patients with CKD of different etiologies. UPP of individual CKD patients (with diabetic kidney disease DKD, (n = 386), IgA nephropathy (n = 743) and vasculitis (n = 150)) and 369 healthy controls were extracted from the Human Urinary Proteome Database which contains >85 000 proteomics datasets analyzed using capillary electrophoresis coupled mass spectrometry. About 80% of the extracted datasets were used as a training and 20% as validation set.
RESULTS
When applying supervised-UMAP to the DKD patient and control datasets, excellent separation with an F1 score of 99.5% ± 0.9% in the training set, and 93.1% ± 3.3% in the independent test set could be observed. Subsequently, this approach was applied to differentiate controls and three kidney diseases (DKD, IgA nephropathy and vasculitis) simultaneously. In the training set an accuracy of up to 98% in DN and controls, and an overall F1 score of 93.7% ± 2.3% (Figure) was achieved. In the independent test set, accuracy decreased as expected to around 90% for controls, 83.8% for IgA nephropathy, 79.2% for DKD and 66.7% for vasculitis. The overall F1 score in the test set is 81.9% ± 2.2%. Of note, controls (n = 369) were consistently classified with the highest accuracy across all groups, the disease with smallest sample size (vasculitis, n = 150) always showed the lowest accuracy. A substantial proportion of vasculitis was classified as IgA nephropathy, which has the largest sample size of n = 743.
For the validation of the pipeline the permutation test was used. Permutation test was repeated 100 times using all the samples of CKD -free controls and three kidney diseases. The resulted scores were normally distributed, with a mean of 32.5% and standard deviation of 1.2%. Compared with the true F1 score, which was calculated as 81.9% from above, the probability of obtaining such a high score by chance is very low (P < 0.01).
CONCLUSION
We show that UMAP combined with supervised machine learning applied to high dimensional peptidomics data, enables distinguishing multiple kidney diseases with good accuracy and with very small standard deviation between multiple train-test splits. To our knowledge, our study is the first of its kind to reduce the complexity of the urinary peptidome to a single point in space, and categorize disease etiology based on the spatial information. The approach presented has the potential to enable non-invasive differential diagnosis of kidney disease etiologies. To improve accuracy of this non-invasive method, inclusion of additional clinical parameters will be tested.
In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 ...expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis.
The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1.
GSNO strongly inhibited serum-induced MC proliferation (-84% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 μmol/L, whereas mRNA levels increased again at concentrations above 500 μmol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (-40%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO.
Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.
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