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OBJECTIVE: Necrotizing crescentic glomerulonephritis (NCGN) is a major contributor to morbidity and mortality in ANCA-associated vasculitis (AAV). Since therapy relies on immunosuppressive agents with potentially severe adverse effects, a reliable non-invasive biomarker of disease activity is needed to guide treatment. METHODS: We used flow cytometry to quantify T cell subsets in blood and urine samples from 95 AAV patients and 8 controls to evaluate their biomarker characteristics. These were compared to soluble markers (MCP-1, sCD163, sCD25, and C5a) measured using multiplex analysis. Available kidney biopsies (n = 21) were classified according to Berden. RESULTS: Patients with active renal AAV showed significantly higher urinary cell counts than those in remission or with extra-renal manifestation or healthy controls. Urinary T cells showed robust discrimination of disease activity with superior performance compared to MCP-1 and sCD163. Patients whose kidney biopsies had been classified as "crescentic" according to Berden classification showed higher urinary T cell counts. Discordant T(reg) cell proportions and CD4(+) / CD8(+) ratio in blood and urine suggested that urinary cells reflect tissue migration rather than mere micro-bleeding. Furthermore, urinary T(reg) and T(H)17 patterns were associated with clinical response and risk of renal relapse. CONCLUSION: Urinary T cells reflect the renal inflammatory milieu in AAV and provide further insights into the pathogenesis of this chronic condition. Their promising potential as noninvasive diagnostic and prognostic biomarkers deserves further exploitation.

Hypoxia describes limited oxygen availability at the cellular level. Myeloid cells are exposed to hypoxia at various bodily sites and even contribute to hypoxia by consuming large amounts of oxygen during respiratory burst. Hypoxia-inducible factors (HIFs) are ubiquitously expressed heterodimeric transcription factors, composed of an oxygen-dependent α and a constitutive β subunit. The stability of HIF-1α and HIF-2α is regulated by oxygen-sensing prolyl-hydroxylases (PHD). HIF-1α and HIF-2α modify the innate immune response and are context dependent. We provide a historic perspective of HIF discovery, discuss the molecular components of the HIF pathway, and how HIF-dependent mechanisms modify myeloid cell functions. HIFs enable myeloid-cell adaptation to hypoxia by up-regulating anaerobic glycolysis. In addition to effects on metabolism, HIFs control chemotaxis, phagocytosis, degranulation, oxidative burst, and apoptosis. HIF-1α enables efficient infection defense by myeloid cells. HIF-2α delays inflammation resolution and decreases antitumor effects by promoting tumor-associated myeloid-cell hibernation. PHDs not only control HIF degradation, but also regulate the crosstalk between innate and adaptive immune cells thereby suppressing autoimmunity. HIF-modifying pharmacologic compounds are entering clinical practice. Current indications include renal anemia and certain cancers. Beneficial and adverse effects on myeloid cells should be considered and could possibly lead to drug repurposing for inflammatory disorders.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1(-/-), Cybb(-/-) or Casp1(-/-)/Cybb(-/-) knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

Inflammation is one underlying contributing factor in the pathology of acute and chronic kidney disorders. Phagocytes such as monocytes, neutrophils and dendritic cells are considered to play a deleterious role in the progression of kidney disease but may also contribute to organ homeostasis. The kidney is a target of life-threatening autoimmune disorders such as the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Neutrophils and monocytes express ANCA antigens and play an important role in the pathogenesis of AAV. Noninvasive in vivo methods that can quantify the distribution of inflammatory cells in the kidney as well as other organs in vivo would be vital to identify the causality and significance of inflammation during disease progression. Here we describe an noninvasive technique to study renal inflammation in rodents in vivo using fluorine ((19)F) MRI. In this protocol we chose a murine ANCA-AAV model of renal inflammation and made use of nanoparticles prepared from perfluoro-5-crown-15-ether (PFCE) for renal (19)F MRI.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis.

CD177 presents antigens in allo- and autoimmune diseases on the neutrophil surface. Individuals can be either CD177-deficient or harbor distinct CD177(neg) and CD177(pos) neutrophil subsets. We studied mechanisms controlling subset-restricted CD177 expression in bimodal individuals. CD177(pos), but not CD177(neg) neutrophils, produced CD177 protein and mRNA. Haplotype analysis indicated a unique monoallelic CD177 expression pattern, where the offspring stably transcribed either the maternal or paternal allele. Hematopoietic stem cells expressed both CD177 alleles and silenced one copy during neutrophil differentiation. ChIP and reporter assays in HeLa cells with monoallelic CD177 expression showed that methylation reduced reporter activity, whereas demethylation caused biallelic CD177 expression. HeLa cell transfection with c-Jun and c-Fos increased CD177 mRNA. Importantly, CD177(pos) human neutrophils, but not CD177(neg) neutrophils, showed a euchromatic CD177 promoter, unmethylated CpGs, and c-Jun and c-Fos binding. We describe epigenetic mechanisms explaining the two distinct CD177 neutrophil subsets and a novel monoallelic CD177 expression pattern that does not follow classical random monoallelic expression or imprinting.

The maintenance of water and electrolyte homeostasis is of enormous importance for the functioning of cells and tissues. A number of therapeutic procedures intentionally or unintentionally influence important regulatory mechanisms of these interdependent balanced systems. Excessive salt intake doesn't only expand the extracellular volume; it can also cause a considerable increase in tonicity. Owing to its insulin-dependent duality of action, glucose can represent an effective or an ineffective osmolyte. This fact has to be considered in patients with diabetic ketoacidosis. Diuretics reduce the volume expansion via renal excretion of sodium (and water); however, in addition to hypokalemia, diuretics can also cause severe alkalosis. Nowadays, hemodialysis is a routine procedure-but even routine procedures can deliver undesirable surprises. Can dialysis cause an increase in calcium levels, or does the procedure remove therapeutically administered radioactive iodine? The current article presents a series of cases we have come across in recent years. These case reports illustrate common, but also rare iatrogenic situations. The discussion of these cases is aimed at raising awareness of the issues involved in a pathophysiological approach to clinical problems.

Atherosclerosis and vasculitis both feature inflammation mediated by neutrophil-endothelial-cell (EC) contact. Neutrophil myeloperoxidase (MPO) can disrupt normal EC function, although the mechanism(s) by which MPO is transferred to EC are unknown. We tested the hypothesis that close, beta2-integrin-dependent neutrophil-EC contact mediates MPO transfer from neutrophils to EC. We used sensitive MPO assays and flow cytometry to detect MPO in EC and demonstrate that EC acquired MPO when contacted by neutrophils directly but not when EC and neutrophils were separated in transwells. The transfer was dependent on neutrophil number, exposure time, and incubation temperature. Transfer occurred in several EC types, increased with endotoxin, was not accompanied by MPO release into the medium and was not abrogated by inhibiting degranulation to secretagogues. Confocal microscopy showed MPO internalization by EC with cytoplasmic and nuclear staining. Neutrophils and EC formed intimate contact sites demonstrated by electron microscopy. Blocking CD11b or CD18 beta2-integrin chains, or using neutrophils from CD11b gene-deleted mice, reduced MPO transfer. EC-acquired MPO was enzymatically active, as demonstrated by its ability to oxidize the fluorescent probe aminophenyl fluorescein in the presence of a hydrogen peroxide source. The data suggest an alternative to EC uptake of soluble MPO, namely the cell contact-dependent, {beta}2-integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis.

We report on a patient with long-standing severe autonomic failure that affected his sympathetic and parasympathetic nervous systems. Antibodies against the ganglionic acetylcholine receptors were detected in the serum. Removal of the antibodies by means of plasma exchange resulted in a dramatic clinical improvement.

The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.