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BACKGROUND AND AIMS The atypical hemolytic uremic syndrome (aHUS) is a complement-mediated (CM) ultrarare disease that manifests as thrombotic microangiopathy (TMA) with preferential small kidney vessels involvement. aHUS is caused by genetic or acquired deregulation of complement (C’) system alternative pathway at the cell membrane surface which produces C’ overactivation and, eventually, membrane attack complex (MAC or C5b9) deposition, pore formation and, hence, vascular cell lysis. Currently, there is not a gold standard diagnostic test for aHUS, neither to monitor patient response to eculizumab therapy. It has been suggested that direct measurement of C5b9 deposits on cultured endothelial cells could be a plausible approach to evaluate the complement functional state in aHUS patients. Here we assess the C5b9 ex vivo deposition test applicability as a tool to diagnose aHUS and the monitoring of the Eculizumab therapy response in patients with aHUS in native, transplanted kidney and hematopoietic stem cell (HSC) transplantation (Tx) associated aHUS. METHOD A total of 14 patients with aHUS in native kidney (n = 10), aHUS recurrence after kidney Tx (n = 3) or HSC transplantation associated aHUS (n = 1), were studied during the acute phase of the disease prior to treatment, or in remission while receiving eculizumab. Chronic C3 glomerulopathy patients (n = 2) and healthy individuals were included as controls (n = 20). Serum samples from every patient to measure the C5b9 deposition on human vascular endothelial cells (HMEC-1; ATCC CRL-3234) were collected. HMEC-1 cells were platted on cover glasses in 24-well plates. Once cells reached confluence, they were treated with 10 µM adenosine diphosphate (ADP) for 10 min to activate the endothelial cells (activated conditions) or with test medium (resting conditions). Afterward, they were incubated with patient or control serum samples diluted 1:2 for 4 h. Cells were fixed with 4% paraformaldehyde solution, and C5b9 was immunodetected (1:500 rabbit polyclonal anti-complement C5b9 (Calbiochem, ref. 204 903)). Images were taken using a FV100 Fluoroview Olympus Confocal Microscope at 40×. Fifteen fields were analyzed to calculate the average staining area of each sample in pixels2 (pxl2) using the ImageJ. RESULTS C5b9 deposition on HMEC-cells using serum samples from healthy controls (n = 20) was quantified in order to establish the normal reference test range. C5b9 deposits staining area mean and standard deviation (m ± SD) on resting cells were 2018.25pxl2 ± 116.54 in healthy controls. Under ADP-activated conditions, C5b9 deposits m ± SD was 4210.51pxl2 ± 158.06 (gray boxes in Fig. 1). As expected, in the acute disease phase aHUS patients had a significant increase of C5b9 deposition (P = 0.01) as compared with controls in both resting and activated conditions (G1 in Fig. 1). The same finding was observed in a patient with aHUS associated with HSC-transplantation and in another aHUS patient who did not respond to Eculizumab (G2 and G3 in Fig. 1, respectively). All patients with aHUS in native kidneys (G4 in Fig. 1) or recurrence of the disease after kidney Tx (G5 in Fig. 1) who responded to eculizumab showed levels of C5b9 deposition comparable to the normal range except for patient 9. C3 glomerulopathy patients showed C5b9 deposition levels comparable to those found in healthy controls (G6 in Fig. 1). CONCLUSION The ex vivo C5b9 deposition test is an auspicious tool to diagnose and monitor aHUS response to eculizumab. We now demonstrated that the ex vivo C5b9 deposition test is also useful for HSC associated aHUS and for aHUS recurrence after kidney transplantation suggesting that it could be a useful diagnostic tool for any type of CM aHUS. These results expand the spectrum of patients that would benefit from a better diagnostic and treatment monitoring procedure.

Background and Aims Distal renal tubular acidosis (dRTA) is a rare disorder characterised by an inability of the distal tubule to secrete acid, leading to metabolic acidosis. Clinical consequences typically include hypokalaemia, hypercalciuria with nephrocalcinosis and/or urolithiasis, as well as bone disease. Treatment with adequate alkali supplementation corrects the acidosis and hypercalciuria, but there are few data on long-term outcome. In 2018, a registry for dRTA was established by the European Society for Paediatric Nephrology, hosted by the European Rare Kidney Disease Reference Network. Here, we present an initial analysis of data in the registry. Method Analysis of data entered into the registry by the cut-off data of 18/11/2020. Results A total of 135 patients had been entered, of which 106 had additional data from an annual follow-up visit. Median age at last visit was 10 years (range 0-54), including 16 adults (>17y). Genetic testing had been performed in 91 subjects and causative variants were reported in 74 (81%). Pertinent clinical details according to genetic group are listed in table 1. Treatment was provided with at least 15 different preparations, containing citrate or bicarbonate, given in 1-10 (median 3) daily doses. Adequate treatment at last follow-up, as judged by a plasma bicarbonate level >21 mmol/l and a urine calcium-creatinine ratio in the age-specific normal range was present in 46% of subjects. There was a trend for higher eGFR and height SDS in subjects with adequate treatment compared to those without, but this was not statistically significant. Conclusion Currently available data demonstrate the difficulties in treating dRTA, with less than half of subjects achieving adequate control of their acidosis. By collecting long-term data, the registry will provide important information on the prognosis and complications of dRTA and to what degree these can be prevented with treatment. Enrollment of further, especially adult patients will contribute to our understanding of this rare disorder.

Background and Aims Due to the unphysiological composition of PD fluids, chronic peritoneal dialysis (PD) induces progressive peritoneal fibrosis, hypervascularization, and vasculopathy. The evolution of the PD membrane and vasculopathy following kidney transplantation (KTx) is largely unknown. Method Arteriolar and peritoneal tissues were obtained from 107 children with chronic kidney disease (CKD5), 72 children on PD (treated with neutral pH PD fluids, with low glucose degradation product content, GDP) and 21 children, who underwent KTx 4-5 weeks after a median 21 months of PD. Specimen underwent standardized digital quantitative histomorphometry. Molecular mechanisms were studied in omental arterioles microdissected from surrounding fat by multi-omics followed by Gene Set Enrichment Analysis (GSEA); key findings were validated in parietal tissues of independent, matched cohorts by quantitative immunohistochemistry (n=15/group). Results Arteriolar transcriptome and proteome GSEA revealed suppression of leucocyte migration and T-cell activation / secretory pathways regulation, of sprouting angiogenesis biological processes and of epithelial proliferation and cell cycle after KTx as compared to PD. Lipid / fatty acid metabolism, autophagy and ATP synthesis pathways were activated. Transcriptome analysis including KTx, PD and CKD5 specifically attributed regulation of arteriolar lipid and fatty acid metabolism to transplantation and comprised 140 transcripts; their regulation was confirmed on the proteome level. Hub gene fatty acid synthase was identified by protein interaction analysis (string-db.org). 15 arteriolar genes activated by PD were inactivated after KTx and included glucose metabolisms and cytoskeleton related transcripts. 24 transcripts and 10 corresponding proteins induced by PD were still active after KTx and associated with biological processes related to TGF-ß signaling, fibrosis and mineral absorption. In line with arteriolar multi-omics findings, peritoneal hypervascularization induced by chronic PD was reversed after Tx to CKD5 level. CD45 positive tissue infiltrating leucocytes count was reduced by 40% and was independently associated with microvessel density in multivariable analysis including PD vintage, daily GDP exposure and recent KTx. Peritoneal lymphatic vessel density, submesothelial thickness, activated fibroblast, fibrin deposit, macrophage and EMT cell counts remained unchanged after KTx compared to PD. Arteriolar lumen to vessel ratios (a marker of vasculopathy) were similar in both groups. Vessel-homeostasis-related proteins in independent, matched cohorts demonstrated increased caspase-3 abundance in peritoneal arterioles after KTx. Arteriolar VEGF-A, thrombospondin, angiopoietin1/2, and hypoxia-inducible factor-1 (HIF-1a) were unchanged, while submesothelial HIF-1a and angiopoietin1/2 were decreased after Tx, favoring vessel maturation. The abundance of the key driver of fibrosis, TGF-ß-effector pSMAD2/3, was unchanged in the peritoneum and arterioles after Tx. Conclusion Our multi-omics analyses of fat covered omental arterioles, not directly exposed to PD fluids, demonstrate inhibition of PD induced immune response and angiogenesis pathways, of glucose metabolism and cytoskeleton regulation to levels similar as seen in children with CKD5. Arteriolar lipid and fatty acid metabolism is selectively altered after KTx. Reversal of low GDP PD induced hypervascularization and inflammation of the parietal peritoneum after KTx, mirror molecular changes in omental arterioles, while profibrotic activity persists after KTx in omental arterioles and in the parietal peritoneum.