Background and AimsSystemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by the development of autoantibodies against multiple antigens, including complement C1q, starter molecule of the classical pathway. Anti-C1q autoantibodies (anti-C1q) are not only a biomarker of disease activity but believed to contribute to the pathogenesis of proliferative lupus nephritis. Previous studies demonstrated that a key immunogenic site of C1q (so-called ‘A08’) shares an identical sequence with Epstein-Barr-Virus (EBV) Nuclear antigen-1, and that anti-C1q can be induced by this EBV antigenic site in vivo.MethodsWe investigated whether an EBV-derived antigen can trigger a cross-reactive anti-C1q response in lupus-prone mice and enhance renal pathology. Mertk-deficient mice, which exhibit a defective clearance of apoptotic cells, were immunized with EBV-derived peptide. Antibody responses against the EBV antigen, intact C1q and the C1q-derived antigenic site A08 were determined, and renal pathology was assessed histologically and by electron microscopy.ResultsThe immunization with EBV antigen led to the generation of antibodies recognizing the C1q-derived antigen A08 in most, and the formation of anti-C1q with binding characteristics as occurring in SLE patients in a substantial subset of mice. Generation of anti-C1q was associated with accelerated mesangioproliferative glomerulonephritis and increased glomerular IgG and complement deposition.ConclusionsOur findings demonstrate that EBV-derived peptides can elicit pathogenic anti-C1q via molecular mimicry, thereby exacerbating renal disease in lupus-prone mice. The data provide mechanistic evidence for how an EBV antigen can accelerate SLE progression, and confirm the concept of anti-C1q being a driver of lupus nephritis.

Focal segmental glomerulosclerosis (FSGS) represents one of the most common etiologies of nephrotic syndrome. In 10% to 20% of cases, it is associated with steroid resistance and has the potential to progress to kidney failure. Compound heterozygote or homozygote mutations in the NUP93 gene have been associated with FSGS-related nephrotic syndrome. To the best of our knowledge, no case of NUP93 related FSGS has presented in the literature with associated ophthalmological anomaly. In this report, we present the case of a 25-year-old White patient who presented with rapidly progressive chronic kidney disease, congenital bilateral optic nerve atrophy and steroid-resistant nephrotic syndrome. Kidney biopsy showed FSGS with more than 80% foot process effacement on electronic microscopy. Genetic testing by means of whole exome sequencing later showed the presence of biallelic class 4, likely pathogenic variants in the NUP93 gene. Although we cannot exclude another cause, such as a genetic defect outside the genomic regions screened by our exome analyses, for the bilateral optic atrophy observed in our patient, this suggests that this association is not coincidental. Our case report highlights the importance of genetic testing in cases of steroid-resistant nephrotic syndrome that present with syndromic congenital features.

Hypertension is a major risk factor for human morbidity and mortality, and the junctional protein paracingulin (CGNL1, JACOP) is required for the development of hypertension in a Dahl salt-sensitive rat model and is linked to human hypertension in genome wide association studies. However, the mechanism through which CGNL1 may regulate hypertension is unknown. Here, we address this question using a mouse model, where hypertension is induced by unilateral nephrectomy and angiotensin II infusion (N+A protocol). Although untreated WT and CGNL1-KO mice showed similar blood pressure, the N+A protocol induced hypertension in WT mice but not in CGNL1-KO mice. We show by immunolocalization and transcriptomic analysis that CGNL1 is expressed throughout the kidney tubules and in the endothelium of blood vessels, but not in smooth muscle. The N+A protocol induced decreased potassium urinary excretion in wild-type (WT), but not in CGNL1-KO mice. Immunoblot analysis shows that the KO of CGNL1 blunted the N+A-induced changes in the expression levels and activation of tubular ion transporters, including the Na/H exchanger 3 (NHE3) and the thiazide-sensitive Na-Cl cotransporter (NCC), and blunted the angiotensin II-dependent changes in the levels and/or activation of AMP-activated protein kinase (AMPK), ERK and myosin light chain. In contrast, myography showed comparable vascular reactivity in thoracic aortas and mesenteric arteries isolated from WT or CGNL1-KO mice. Together, these results suggest the KO of CGNL1 attenuates hypertension by uncoupling angiotensin II signaling in kidney tubule cells, indicating a novel pathway of regulation of signaling by a junctional protein.NEW & NOTEWORTHY The knock-out of paracingulin (CGNL1) prevents the development of hypertension in a unilateral nephrectomy/angiotensin II infusion model (N+A) in mice and this antihypertensive effect likely depends on uncoupling of angiotensin II from stimulation of sodium transporter activity in kidney tubules rather than on alteration of resistance blood vessel contractility.

INTRODUCTION:Metabolic alterations are recognized as key features of kidney injury, but their causal role in kidney repair remains debatable. Here, we investigate the role of phosphoenolpyruvate carboxykinase 1 (PCK1), an enzyme involved in gluconeogenesis and cataplerosis (removal of tricarboxylic acid (TCA) cycle intermediates from the mitochondrial matrix) in kidney disease progression. METHODS:We used mice with kidney tubular cell-specific deletion or overexpression of the PCK1 enzyme, and different models of kidney injury such as ischemia-reperfusion injury or cis-platin-induced nephropathy. Furthermore, we measured metabolites in kidney biopsy tissue from patients with stage 3b/4 chronic kidney disease (CKD). RESULTS:Using flux analysis, we confirm that cataplerosis and the TCA cycle are blocked by PCK1 deficiency. This results in injured mitochondria leading to inflammation, tubular injury and impaired tubular cell repair. Inversely, maintaining PCK1 function in different models of kidney injury preserves kidney structure, improves TCA cycle metabolite clearance and increase ATP production. In kidney biopsies from different patient cohorts, we confirm the correlation between PCK1 loss, mitochondrial injury and a failed tubular cell repair phenotype. Furthermore, in CKD, accumulation of TCA cycle metabolites is consistent with disrupted cataplerosis. CONCLUSIONS:Overall, we demonstrate that PCK1 loss in kidney tubular cells leads to decreased respiration and the accumulation of TCA cycle metabolites. Maintenance of cataplerosis is an important factor of tubular physiology and repair, with PCK1 serving as a causal and potential therapeutic target in this process. PCK1 restoration enhances mitochondrial health, limiting progression to inflammation and fibrosis.

IntroductionThe impact of hypertension (HT) of living donors on later kidney graft function remains insufficiently understood.MethodsWe retrospectively reviewed recipients of living donor (LD) kidney transplant (KTX) at a single tertiary center from January 2003 to December 2021 with a follow-up until December 2022. LD blood pressure (BP) values obtained with office measurement and 24 h ambulatory BP monitoring (ABPM) were the main predictors, while recipient estimated glomerular filtration rate (eGFR) and proportion of kidney fibrosis on biopsies were the outcomes. Multivariate analyses were adjusted for pre-transplant donor characteristics: age, sex, ethnicity, body mass index (BMI) and eGFR.Results212 LD KTX recipients were included, with mean age 51 years. 133 were women (62.7%). 73 (34.4%) LD were hypertensive based on office BP. In a sub-group of 112 LD with ABPM, 64 (57.1%) were hypertensive. Office systolic blood pressure (SBP) was negatively associated with eGFR at 6 months, 1 year, 5 years and 10 years (p < 0.05). Office SBP was positively associated with kidney fibrosis at 1 year (p < 0.05). Those associations were not significant after multivariate adjustment.DiscussionIn conclusion, while an adverse impact of LD HT on later kidney function and fibrosis was measured, this effect seemed negligible after accounting for other more relevant clinical characteristics.

Chronic kidney disease (CKD) is a growing public health crisis, affecting over 10% of the global population and significantly increasing mortality and morbidity. Irrespective of its underlying cause, tubulointerstitial fibrosis (TIF) is a hallmark of CKD progression, with myofibroblasts being the primary effectors of renal fibrosis. Here, we show that 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a critical driver of pathogenic myofibroblast differentiation and fibrosis in CKD. Using genetic deletion and pharmacological inhibition of 11beta-HSD1 in mouse models, we demonstrate a marked reduction in TIF severity and improved renal function, linked to the suppression of a regulatory myofibroblast (Reg-MF) subpopulation. Single-cell and spatial transcriptomics data reveal that 11beta-HSD1 is essential for the activation and expansion of Reg-MFs, which is conserved across species and predicts worse outcomes in CKD patients and kidney allograft recipients. These findings establish a direct link between 11beta-HSD1 activity and renal fibrogenesis, highlighting its role during the transition from pericytes to pathogenic Reg-MFs. Our results support 11beta-HSD1 inhibition as a promising therapeutic strategy to mitigate CKD progression, offering both mechanistic insights and translational potential for improving patient outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Acute kidney injury has been recognized as a frequent complication of severe COVID-19 early in the pandemic1 with histology mostly revealing acute tubular injury.2 Later, collapsing glomerulopathy was identified as a peculiar manifestation of SARS-CoV-2 and termed COVID-19 associated nephropathy (COVAN).3–5 COVAN is morphologically indistinguishable from HIV-associated nephropathy and affects, with few exceptions, persons of African ancestry with high-risk APOL1 genotype.6 In addition to these two well-recognized renal manifestations of COVID-19, numerous case reports and case series have described a variety of other renal diseases, mostly glomerulopathies, manifesting in temporal association with SARS-CoV-2 infection.

The application of single-cell technologies in clinical nephrology remains elusive. We generated an atlas of transcriptionally defined cell types and cell states of human kidney disease by integrating single-cell signatures reported in the literature with newly generated signatures obtained from 5 patients with acute kidney injury. We used this information to develop kidney-specific cell-level information ExtractoR (K-CLIER), a transfer learning approach specifically tailored to evaluate the role of cell types/states on bulk RNAseq data. We validated the K-CLIER as a reliable computational framework to obtain a dimensionality reduction and to link clinical data with single-cell signatures. By applying K-CLIER on cohorts of patients with different kidney diseases, we identified the most relevant cell types associated with fibrosis and disease progression. This analysis highlighted the central role of altered proximal tubule cells in chronic kidney disease. Our study introduces a new strategy to exploit the power of single-cell technologies toward clinical applications.

CEMIP (cell migration‐inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions.

MRI T1-mapping is an important non-invasive tool for renal diagnosis. Previous work shows that ΔT1 (cortex-medullary difference in T1) has significant correlation with interstitial fibrosis in chronic kidney disease (CKD) allograft patients. However, measuring cortico-medullary values by manually drawing ROIs over cortex and medulla (a gold standard method) is challenging, time-consuming, subjective and requires human training. Moreover, such subjective ROI placement may also affect the work reproducibility. This work proposes a deep learning-based 2D U-Net (RCM U-Net) to auto-segment the renal cortex and medulla of CKD allograft kidney T1 maps. Furthermore, this study presents a correlation of automatically measured ΔT1 values with eGFR and percentage fibrosis in allograft kidneys. Also, the RCM U-Net correlation results are compared with the manual ROI correlation analysis. The RCM U-Net has been trained and validated on T1 maps from 40 patients (n = 2400 augmented images) and tested on 10 patients (n = 600 augmented images). The RCM U-Net segmentation results are compared with the standard VGG16, VGG19, ResNet34 and ResNet50 networks with U-Net as backbone. For clinical validation of the RCM U-Net segmentation, another set of 114 allograft kidneys patient’s cortex and medulla were automatically segmented to measure the ΔT1 values and correlated with eGFR and fibrosis. Overall, the RCM U-Net showed 50% less Mean Absolute Error (MAE), 16% better Dice Coefficient (DC) score and 12% improved results in terms of Sensitivity (SE) over conventional CNNs (i.e. VGG16, VGG19, ResNet34 and ResNet50) while the Specificity (SP) and Accuracy (ACC) did not show significant improvement (i.e. 0.5% improvement) for both cortex and medulla segmentation. For eGFR and fibrosis assessment, the proposed RCM U-Net correlation coefficient (r) and R-square (R2) was better correlated (r = -0.2, R2 = 0.041 with p = 0.039) to eGFR than manual ROI values (r = -0.19, R2 = 0.037 with p = 0.051). Similarly, the proposed RCM U-Net had noticeably better r and R2 values (r = 0.25, R2 = 0.065 with p = 0.007) for the correlation with the renal percentage fibrosis than the Manual ROI results (r = 0.3, R2 = 0.091 and p = 0.0013). Using a linear mixed model, T1 was significantly higher in the medulla than in the cortex (p<0.0001) and significantly lower in patients with cellular rejection when compared to both patients without rejection and those with humoral rejection (p<0.001). There was no significant difference in T1 between patients with and without humoral rejection (p = 0.43), nor between the types of T1 measurements (Gold standard manual versus automated RCM U-Net) (p = 0.7). The cortico-medullary area ratio measured by the RCM U-Net was significantly increased in case of cellular rejection by comparison to humoral rejection (1.6 +/- 0.39 versus 0.99 +/- 0.32, p = 0.019). In conclusion, the proposed RCM U-Net provides more robust auto-segmented cortex and medulla than the other standard CNNs allowing a good correlation of ΔT1 with eGFR and fibrosis as reported in literature as well as the differentiation of cellular and humoral transplant rejection. Therefore, the proposed approach is a promising alternative to the gold standard manual ROI method to measure T1 values without user interaction, which helps to reduce analysis time and improves reproducibility.

ABSTRACT Background The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). Methods We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. Results In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. Conclusions The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

Background & Aims: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepato-cellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored.Methods: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC.Results: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line -derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment.Conclusions: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract Membranous nephropathy (MN) is determined by deposition of autoantibodies targeting glomerular podoyctes antigens. MN progression to renal failure remains a central issue: antibodies modifying the inflammatory course after the first antibody hit may be involved. We investigated sera of MN patient using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 258 proteins reactive with MN sera were identified. We focused on Formin-like 1 (FMNL1) protein expressed by macrophages. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patient with orthogonal methodologies (ELISA) and were observed co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, strengthening the concept that, autoantibodies directed to cells of the tissue repair, might have a definite role in determining the disease outcome. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, ie. idiopathic and genetic FSGS, IgAGN, stressing the non-specificity for MN and suggesting their involvement in glomerular conditions affecting many patients worldwide.

Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.

Background Nephrotic syndrome (NS) is characterized by massive sodium chloride retention. Along the kidney tubule, sodium and chloride reabsorption are coupled via a combination of transcellular and paracellular transport pathways. The mechanism of sodium retention in NS has been extensively studied, but the associated chloride transport pathway has not been elucidated. Methods To investigate the pathway of chloride retention in NS, we assessed the expression levels of both paracellular and transcellular components of chloride transport in the CD of POD-ATTAC mice and PAN rats, two rodent models of NS. We also used cultured mouse cortical collecting duct cells to see how overexpression or silencing of claudin-4 affect paracellular permeability. Finally, human renal biopsies were used to confirm our in vivo results. Results In control animals, claudin-4 was expressed at low levels in collecting duct (CD). In POD-ATTAC mice and PAN rats, claudin-4 expression was strongly increased in CD beta-intercalated cells (B-IC) and to a lesser extent in CD principal cells and was also induced in connecting tubules. Similarly, we found that claudin-4 was expressed at low levels in normal human kidneys and was dramatically increased in CD cells of nephrotic human kidneys (focal and segmental glomerulosclerosis). In parallel, the expression of pendrin, which exchanges chloride for bicarbonates in B-IC, was decreased in nephrotic compared to control animals. However, the increase in claudin-4 expression observed in NS is likely independent of pendrin abundance. Increased claudin-4 abundance is coupled with increased ENaC-dependent sodium transport. Overexpression or silencing of claudin-4 in mCCD cl1 cells confirmed the preferential permeability of claudin-4 to chloride over sodium. Conclusions These results suggest that during NS, transcellular Cl-/HCO - transport decreases while paracellular chloride transport via claudin-4 may increase along the collecting system. Paracellular chloride permeability may constitute a chloride shunt that favors Na + reabsorption and opposes K + secretion along the CD in NS. Significance Statement Nephrotic syndrome is a common disease characterized by massive proteinuria, hypoalbuminemia and edema due to renal sodium-chloride retention. We demonstrate for the first time an induction of claudin-4 expression indicating a partial shift from transcellular to paracellular chloride transport in the renal collecting system of nephrotic rodents. We confirmed the increased expression of claudin-4 in kidney biopsies of nephrotic patients, highlighting the translational significance of these results. Whether the paracellular pathway may represent a novel target to treat edema in nephrotic syndrome remains to be elucidated.

Acute interstitial nephritis is characterized by renal inflammation and interstitial edema. The clinical presentation is pauci-symptomatic and often non-specific. Acute interstitial nephritis typically presents with acute renal failure, alone or with fever, eosinophilia, hematuria, sterile pyuria and small range proteinuria. An early diagnosis is crucial to prevent the morbidity and mortality associated with renal function decline. The most frequent etiology of this disease is drug-induced. A kidney biopsy is not systematically required to establish the diagnosis. It should be considered in the absence of renal function improvement 5 to 7 days after withdrawal of the causal agent. Although the benefits of glucocorticoid treatment have not been proven to date, its use may be associated with a better kidney function recovery.

Numerous cases of glomerulonephritis manifesting shortly after SARS-CoV-2 vaccination have been reported, but causality remains unproven. We studied the association between mRNA-based SARS-CoV-2 vaccination and new-onset glomerulonephritis using a nationwide retrospective cohort and case-cohort design. Data from all Swiss pathology institutes processing native kidney biopsies served to calculate incidence of IgA nephropathy, pauci-immune necrotizing glomerulonephritis, minimal change disease and membranous nephropathy. The observed incidence during the vaccination campaign (Jan to Aug 2021) was not different from the expected incidence based on the years 2015 to 2019 (incidence rate ratio 0.86, 95%-credible interval 0.73–1.02) and did not cross the upper boundary of the 95% credible interval for any month. Among 111 patients aged >18 years with newly diagnosed glomerulonephritis between January and August 2021, 38.7% had received at least one vaccine dose before biopsy, compared to 39.5% of the general Swiss population matched for age and calendar-time. The estimated risk ratio for the development of new-onset biopsy-proven glomerulonephritis was 0.97 (95% CI 0.66–1.42, P =0.95) in vaccinated vs. unvaccinated individuals. Patients with glomerulonephritis manifesting within 4 weeks after vaccine did not differ clinically from the rest of the cohort. Results were consistent across all types of glomerulonephritis with the possible exception of minimal change disease. In conclusion, vaccination against SARS-CoV-2 was not associated with new-onset glomerulonephritis in these two complementary studies. Most temporal associations between SARS-CoV-2 vaccination and glomerulonephritis are likely coincidental.### Competing Interest StatementAll authors have completed the ICMJE uniform disclosure form at [www.icmje.org/disclosure-of-interest/][1]. I. F. reports holding stocks from Pfizer. All other authors declare no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.### Funding StatementThis study did not receive external funding.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics Committee of Eastern Switzerland gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesData will be made available on reasonable request to the corresponding author, after approval by the local ethics committee. [1]: http://www.icmje.org/disclosure-of-interest/