Robust Rat and Mouse Models of Bilateral Renal Ischemia Reperfusion Injury.

BACKGROUND/AIM: Acute and chronic kidney diseases are a major contributor to morbidity and mortality worldwide, with no specific treatments currently available for these. To enable understanding the pathophysiology of and testing novel treatments for acute and chronic kidney disease, a suitable in vivo model of kidney disease is essential. In this article, we describe two reliable rodent models (rats and mice) of efficacious kidney injury displaying acute to chronic kidney injury progression, which is also reversible through novel therapeutic strategies such as ischemic preconditioning (IPC). MATERIALS AND METHODS: We utilized adult male Lewis rats and adult male wildtype (C57BL/6) mice, performed a midline laparotomy, and induced warm ischemia to both kidneys by bilateral clamping of both renal vascular pedicles for a set time, to mimic the hypoxic etiology of disease commonly found in kidney injury. RESULTS: Bilateral ischemia reperfusion injury caused marked structural and functional kidney injury as exemplified by histology damage scores, serum creatinine levels, and kidney injury biomarker levels in both rodents. Furthermore, this effect displayed a dose-dependent response in the mouse model. CONCLUSION: These rodent models of bilateral kidney IRI are reliable, reproducible, and enable detailed mechanistic study of the underlying pathophysiology of both acute and chronic kidney disease. They have been carefully optimised for single operator use with a strong track record of training both surgically trained and surgically naïve operators.

Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Tissue-specific transcriptional programming of macrophages controls the microRNA transcriptome targeting multiple functional pathways.

Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming, and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue-specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6 deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, miR-223, and miR-203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.

Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection.

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

Randomized Trial on the Effect of an Oral Spleen Tyrosine Kinase Inhibitor in the Treatment of IgA Nephropathy.

Introduction: We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of patients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells. Methods: This study was a double-blind, randomized, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomized to receive placebo, fostamatinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors. The primary end point was reduction of proteinuria. Secondary end points included change from baseline in estimated glomerular filtration rate (eGFR) and kidney histology. Results: Although we could not detect significant reduction in proteinuria with fostamatinib overall, in a predetermined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27%, and 36% in the placebo, fostamatinib 100 mg, and 150 mg groups, respectively) in patients with baseline urinary protein-to-creatinine ratios (UPCR) more than 1000 mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well-tolerated. Side effects included diarrhea, hypertension, and increased liver enzymes. Thirty-nine patients underwent repeat biopsy showing reductions in SYK staining associated with therapy at low dose (-1.5 vs. 1.7 SYK+ cells/glomerulus in the placebo group, P < 0.05). Conclusions: There was a trend toward reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 mg/g. Further study may be warranted.

UK Kidney Association Clinical Practice Guideline: Sodium-Glucose Co-transporter-2 (SGLT-2) Inhibition in Adults with Kidney Disease 2023 UPDATE.

Large placebo-controlled trials have demonstrated kidney and cardiovascular clinical benefits of SGLT-2 inhibitors. Data from the EMPA-KIDNEY and DELIVER trials and associated meta-analyses triggered an update to the UK Kidney Association Clinical Practice Guideline on Sodium-Glucose Co-transporter-2 (SGLT-2) Inhibition in Adults with Kidney Disease. We provide a summary of the full guideline and highlight the rationale for recent updates. The use of SGLT-2 inhibitors in people with specific medical conditions, including type 1 diabetes, kidney transplants, and people admitted to hospital with heart failure is also considered, along with Recommendations for future research and Recommendations for implementation. A full "lay" summary of the guidelines is provided as an appendix to ensure that these guidelines are accessible and understandable to people who are not medical professionals.

Identification and detection of microRNA kidney disease biomarkers in liquid biopsies.

PURPOSE OF REVIEW: MicroRNAs (miRNAs) are emerging rapidly as a novel class of biomarkers of major organ disorders, including kidney diseases. However, current PCR-based detection methods are not amenable to development for high-throughput, cost-effective miRNA biomarker quantification. RECENT FINDINGS: MiRNA biomarkers show significant promise for diagnosis and prognosis of kidney diseases, including diabetic kidney disease, acute kidney injury, IgA nephropathy and delayed graft function following kidney transplantation. A variety of novel methods to detect miRNAs in liquid biopsies including urine, plasma and serum are being developed. As miRNAs are functional transcripts that regulate the expression of many protein coding genes, differences in miRNA profiles in disease also offer clues to underlying disease mechanisms. SUMMARY: Recent findings highlight the potential of miRNAs as biomarkers to detect and predict progression of kidney diseases. Developing in parallel, novel methods for miRNA detection will facilitate the integration of these biomarkers into rapid routine clinical testing and existing care pathways. Validated kidney disease biomarkers also hold promise to identify novel therapeutic tools and targets. VIDEO ABSTRACT: http://links.lww.com/CONH/A43.

The ecology of palm genomes: repeat-associated genome size expansion is constrained by aridity.

Genome size varies 2400-fold across plants, influencing their evolution through changes in cell size and cell division rates which impact plants' environmental stress tolerance. Repetitive element expansion explains much genome size diversity, and the processes structuring repeat 'communities' are analogous to those structuring ecological communities. However, which environmental stressors influence repeat community dynamics has not yet been examined from an ecological perspective. We measured genome size and leveraged climatic data for 91% of genera within the ecologically diverse palm family (Arecaceae). We then generated genomic repeat profiles for 141 palm species, and analysed repeats using phylogenetically informed linear models to explore relationships between repeat dynamics and environmental factors. We show that palm genome size and repeat 'community' composition are best explained by aridity. Specifically, Ty3-gypsy and TIR elements were more abundant in palm species from wetter environments, which generally had larger genomes, suggesting amplification. By contrast, Ty1-copia and LINE elements were more abundant in drier environments. Our results suggest that water stress inhibits repeat expansion through selection on upper genome size limits. However, elements that may associate with stress-response genes (e.g. Ty1-copia) have amplified in arid-adapted palm species. Overall, we provide novel evidence of climate influencing the assembly of repeat 'communities'.

Exploration of a panel of urine biomarkers of kidney disease in two paediatric cohorts with Type 1 diabetes mellitus of differing duration.

BACKGROUND: The pathogenesis of diabetic kidney disease (DKD) is complex and involves both glomerular and tubular dysfunction. A global assessment of kidney function is necessary to stage DKD, a progressive kidney disease that is likely to begin in childhood. The present study evaluated whether kidney injury biomarkers identified as early DKD biomarkers in adults have any prognostic value in the very early stages of childhood diabetes. METHODS: We measured urine free Retinol-binding protein 4 (UfRBP4), albumin (UAlb), Kidney injury molecule-1 (KIM-1) and the microRNAs miR-155, miR-126 and miR-29b in two cohorts of paediatric T1DM patients without evidence of DKD, but with diabetes of short-duration, ≤ 2.5 years (SD, n = 25) or of long-duration, ≥ 10 years (LD, n = 29); non-diabetic siblings (H, n = 26) were recruited as controls. A p value < 0.05 was considered significant for all results. RESULTS: UfRBP4 and UAlb were not significantly different across the three groups. No differences were found in KIM-1 excretion between any of the three groups. UfRBP4 was correlated with UAlb in all three groups (r 0.49; p < 0.001), whereas KIM-1 showed no correlation with albumin excretion. Among microRNAs, miR-29b was higher in all diabetic children compared with the H control group (p = 0.03), whereas miR-155 and miR-126 were not significantly different. No differences were found between the SD and LD groups for all three microRNAs. No associations were identified between these biomarkers with sex, age, BMI, eGFR, T1DM duration or glycaemic control. CONCLUSIONS: UfRBP4, KIM-1, miR-155, and miR-126 were unaffected by the presence and duration of diabetes, whereas miR-29b showed a modest elevation in diabetics, regardless of duration. These data support the specificity of a panel of urine biomarkers as DKD biomarkers, rather than any relationship to diabetes per se or its duration, and not as early DKD biomarkers in a paediatric setting.

24-h Glycaemic profiles in peritoneal dialysis patients and non-dialysis controls with advanced kidney disease.

BACKGROUND: For patients on peritoneal dialysis (PD), the deleterious effects of high concentrations of dialysate glucose on the peritoneal membrane are well-documented. Systemic effects of peritoneally absorbed glucose are more poorly defined. Using continuous glucose monitoring (CGM), we aimed to describe 24-h glycaemic profiles of PD patients without diabetes and compare with non-dialysis controls with stage 5 chronic kidney disease (CKD-5). METHODS: In this cross-sectional, case-control study, 15 patients on PD (9 automated PD (APD) and 6 continuous ambulatory PD (CAPD)) and 16 CKD-5 controls underwent 72 h of CGM and metabolic profiling. CGM was used to derive average glucose concentrations and within-participant standard deviation (SD) of glucose. Data were analysed for the whole 72-h monitoring period and as daytime (09.00 to 21.00) and night-time (21.00 to 09.00). RESULTS: Average glucose concentrations and within-participant SD of glucose for the whole monitoring period were not different between the three groups (p ≥ 0.5). Daytime average glucose concentrations were also similar across the three groups (p = 0.729). APD was associated with a significantly higher nocturnal glucose than CAPD (5.25 mmol/L ± 0.65 vs. 4.28 ± 0.5, p = 0.026). A significant drop in nocturnal glucose compared with daytime average seen in both CAPD patients and controls was absent in APD patients. CONCLUSIONS: Systematically different glycaemic patterns were observed in non-diabetic APD and CAPD patients, including an absence of physiological nocturnal glucose dipping in patients on APD. Comprehensive CGM data sets highlight subtleties not appreciated by traditional metabolic biomarkers; this has implications when choosing the most appropriate outcome measures in future research addressing the metabolic impact of PD.

An exploratory study of the relationship between systemic microcirculatory function and small solute transport in incident peritoneal dialysis patients.

BACKGROUND: The peritoneal capillary endothelium is widely considered to be the most influential structure in dictating the rate of small solute transport (SST) during peritoneal dialysis (PD). PD patients are at significant risk of systemic microcirculatory dysfunction. The relationship between peritoneal and systemic microcirculations in patients new to PD has not been well studied. We hypothesised that for patients on PD for less than 6 months, dysfunction in the systemic microcirculation would be reflected in the rate of SST. METHODS: We recruited 29 patients to a cross-sectional, observational study. Rate of SST was measured using a standard peritoneal equilibration test. Laser Doppler Flowmetry was used to measure response to physical and pharmacological challenge (post-occlusive hyperaemic response and iontophoretic application of vasodilators) in the cutaneous microcirculation. Sidestream Darkfield imaging was used to assess sublingual microvascular density, flow and endothelial barrier properties. RESULTS: We found no moderate or strong correlations between any of the measures of systemic microcirculatory function and rate of SST or albumin clearance. There was however a significant correlation between dialysate interleukin-6 concentrations and both SST (rs = 0.758 p ≤ 0.0001) and albumin clearance (rs = 0.53, p = 0.01). CONCLUSIONS: In this study, systemic microvascular dysfunction did not significantly influence the rate of SST even early in patients PD careers. In conclusion, this study demonstrates that intraperitoneal factors particularly inflammation have a far greater impact on rate of SST than systemic factors.

MicroRNAs and their delivery in diabetic fibrosis.

The global prevalence of diabetes mellitus was estimated to be 463 million people in 2019 and is predicted to rise to 700 million by 2045. The associated financial and societal costs of this burgeoning epidemic demand an understanding of the pathology of this disease, and its complications, that will inform treatment to enable improved patient outcomes. Nearly two decades after the sequencing of the human genome, the significance of noncoding RNA expression is still being assessed. The family of functional noncoding RNAs known as microRNAs regulates the expression of most genes encoded by the human genome. Altered microRNA expression profiles have been observed both in diabetes and in diabetic complications. These transcripts therefore have significant potential and novelty as targets for therapy, therapeutic agents and biomarkers.

miR-141 mediates recovery from acute kidney injury.

Acute kidney injury (AKI) is a global clinical problem characterised by a sudden decline in renal function and mortality as high as 60%. Current AKI biomarkers have limited ability to classify disease progression and identify underlying pathological mechanisms. Here we hypothesised that alterations in urinary microRNA profiles could predict AKI recovery/nonrecovery after 90 days, and that injury-specific changes would signify microRNA mediators of AKI pathology. Comparison of urinary microRNA profiles from AKI patients with controls detected significant injury-specific increases in miR-21, miR-126 and miR-141 (p < 0.05) and decreases in miR-192 (p < 0.001) and miR-204 (p < 0.05). Expression of miR-141 increased in renal proximal tubular epithelial cells (PTECs) under oxidative stress in vitro and unilateral ischaemic reperfusion injury in vivo. Forced miR-141 expression in the presence of H2O2 increased PTEC death and decreased cell viability. Of nine messenger RNA targets with two or more miR-141 3'-untranslated region binding sites, we confirmed protein tyrosine phosphatase receptor type G (PTPRG) as a direct miR-141 target in PTECs. PTPRG-specific siRNA knockdown under oxidative stress increased PTEC death and decreased cell viability. In conclusion, we detected significant alterations in five urinary microRNAs following AKI, and identified proximal tubular cell PTPRG as a putative novel therapeutic target.

Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis.

BACKGROUND: Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis. METHODS: Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing. RESULTS: A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1-S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters ("new PTC clusters") were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages. CONCLUSIONS: These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.

Detection of urinary microRNA biomarkers using diazo sulfonamide-modified screen printed carbon electrodes.

This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA (miRNA) quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects. Aberrant miRNA expression has been observed in several major human disorders, and we have identified a urinary miRNA signature for DKD. MiRNAs therefore have considerable promise as disease biomarkers, and techniques to quantify these transcripts from clinical samples have significant clinical and commercial potential. Current RT-qPCR-based methods require technical expertise, and more straightforward methods such as electrochemical detection offer attractive alternatives. We describe a method to detect urinary miRNAs using diazo sulfonamide-modified screen printed carbon electrode-based biosensors that is amenable to parallel analysis. These sensors showed a linear response to buffered miR-21, with a 17 fM limit of detection, and successfully discriminated between urine samples (n = 6) from DKD patients and unaffected control subjects (n = 6) by differential miR-192 detection. Our technique for quantitative miRNA detection in liquid biopsies has potential for development as a platform for non-invasive high-throughput screening and/or to complement existing diagnostic procedures in disorders such as DKD.

Phytochrome A elevates plant circadian-clock components to suppress shade avoidance in deep-canopy shade.

Shade-avoiding plants can detect the presence of neighboring vegetation and evoke escape responses before canopy cover limits photosynthesis. Rapid stem elongation facilitates light foraging and enables plants to overtop competitors. A major regulator of this response is the phytochrome B photoreceptor, which becomes inactivated in light environments with a low ratio of red to far-red light (low R:FR), characteristic of vegetational shade. Although shade avoidance can provide plants with a competitive advantage in fast-growing stands, excessive stem elongation can be detrimental to plant survival. As such, plants have evolved multiple feedback mechanisms to attenuate shade-avoidance signaling. The very low R:FR and reduced levels of photosynthetically active radiation (PAR) present in deep canopy shade can, together, trigger phytochrome A (phyA) signaling, inhibiting shade avoidance and promoting plant survival when resources are severely limited. The molecular mechanisms underlying this response have not been fully elucidated. Here, we show that Arabidopsis thaliana phyA elevates early-evening expression of the central circadian-clock components TIMING OF CAB EXPRESSION 1 (TOC1), PSEUDO RESPONSE REGULATOR 7 (PRR7), EARLY FLOWERING 3 (ELF3), and ELF4 in photocycles of low R:FR and low PAR. These collectively suppress stem elongation, antagonizing shade avoidance in deep canopy shade.

Determination of a microRNA signature of protective kidney ischemic preconditioning originating from proximal tubules.

Ischemic preconditioning (IPC) is effective in limiting subsequent ischemic acute kidney injury in experimental models. MicroRNAs are an important class of post-transcriptional regulator and show promise as biomarkers of kidney injury. We evaluated the time- and dose-dependence of benefit from IPC in a rat model of functional (bilateral) ischemia-reperfusion injury (IRI). We found optimal protection from subsequent injury following short, repetitive sequences of preconditioning insult. We subsequently used hybridization array and microRNA sequencing to characterize microRNA signatures of protective IPC and of IRI. These approaches identified a profile of microRNA changes consequent on IRI, that were limited by prior IPC. To localize these signals within the kidney, we used laser capture microdissection and RT-qPCR to measure microRNA abundance in nephron segments, pinpointing microRNA changes principally to glomeruli and proximal tubules. Our data describe a unique microRNA signature for IRI in the rat kidney. Pulsatile IPC reduces kidney damage following IRI and diminishes this microRNA signal. We have also identified candidate microRNAs that may act as biomarkers of injury and therapeutic targets in this context.

Differential expression of microRNA miR-150-5p in IgA nephropathy as a potential mediator and marker of disease progression.

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases.

Molecular detection of SARS-CoV-2 using a reagent-free approach.

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost. Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.