Renal transplants increase in size and function in keeping with compensatory renal hypertrophy.

AIM: To evaluate changes in allograft kidney length in renal transplant recipients and the relationship with estimated glomerular filtration rate (eGFR). METHODS: This single-centre retrospective study of renal transplant recipients was conducted at Flinders Medical Centre (FMC) from January 2007 to June 2020. Donor and recipient details, renal allograft length from transplant ultrasounds at 0, 1, 3, 6 and 12 months were collected. The association between compensatory renal hypertrophy (CRH) and eGFR and its magnitude was analysed using multivariate multilevel mixed-effects linear regression models. RESULTS: A total of 183 renal transplant recipients were studied. 100 of 175 recipients (62.9%) demonstrated an increase in renal length defined as any increase in maximal longitudinal diameter on serial ultrasounds. Twenty-three recipients (13.1%) had no change in transplant length and 42 recipients (24%) had a decrease in length. The mean increase in kidney length over the first 12 months was 0.57 cm. Ninety of 156 (57.7%) recipients with a renal ultrasound within a month post-transplant demonstrated a mean increase kidney length of 0.3 cm. Multivariate analysis demonstrated that eGFR increased by 2.5 mL/min/1.73 m2 (95% CI 0.72- 4.4; p = .006) with every 1 cm increase in kidney length. Absolute changes in kidney length did not demonstrate any statistically significant correlation with eGFR in both complete case and multiple imputation analysis. CONCLUSION: An increase in transplant kidney length is common in renal transplant recipients and is associated with enhanced eGFR. However, further studies need to be performed to study the association of absolute change in kidney length and eGFR.

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection.

Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Protocol for the Flinders Kidney Health Registry: patient outcomes of kidney cancers and nephrectomies.

BACKGROUND: Kidney cancer accounts for 2% of new cancers diagnosed in Australia annually. Partial and radical nephrectomy are the treatment of choice for kidney cancer. Nephrectomy is also performed for living donor kidney transplantation. Nephrectomy is a risk factor for new-onset chronic kidney disease (CKD) or deterioration of pre-existing CKD. Understanding the risk factors for new-onset or deterioration of existing CKD after nephrectomy is important in developing preventive measures to provide better care for these patients. There is also a need to understand the incidence, natural history, management trends, and sequelae of radiofrequency ablation as well as surveillance of small renal cancers or small renal masses (SRMs). Clinical registries are critical in providing excellent patient-centre care and clinical research as well as basic science research. Registries evaluate current practice and guide future practice. The Flinders Kidney Health Registry will provide the key information needed to assess various treatment outcomes of patients with kidney cancer and patients who underwent nephrectomy for other reasons. The registry aims to provide clinical decision makers with longitudinal data on patient outcomes, health systems performance, and the effect of evolving clinical practice. The registry will also provide a platform for large-scale prospective clinical studies and research. METHODS: Patients above the age of 18 undergoing nephrectomy or radiofrequency ablation for any indication and patients with SRMs will be included in the registry. Demographic, clinical and quality of life data will be collected from hospital information systems and directly from the patient and/or caregiver. DISCUSSION: The Registry will report a summary of patient characteristics including indication for treatment, clinical risk profiles, surgical and oncological outcomes, the proportion of patients who progress to CKD and end stage kidney disease, quality of life post treatment as well as other relevant outcomes for all patients who have undergone nephrectomy for any indication, ablation or surveillance for SRMs. The registry will record the follow-up practice after nephrectomy and patient on active surveillance, which will help to develop and enhance a best practice protocol. The collected prospective data will provide a platform for ongoing patient-orientated research and improve patient-centred healthcare delivery.

Prostate cancer detection: a systematic review of urinary biosensors.

BACKGROUND: Current diagnostic methods for prostate cancer are invasive and lack specificity towards aggressive forms of the disease, which can lead to overtreatment. A new class of non-invasive alternatives is under development, in which urinary biomarkers are detected using biosensing devices to offer rapid and accurate prostate cancer diagnosis. These different approaches are systematically reviewed and their potential for translation to clinical practice is evaluated. METHODS: A systematic review of the literature was performed in May 2021 using PubMed Medline database, Embase, and Web of Science. The objective was to review the structural designs and performance of biosensors tested on urine samples from patients with prostate cancer. RESULTS: A total of 76 records were identified. After screening and eligibility, 14 articles were included and are discussed in this paper. The biosensors were discussed based on the target biomarkers and detection technologies used, as well as the results of the clinical studies. Most of the works reported good discrimination between patients with prostate cancer and controls. CONCLUSIONS: This review highlights the potential of urinary biosensors for non-invasive prostate cancer detection. However, clinical studies have so far only been conducted on small cohorts of patient, with large scale trials still needed to validate the proposed approaches. Overall, the consensus arising from the proof of concepts studies reviewed here, is that an adequate combination of biomarkers into multiplex biosensor platforms is required to achieve accurate diagnostic tests. Furthermore, whether such devices can discriminate between aggressive and indolent cancer has not yet been addressed, because it entails optimized biomarkers panels and long-term clinical trials.

Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Selective Microfluidic Capture and Detection of Prostate Cancer Cells from Urine without Digital Rectal Examination.

Urine-based biomarkers have shown suitable diagnostic potential for prostate cancer (PCa) detection. Yet, until now, prostatic massage remains required prior to urine sampling. Here, we test a potential diagnostic approach using voided urine collected without prior digital rectal examination (DRE). In this study, we evaluated the diagnostic performance of a microfluidic-based platform that combines the principle of photodynamic diagnostic with immunocapture for the detection of PCa cells. The functionality and sensitivity of this platform were validated using both cultured cells and PCa patient urine samples. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) demonstrated this platform had a detection limit of fewer than 10 cells per 60 µL and successfully validated the presence of a PCa biomarker in the urine of cancer patients without prior DRE. This biosensing platform exhibits a sensitivity of 72.4% and a specificity of 71.4%, in suitable agreement with qRT-PCR data. The results of this study constitute a stepping stone in the future development of noninvasive prostate cancer diagnostic technologies that do not require DRE.

Arginine Metabolites as Biomarkers of Myocardial Ischaemia, Assessed with Cardiac Magnetic Resonance Imaging in Chronic Kidney Disease.

(1) Background: Cardiovascular disease (CVD) is the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Myocardial oxygenation and perfusion response to stress, using oxygen-sensitive cardiovascular magnetic resonance (OS-CMR) and stress T1 mapping respectively, are impaired in CKD patients with and without known coronary artery disease (CAD). Endothelial dysfunction, assessed by circulating levels of asymmetric dimethylarginine (ADMA) and homoarginine (HMA), promotes atherosclerosis. We hypothesized that in CKD patients, worsening endothelial dysfunction is associated with worsening myocardial oxygenation and perfusion as assessed by change in OS-CMR signal intensity (Δ OS-CMR SI) and stress T1 (ΔT1) values. (2) Methods: 38 patients with advanced CKD underwent cardiovascular magnetic resonance (CMR) scanning at 3 Tesla. OS-CMR and T1 mapping images were acquired both at rest and after adenosine stress and analyzed semi-quantitatively. Serum ADMA and HMA concentrations were assessed using mass spectrometry. (3) Results: There was no significant correlation between Δ OS-CMR SI and ADMA or HMA. Interestingly, there was a significant negative correlation seen between Δ T1 and ADMA (r = -0.419, p = 0.037, n = 30) but not between Δ T1 and HMA. (4) Conclusions: Stress T1 response is impaired in CKD patients and is independently associated with higher circulating ADMA concentrations.

Cancer cell detection device for the diagnosis of bladder cancer from urine.

Bladder cancer is common and has one of the highest recurrence rates. Cystoscopy, the current gold standard diagnosis approach, has recently benefited from the introduction of blue light assisted photodynamic diagnostic (PDD). While blue light cystoscopy improves diagnostic sensitivity, it remains a costly and invasive approach. Here, we present a microfluidic-based platform for non-invasive diagnosis which combines the principle of PDD with whole cell immunocapture technology to detect bladder cancer cells shed in patient urine ex vivo. Initially, we demonstrate with model cell lines that our non-invasive approach achieves highly specific capture rates of bladder cancer cells based on their Epithelial Cell Adhesion Molecule expression (>90%) and detection by the intensity levels of Hexaminolevulinic Acid-induced Protoporphyrin IX fluorescence. Then, we show in a pilot study that the biosensor platform successfully discriminates histopathologically diagnosed cancer patients (n = 10) from non-cancer controls (n = 25). Our platform can support the development of a novel non-invasive diagnostic device for post treatment surveillance in patients with bladder cancer and cancer detection in patients with suspected bladder cancer.

Plasma enabled devices for the selective capture and photodynamic identification of prostate cancer cells.

Prostate cancer is the second most common cancer in men and the second leading cause of male cancer deaths. The current blood test for detecting prostate cancers measures prostate-specific antigen. It has many limitations including a very high rate of false positives. Herein, prostate-specific membrane antigen (PSMA) based immunocapture and hexaminolevulinate (HAL) based photodetection are integrated into a new diagnostic device designed to selectively identify whole prostate cancer cells from voided urine with the aim of providing an accurate noninvasive alternative to current diagnosis methods. Prestained, prostate cancer cells spiked in urine samples at concentrations ranging from 1500 to 2000 cells/ml were captured with 89% sensitivity and 95% specificity. HAL, a cancer specific photosensitizer, was then used to circumvent the need for prestaining. Optimum HAL incubation conditions were identified (50 µM at 37 °C for 2 h) where the mean HAL-induced fluorescence intensity of LNCaP cells was three times that of healthy PNT2 cells, thus providing an independent way to discriminate captured cancer cells from background metabolites. Combining anti-PSMA immunocapture with HAL-induced fluorescent detection, 86% sensitivity and 88% selectivity were achieved, thereby proving the validity of the dual-method for the selective photospecific detection of prostate cancer cells.

MicroRNA-Related Genetic Variants Are Associated With Diabetic Retinopathy in Type 1 Diabetes Mellitus.

Purpose: Few studies have explored the association of genetic variants in microRNA genes and binding sites with diabetic retinopathy (DR) in type 1 diabetes. We conducted a genome-wide scan for single nucleotide polymorphisms (SNPs) in these genes by using data from a genome-wide association study (GWAS). Methods: All known SNPs were imputed from our GWAS data (n = 325) of DR cases and diabetic controls (no DR). Relevant SNPS were extracted using miRNASNP and PolymiRTS (version 2) databases. χ2 tests and logistic regression (adjusting for age, sex, duration of diabetes, HbA1c, and hypertension) were used to test the association between the imputed SNPs and DR phenotypes (any DR, nonproliferative DR [NPDR], proliferative DR [PDR], diabetic macular edema [DME], and sight-threatening DR defined as PDR, severe NPDR, or clinically significant macula edema [CSME]) compared with diabetic controls. Top-ranking SNPs were genotyped in a larger cohort (N = 560) to confirm their association with DR. Results: Three SNPs (rs10061133, rs1049835, rs9501255) were selected and genotyped in the final cohort. Rs10061133 in MIR449b was protective of sight-threatening DR (odds ratio [OR] = 0.32, P = 3.68 × 10-4) and PDR (OR = 0.30, P = 8.12 × 10-4), and the associations became more significant as the cohort increased in size. Conclusions: Rs10061133 in MIR449b is significantly associated with a decreased risk of PDR and sight-threatening DR in Caucasian patients with type 1 diabetes. This can guide future studies on genetic risk profiling and on developing microRNA-related therapies for sight-threatening DR.

In order for the light to shine so brightly, the darkness must be present-why do cancers fluoresce with 5-aminolaevulinic acid?

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection and photodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IX phenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies.

Compensatory renal hypertrophy following nephrectomy: When and how?

Following surgical removal of one kidney, the other enlarges and increases its function. The mechanism for the sensing of this change and the growth is incompletely understood but begins within days and compensatory renal hypertrophy (CRH) is the dominant contributor to the growth. In many individuals undergoing nephrectomy for cancer or kidney donation this produces a substantial and helpful increase in renal function. Two main mechanisms have been proposed, one in which increased activity by the remaining kidney leads to hypertrophy, the second in which there is release of a kidney specific factor in response to a unilateral nephrectomy that initiates CRH. Whilst multiple growth factors and pathways such as the mTORC pathway have been implicated in experimental studies, their roles and the precise mechanism of CRH are not defined. Unrestrained hypoxia inducible factor activation in renal cancer promotes growth and may play an important role in driving CRH.

Mitochondrial haplogroups are not associated with diabetic retinopathy in a large Australian and British Caucasian sample.

Mitochondrial haplogroups H1, H2 and UK have previously been reported to be associated with proliferative diabetic retinopathy (PDR) in Caucasian patients with diabetes. We aimed to replicate this finding with a larger sample and expand the analysis to include different severities of DR, and diabetic macular edema (DME). Caucasian participants (n = 2935) with either type 1 or type 2 diabetes from the Australian Registry of Advanced Diabetic Retinopathy were enrolled in this study. Twenty-two mitochondrial single nucleotide polymorphisms were genotyped by MassArray and haplogroups reconstructed using Haplogrep. Chi square tests and logistic regressions were used to test associations between haplogroup and DR phenotypes including any DR, non-proliferative DR (NPDR), proliferative DR (PDR) and DME. After stratifying the samples in type 1 and type 2 diabetes groups, and adjusting for sex, age, diabetes duration, concurrent HbA1c and hypertension, neither haplogroups H1, H2, UK, K or JT were associated with any DR, NPDR, PDR or DME.

Circulating and Urinary miR-210 and miR-16 Increase during Cardiac Surgery Using Cardiopulmonary Bypass - A Pilot Study.

A pilot study to measure and compare blood and urine microRNAs miR-210 and miR-16 in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and off-pump coronary artery bypass grafting surgery. Frequent serial blood and urine samples were taken from patients undergoing cardiac surgery with CPB (n = 10) and undergoing off-pump cardiac surgery (n = 5) before, during, and after surgery. Circulating miR-210 and miR-16 levels were determined by relative quantification real-time polymerase chain reaction. Levels of plasma-free haemoglobin (fHb), troponin-T, creatine kinase, and creatinine were measured. Perioperative serum miR-210 and miR-16 were elevated significantly compared to preoperative levels in patients undergoing cardiac surgery with CPB (CPB vs. Pre Op and Rewarm vs. Pre Op; p < .05 for both). There were increases of greater than 200% in miR-210 levels during rewarming and immediately postoperatively and a 3,000% increase in miR-16 levels immediately postoperatively in urine normalized to urinary creatinine concentration. Serum levels of miR-16 were relatively constant during off-pump surgery. miR-210 levels increased significantly in off-pump patients perioperatively (p < .05 Octopus on vs. Pre Op); however, the release was less marked when compared to cardiac surgery with CPB. A significant association was observed between both miR-16 and miR-210 and plasma fHb when CPB was used (r = -.549, p < .0001 and r = -.463, p < .0001 respectively). Serum and urine concentrations of hypoxically regulated miR-210 and hemolysis-associated miR-16 increased in cardiac surgery using CPB compared to off-pump surgery. These molecules may have utility in indicating severity of cardiac, red cell, and renal injury during cardiac surgery.

A platform for selective immuno-capture of cancer cells from urine.

Urothelial cancers are amongst the 10 most common types of cancer and represent a major health problem worldwide. Current urinary diagnostic tests for urothelial cancer are expensive and have limited sensitivity and specificity. In this work, proofs of concept for a selective cancer cell capture platform are presented with the aim to achieve the first generation of specific urinary tests for the detection of cancer cells in urine specimen. The unique reactivity of plasma deposited polyoxazoline was used to covalently bind cancer specific antibodies in microchannels. Cancer cells dispersed in patient urine were successfully captured with up to 99% selectivity and 100% sensitivity over a wide range of cell concentrations. The streamlined two steps preparation process of the capture platform represents an important advance in medical diagnostics, with broader potential applications.

Lack of appropriate investigations in making a diagnosis of syndrome of inappropriate antidiuretic hormone.

The syndrome of inappropriate antidiuretic hormone (SIADH) is reported as the most common cause of hyponatraemia. This retrospective cross-sectional study evaluated the diagnosis of SIADH in 110 hospitalised patients in an Australian tertiary hospital with reference to recently published clinical guidelines. Investigation of SIADH was incomplete in all but 20% of cases. Adrenal insufficiency and hypothyroidism were not excluded in a significant number of cases.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.