Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in renal excretion of 2,8-dihydroxyadenine ...(DHA) in excessive amounts, leading to kidney stones and crystal nephropathy with associated inflammation and fibrosis. The effects of other kidney stone constituents, such as calcium oxalate and uric acid, have been thoroughly studied in cell culture models, both in monolayer and three-dimensional (3D) assays. However, the effect of DHA in cell culture models has not yet been investigated. This study aimed to establish a comprehensive cell culture model to investigate DHA crystal-induced kidney injury and to identify therapeutic targets for clinical intervention. Method Three lines of kidney cells (MDCK, HK-2, and HEK293) were utilized in monolayer and 3D assays, employing both liquid-liquid interface (LLI) and “on-top” of Matrigel culture models. DHA exposure, matching the quantities found in the urine of untreated patients with APRT deficiency, was used in conjunction with siRNA-mediated APRT gene knockdown to assess the changes in cellular phenotypes. Assessments included cell viability and migration assays, RT-PCR, western blotting, phase-contrast microscopy, and immunostaining. Paraffin-embedded kidney tissue samples from patients with APRT deficiency and healthy controls were obtained from the Landspitali National University Hospital Biobank (Icelandic IRB permit: VSN 21-117-V1) and phenotypically analyzed using immunohistochemistry (IHC) staining for collagen I (Col I), collagen III (Col III), and APRT. Results After 72 h of incubation with DHA at concentrations of 60, 120, 240, and 480 μg/mL, the viability of all cell lines decreased. Proliferation was evaluated using a scratch assay after treatment with DHA at two different concentrations (120 and 480 μg/mL) for 24 h, and all cell lines showed decreased migration at the highest concentration. Enhanced CD44 expression was observed in both HEK293 and MDCK cells with increasing DHA concentration. In a 3D environment “on-top” of Matrigel, MDCK cells maintained polarized structures despite accumulation of DHA and did not show an increased EMT phenotype compared with TGFβ-treated cells. HEK293 and HK-2 cells formed solid colonies with DHA accumulation within the colony. MDCK cells formed a polarized cell layer grown on Transwell polyester membranes and demonstrated trans-epithelial electrical resistance (TEER) in LLI, which decreased when the cells were treated with DHA at 120 and 480 μg/mL. APRT expression was significantly reduced in all cell lines after successful knockdown. Analysis of kidney tissue specimens using IHC showed increased expression of Col III in patients with APRT deficiency compared to healthy controls. Enhanced Col I expression was also observed in these patients, which, together with Col III, was consistent with increased fibrosis. Conclusion This study revealed that incubation with varying concentrations of DHA led to decreased viability and impaired migration in all cell lines tested. Increased CD44 expression suggests potential crystal binding to renal tubular epithelial cells when exposed to increasing DHA concentrations. In a 3D environment, MDCK cells maintained polarized structures with DHA accumulation without enhancement of the EMT phenotype. Treatment with DHA resulted in a decrease in TEER, indicating a defective barrier function. Increased expression of Col III and Col I was observed in kidney tissue samples from APRT-deficient patients, suggesting increased fibrosis. Overall, these findings highlight the impact of DHA on cell viability, proliferation, EMT, and barrier function, highlighting the role of DHA in kidney fibrosis in patients with APRT-deficiency.
Abstract Background and Aims Nephrogenic diabetes insipidus (NDI) is a known side effect of lithium treatment. Lithium has also been associated with the development of chronic kidney disease (CKD). ...The aims of this study were to examine the risk factors for NDI among persons on lithium treatment and the association of NDI and other risk factors with the development of chronic kidney disease (CKD). Method This was a retrospective cohort study of all persons in Iceland using lithium in the years 2003-2018. Lithium exposure was defined as at least one lithium prescription or at least one serum lithium measurement with a detectable lithium level. Patients with affective disorders (ICD-10 codes F30-F39) attending the outpatient clinics of Landspitali–The National University Hospital Mental Health Services in 2014-2016, without lithium exposure, served as controls. NDI was defined as concomitant serum sodium (SNa) >144 mmol/L and urine specific gravity (SG) <1.005; individuals with no SNa measurement after 2003 were excluded from the analysis. CKD stages 3-5 were defined according to the KDIGO guidelines for CKD as estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m2. The eGFR was calculated from serum creatinine (SCr) using the CKD-EPI equation. The analysis of incident CKD was limited to the period when standardized SCr measurements had become available, in 2008-2018. Individuals with <2 SCr measurements after 2008 and those with CKD stages 3-5 before 2008 were excluded. In addition, this analysis specifically examined the relationship between the mean serum lithium concentration and the development of CKD, comparing those not exposed to lithium to 3 groups of lithium users with a mean serum lithium concentration of 0.3-0.59, 0.6-0.79 and 0.8-0.99 mmol/L. Acute kidney injury (AKI) was defined according to the SCr component of the KDIGO criteria for AKI, and other comorbid conditions were defined based on ICD-9 and ICD-10 diagnosis codes. Multivariable Cox regression with time-dependent covariables was used for risk assessment. Results In the analysis of the development of NDI, the group exposed to lithium consisted of 2252 individuals, of whom 58 (2.6%) met the criteria for NDI compared with 13 (1.0%) of the 1272 controls. When compared with those without lithium exposure. the risk of NDI was lithium concentration dependent, with a hazard ratio (HR) of 1.22 (95% CI, 0.50–2.98), 5.52 (95% CI, 2.81–10.85) and 10.03 (95% CI, 4.51–22.27) for groups with a mean lithium concentration of 0.3–0.59, 0.6–0.79 and 0.8–0.99 mmol/L, respectively. A previous diagnosis of CKD was also a significant risk factor (HR 3.00 95% CI, 1.50–6.12). In the CKD analysis, the group of lithium users comprised 1401 individuals. Of those, 192 (13.7%) developed CKD, compared with 39 (2.8%) of the 1406 controls. Analysis adjusted for initial eGFR showed that NDI was a significant risk factor for CKD (HR 1.90 95% CI, 1.03-3.54). Other significant risk factors were mean lithium concentration with HR of 1.25 (95% CI, 0.82–1.91), 2.84 (95% CI, 1.94–4.15) and 5.11 (95% CI, 3.22–8.07) for the groups with a mean lithium concentration of 0.6–0.79 and 0.8–0.99 mmol/L, respectively. Other significant risk factors were age per year (HR 1.03 95% CI, 1.02–1.05), diabetes (HR 1.62 (95% CI, 1.08–2.46) and history of AKI (HR 1.92 95% CI, 1.33–2.77). Conclusion Lithium use is associated with NDI in a concentration-dependent manner. Long-term lithium treatment is associated with a risk of CKD, which also is concentration dependent, in patients with bipolar and unipolar mood disorders. NDI may be an important risk factor for CKD.
Abstract Background and Aims Calciphylaxis is a rare and serious medical condition that primarily affects patients with end-stage kidney disease (ESKD). The disorder is characterized by calcium ...deposition in blood vessels and soft tissues, resulting in skin ulceration and tissue necrosis. The aim of the study was to investigate the incidence, risk factors, and outcomes of calciphylaxis in Iceland. Method This was a retrospective case-control study that included individuals diagnosed with calciphylaxis in 1990-2022. Patients were identified by searching electronic medical records (EMR) for the ICD-10 diagnosis codes E83.5 and L94.2, SNOMED histology codes T01000-T03900 in combination with M55400, M55500 or M54000-M54200, documented sodium thiosulfate (STS) therapy, and by consultation with practicing nephrologists. Data on medical history, medication use, and survival were obtained from EMR. A control group of ESKD patients was matched (1:1) based on age, sex, and duration of dialysis. Total annual incidence was calculated per 1,000,000 population of Iceland each year, and for ESKD patients per 10,000 dialysis patients at the end of each year. Poisson regression was used to estimate changes in incidence. Groups were compared using non-parametric tests and survival estimated by Kaplan-Meier method and Cox regression. Results Twenty-one cases of calciphylaxis were identified, of which 18 (86%) had ESKD. The average incidence was 2/1,000,000/year. For ESKD patients, the median (interquartile range, IQR) age was 69 (66-75) years, and 50% were women. Three patients (17%) had not yet initiated dialysis at the time of diagnosis and the median duration of dialysis was 0.12 (IQR 0.0-2.8) years. The average incidence of calciphylaxis was 75 per 10,000 dialysis patients/year, increasing by 9.0% annually (Fig. 1). No significant differences between calciphylaxis cases and controls were observed for comorbid diseases or other risk factors. The median body mass index of cases was 29.5 (IQR 27.1-33.7) compared with 27.6 (IQR 24.3-30.6) kg/m2 in controls (p=0.20), and 50% of cases and 22% of controls (p=0.16) were on warfarin treatment at diagnosis. Fifteen patients received treatment with sodium thiosulfate (STS). Three of 6 patients received STS therapy before 2016 and all 12 patients after 2016. One-year mortality of ESKD patients with calciphylaxis was 44%. The median survival was 134 days in 1990-2016, increasing to 644 days after 2016. Survival difference between cases and controls was significant (p=0.04) by log rank test, while adjusted Cox regression did not show a statistically significant association between calciphylaxis and death (HR 2.0 (95% CI, 0.67-6.1)). Conclusion The incidence of calciphylaxis in Iceland seems to be increasing and is high compared to incidence rates reported in other countries. Survival, although poor, appears to have improved in recent years. The small sample size is a limitation and further research will require collaborative efforts to generate large patient samples.
Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare disorder of purine metabolism, characterized by urinary excretion of the poorly soluble 2,8-dihydroxyadenine ...(DHA), leading to nephrolithiasis and chronic kidney disease (CKD). Treatment with xanthine oxidoreductase (XOR) inhibitor, allopurinol or febuxostat, reduces DHA excretion and slows or halts CKD progression. The aim of the study was to optimize and validate an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)-based assay for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma, for monitoring of pharmacotherapy in patients with APRT deficiency. Method The UPLC-MS/MS-based assay was optimized employing the chemometric approach design of experiments, using the software Modde Pro 13 (Sartorius, Umeå, Sweden). The assay was validated and used for absolute quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in plasma samples from both untreated and treated APRTd patients and healthy controls. Results Accuracy and precision were within ±15% for all analytes, and the analytes were stable in plasma for 12 months at −80°C. The median (range) concentration of DHA in plasma samples from six APRT deficiency patients was 204.0 (187.2–315.8) ng/mL when they were untreated and 69.2 (below the limit of quantification BLQ–131.6) ng/mL when treated with allopurinol (400 mg/day), and BLQ when febuxostat (80 mg/day) was the therapeutic agent. The median (range) plasma adenine concentration was 238.1 (218–502) ng/mL in the untreated patients, and 561.1 (418–2104) and 856.0 (726–1711) ng/mL in those on treatment with allopurinol and febuxostat, respectively. DHA was not detected in plasma samples from healthy controls. The median (range) plasma concentration of allopurinol, oxypurinol and febuxostat in patients receiving XOR inhibitor therapy was 1657 (BLQ-3266) ng/mL, 8102 (4778–34906) ng/mL and 109 (BLQ-1657) ng/mL, respectively. Conclusion The UPLC-MS/MS-based assay provides accurate and precise quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma. Measurement of plasma DHA is being implemented as a diagnostic test for APRT deficiency and the full assay for monitoring of XOR inhibitor treatment in patients with the disorder.
Abstract Background and Aims International consensus supports the development of standardized protocols for measured glomerular filtration rate (mGFR) to facilitate the adoption of mGFR determination ...in both clinical and research settings. Method The European Kidney Function Consortium (EKFC) convened an international group of mGFR experts. The working group performed an extensive literature search to inform the development of recommendations for mGFR determination using one-compartment plasma clearance models and iohexol as the exogenous filtration marker. Iohexol was selected as it is non-radio labeled, inexpensive, widely available, and can be assayed at a central laboratory. Other commonly used non-radio labeled tracers have been (inulin) or are soon to be (iothalamate) discontinued. A plasma clearance model was selected as it requires no timed urine collections. A one-compartment model was preferred to a two compartment model as it requires fewer samples. The resulting recommendations were based on published evidence complemented by expert opinion. Results Recommendations include practical advice for patients and health professionals, preparation, administration and safety aspects of iohexol, laboratory analysis, blood sample collection and sample timing for both multiple and single sample protocols, description of the mGFR mathematical calculations as well as implementation strategies (Fig.). EKFC will provide materials such as patient information sheets, standard operating procedures, a study protocol template, and support for mGFR calculation. Conclusion Members of the EKFC have evaluated the evidence and formulated recommendations that include all aspects of iohexol plasma clearance measurement using a one compartment model. *Author list on behalf of the European Kidney Function Consortium (EKFC).
We evaluated the 15-year kidney allograft survival in patients with primary glomerulonephritis and determined if the risk of graft loss varied with donor source within each glomerulonephritis group.
...Using data from the European Renal Association-European Dialysis and Transplant Association Registry, Kaplan-Meier, competing risk, and Cox regression analyses were performed on adult, first kidney transplant recipients during 1991 to 2010 (n = 14 383). Follow-up was set to December 31, 2011. Adjustments for pretransplant dialysis duration, sex, country, and transplant era were made. "Death-adjusted graft survival" was assessed in patients with glomerulonephritis and compared with those with autosomal dominant polycystic kidney disease (ADPKD), in which the native kidney disease cannot recur. Additionally, death-adjusted graft survival was compared between living and deceased donor transplants within each glomerulonephritis group.
All glomerulonephritides had a 15-year death-adjusted graft survival probability above 55%. The 15-year risk of death-adjusted graft failure compared to ADPKD ranged from 1.17 (95% confidence interval 95% CI, 1.05-1.31) for immunoglobulin A nephropathy to 2.09 (95% CI, 1.56-2.78) for membranoproliferative glomerulonephritis type II. The expected survival benefits of living over deceased donor transplants were not present in membranoproliferative glomerulonephritis type I (adjusted hazard ratios HRa, 1.08; 95% CI, 0.73-1.60) or type II (HRa, 0.90; 95% CI, 0.32-2.52) but present in immunoglobulin A nephropathy (HRa, 0.74; 95% CI, 0.59-0.92), membranous nephropathy (HRa, 0.47; 95% CI, 0.29-0.75), and focal segmental glomerulosclerosis (HRa, 0.69; 95% CI, 0.45-1.06).
This large European study shows favorable long-term kidney graft survival in all primary glomerulonephritides, although this remains lower than graft survival in ADPKD, and confirms that the reluctance to use living donors in some primary glomerulonephritides remains unfounded. These data will further inform prospective renal transplant recipients and donors during pretransplant counselling.
Background Population mean GFR is lower in older age, but it is unknown whether healthy aging is associated with preserved rather than lower GFR in some individuals. Methods We investigated the ...cross-sectional association between measured GFR, age, and health in persons aged 50–97 years in the general population through a meta-analysis of iohexol clearance measurements in three large European population-based cohorts. We defined a healthy person as having no major chronic disease or risk factors for CKD and all others as unhealthy. We used a generalized additive model to study GFR distribution by age according to health status. Results There were 935 (22%) GFR measurements in persons who were healthy and 3274 (78%) in persons who were unhealthy. The mean GFR was lower in older age by −0.72 ml/min per 1.73 m 2 per year (95% confidence interval 95% CI, −0.96 to −0.48) for men who were healthy versus −1.03 ml/min per 1.73 m 2 per year (95% CI, −1.25 to −0.80) for men who were unhealthy, and by −0.92 ml/min per 1.73 m 2 per year (95% CI, −1.14 to −0.70) for women who were healthy versus −1.22 ml/min per 1.73 m 2 per year (95% CI, −1.43 to −1.02) for women who were unhealthy. For healthy and unhealthy people of both sexes, both the 97.5th and 2.5th GFR percentiles exhibited a negative linear association with age. Conclusions Healthy aging is associated with a higher mean GFR compared with unhealthy aging. However, both the mean and 97.5 percentiles of the GFR distribution are lower in older persons who are healthy than in middle-aged persons who are healthy. This suggests that healthy aging is not associated with preserved GFR in old age.
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