Markers Of Damage Research Articles

Effects of Omega-3 Supplementation on the Delayed Onset Muscle Soreness after Cycling High Intensity Interval Training in Overweight or Obese Males

People with overweight or obesity preferred high-intensity interval training (HIIT) due to the time-efficiency and pleasure. However, HIIT leads to delayed onset muscle soreness (DOMS). The present study aimed to investigate the effects of omega-3 supplementation on DOMS, muscle damage, and acute inflammatory markers induced by cycling HIIT in untrained males with overweight or obesity. A randomized, double-blinded study was used in the present study. Twenty-four males with a sedentary lifestyle were randomly assigned to either receive omega-3 (O3) (4 g fish oil) or placebo (Con). Subjects consumed the capsules for 4 weeks and performed cycling HIIT at the 4th week. After 4 weeks-intervention, the omega-3 index of O3 group increased by 52.51% compared to the baseline. All subjects performed HIIT at 4th week. The plasma creatine kinase (CK) level of Con group increased throughout 48h after HIIT. While the CK level of O3 group increased only immediately and 24h after HIIT and decreased at 48h after HIIT. The white blood cell count (WBC) of Con group increased immediately after the HIIT, while O3 group did not show such increase. There was no change of CRP in both groups. O3 group had a higher reduction of calf pain score compared to Con group. O3 group also showed a recovery of leg strength faster than Con group. Omega-3 supplementation for 4 weeks lower increased CK level, reduced calf pain score, and recovery leg strength, DOMS markers after cycling HIIT.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

ABSTRACT The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.

An analysis of urodynamic parameters in diabetic and nondiabetic women.

Diabetes is highly prevalent worldwide, with an estimated 536 million living with diabetes in 2021, and that number projected to increase to 783 million by 2045. Diabetic bladder dysfunction is thought to affect up to 60%-90% of individuals with diabetes and can significantly impact quality of life. Despite the prevalence of diabetic bladder dysfunction, the exact pathophysiological mechanism, and resulting clinical presentation, remains debated. Our objective was to compare urodynamic parameters between diabetic and nondiabetic women, assessing the impact of various markers of diabetes severity on bladder function. A retrospective chart review was conducted on female patients aged 18 and above who underwent urodynamic studies at a single tertiary care university hospital system from 2014 to 2020. Patients were categorized based on diabetes status, and diabetes severity including duration of disease, hemoglobin A1c levels, insulin dependence, and markers of end-organ dysfunction. Urodynamic variables, including compliance, bladder voided efficiency, bladder contractility index, postvoid residual, maximum flow rate, capacity, voided volume, and detrusor overactivity, were assessed by two independent reviewers. Statistical analyses were performed to assess the impact of diabetes and diabetic severity on urodynamic parameters. A total of 652 female patients were included in the study, of which, 152 (23.3%) had diabetes, with an average duration of diagnosis of 82.3 months. Diabetic women were older and had higher body mass index compared to nondiabetic women. Diabetic retinopathy and neuropathy were present in 18% and 54.6% of diabetic patients, respectively. Significant differences in urodynamic parameters were observed between diabetic and nondiabetic women, with diabetic women showing higher rates of detrusor overactivity (p = 0.01), particularly associated with increasing BMI (p = 0.03). However, classic markers of diabetes severity including duration, as well as markers of end-organ damage, showed mixed associations with urodynamic changes. Despite the prevalence of diabetic bladder dysfunction and its impact on patient quality of life, the exact mechanisms and clinical presentation remain elusive. Our study highlights the significant differences in urodynamic parameters between diabetic and nondiabetic women, emphasizing the need for further research into the relationship between diabetes and diabetic bladder dysfunction.

Biochemical markers of neuro-myocardial damage after pulmonary vein isolation: the effect of different energy sources

Abstract Background Pulmonary vein isolation (PVI) by thermal energy, both radiofrequency (RF) and cryoenergy(Cryo), is associated with a significant release of myocardial and collateral neural injury markers. Damage to periatrial ganglionated plexi also results in a substantial change in cardiac autonomic regulations. On the contrary, pulsed electric field (PEF) energy tends to spare cardiac neural tissue. Purpose To quantify the impact of different ablation energy sources that are used for PVI on neuro-myocardial damage by the assessment of specific biomarkers. Methods A study enrolled 85 patients with paroxysmal atrial fibrillation who underwent PVI (age: 61 ± 12 years, 74% males). In 23 patients, RF energy was delivered by the irrigated-tip catheter, while 30 patients were ablated using a second-generation cryoballoon catheter. In 32 patients, PVI was performed using a multipolar pentaspline catheter and PEF energy. Peripheral venous blood samples for assessing the plasmatic levels of S100 calcium-binding protein B (S100B), neuron-specific enolase (NSE), high-sensitivity troponin I and T (hsTnI and hsTnT) were obtained before, immediately after the PVI, and the next day (usually 18-24 hours after the procedure). Study groups were compared regarding absolute biomarker levels and hsTnT/S100B ratio (a surrogate of cardioselectivity). Results Following successful PVI, a significant increase in plasma levels of all biomarkers was observed: DhsTnT = 1040±434 ng/L, 1731±830 ng/L and 1010±397 ng/L; DhsTnI = 1504±918 ng/L, 16464±11911 ng/L and 10789±7589 ng/L; DS100B: 0.04±0.02 mg/L, 0.03±0.04 mg/L and 0.14±0.08 mg/L; DNSE: 34±72 ng/mL, 27±53 ng/mL and 45±52 ng/mL (all with P <0.001) for RF, PEF and Cryo groups, respectively. A direct comparison of maximal biomarker levels between study groups is depicted in the Figure. The hsTnT/S100B ratio was 23±14, 12±6, and 6±3 in PEF, RF, and Cryo groups, respectively (ANOVA P <0.01), indicating that PEF was 1.9-times and 3.8-times more cardioselective than RF and Cryo, respectively. Following the PVI, the heart rate accelerated by 2±8, 13±14, and 9±11 bpm in the PEF, RF, and Cryo groups, respectively. No correlation was observed between S100B release and HR acceleration (R = 0.07, P = 0.55). Conclusion Compared to thermal energy sources, PEF energy induces more specific damage to cardiomyocytes than cardiac nerves. There was no correlation between S100B release and HR acceleration.

Longitudinal plasma proteomic analysis of 1117 hospitalized patients with COVID-19 identifies features associated with severity and outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.

#1554 The association between gut-derived metabolites and biomarkers of renal and cerebro-vascular damage in type 2 diabetes mellitus patients

Abstract Background and Aims Due to its numerous complications, type 2 diabetes mellitus (T2DM) has a major impact on the global economic burden and overall mortality. This condition modifies the metabolic microenvironment in targeted organs, such as the kidney and the brain, by its hyperglycemic nature. Gut microbiota seems to have significant interference with the renal-cerebral axis, especially in a dysbiosis state. The aim of the study was to quantify gut derived metabolites by metabolomic techniques and to evaluate their association with markers of endothelial, podocyte and proximal tubule damage and with cerebro-vascular hemodynamic indices. Method The metabolomic profiling was realized on serum and urine samples originated from 90 T2DM patients, divided into three subgroups, such as the following: 30 normal to mildly-, 30 moderately-, and 30 severely increased albuminuria, respectively, and 20 healthy subjects. The samples were examined by using untargeted and targeted ultra-high performance liquid chromatography coupled with electrospray-ionization quadroupole time-of-flight mass spectrometry (UHPLC-QTOF-ESI+-MS), in order to obtain the metabolic profile. The markers of renal damage such as synaptopodin (SP), podocalyxin (PD), N-acetyl-β-D-glucosaminidase (NAG), kidney injury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1) were assessed by ELISA technique. The hemodynamic cerebro-vascular indices were obtained by high resolution Doppler ultrasound of common carotid arteries (CCAs), internal carotid arteries (ICAs), and middle cerebral arteries (MCAs), bilaterally. The correlations between the metabolites, the markers of renal damage, and neurosonological indices were realized by univariable and multivariable linear regression analyses. Results UHPLC-QTOF-ESI+-MS targeted analysis and subsequent statistical assessments revealed a subset of metabolites with a specific pattern for the normal to mildly increased albuminuria subgroup. The metabolites identified as significant were, in serum: arginine (sArg), hippuric acid (sHA), indoxyl sulfate (sIS), butenoylcarnitine (sBCA), sorbitol (sSorb); and in urine, arginine (uArg), butenoylcarnitine (uBCA), indoxyl sulfate (uIS) and p-cresyl sulfate (uPCS). The univariable linear regression analysis revealed that sArg correlated positively with the breath-holding index (BHI) and negatively with intima media thickness (IMT) in ACI, MCP-1 and ICAM-1; sIS, sSorb, and sBCA correlated inversely with these parametes. The multivariable linear regression analysis displayed the specific correlations between metabolites, markers of endothelial dysfunction, and neurosonological indices, such as follows: sArg correlated with IMT and ICAM-1; sIS followed a predictive pattern along with resistance index (RI) in ACIs and ICAM-1; sBCA correlated with IMT and MCP-1 and sSorb correlated with ICAM-1 and BHI. The urine metabolites were correlated with markers of podocyte and proximal tubule dysfunction: uIS and uBCA were correlated with PD and urinary albumin to-creatinin ratio (uACR), and uPCR correlated with KIM-1 and uACR. Conclusion This study revealed a set of metabolites potentially derived from the intestinal microflora which may act as biomarkers for the early detection of both CMA and DKD in T2DM patients. SArg, sIS and, sBCA are indicators of renal endothelial dysfunction. Moreover, sArg and sIS reflect incipient common and internal carotid artery atherosclerosis, suggesting vascular remodeling at these levels. SSorb correlated with BHI, indicating blood-brain barrier dysfunction and CMA development. In urine, uBCA and uIS are indicators of podocytopathy, whereas uPCS is associated with proximal tubule damage in incipient DKD.

#553 Renal tubular damage is an independent risk factor for all-cause and cardiovascular mortality in a community-based population: the Takahata Study

Abstract Background and Aims Renal tubulointerstitial damage is a risk for renal failure. However, the association between renal tubulointerstitial damage and all-cause and cardiovascular mortality in the general population remains unclear. In this prospective cohort study, we examined this point using urinary β2-microglobulin/creatinine ratio (UBCR) as a marker of renal tubulointerstitial damage in the general Japanese population. Methods We examined the association between UBCR values at entry and all-cause and cardiovascular mortality during the follow-up period (median 9.2 years) in 3 427 residents (mean age 62.5 years, 45.1% male, mean eGFR 81.6 ml/min/1.73 m², ≥1+ protein 4.4%) of Takahata Town in Japan. Results Study participants were divided into two groups (low < 300 μg/gCre, high ≥300 μg/gCre) based on UBCR at entry. Kaplan-Meier analysis showed a significantly higher incidence of all-cause and cardiovascular mortality in the high UBCR group (Log-rank P < 0.01). A multivariate Cox proportional hazards analysis adjusted for age, sex, eGFR, urinary protein level, comorbidities, smoking status, and other background factors showed a hazard ratio of 1.47 (95% CI 1.09-2.00, P = 0.01) for all-cause mortality and a hazard ratio of 1.82 (95% CI 1.06-3.12, P = 0.03) for cardiovascular death in the high-UBCR group. The addition of high UBCR to conventional risk factors, including eGFR and urinary protein level, significantly improved the net reclassification improvement (NRI) for all-cause and cardiovascular mortality (P < 0.01). Conclusions UBCR, a marker of tubulointerstitial damage, was identified as a risk factor for all-cause and cardiovascular mortality in the general population independent of eGFR and urinary protein levels.

#1141 Chronic kidney disease in inflammatory bowel disease: systematic review and meta-analysis

Abstract Background and Aims Chronic kidney disease (CKD) is defined by a reduction in kidney function (estimated glomerular filtration rate < 60 mL/min/1.73 m2) and/or damage markers that are present for at least three months, regardless of the underlying aetiology. Case reports and cohort studies suggest that inflammatory bowel disease (IBD is associated with CKD. The extent and magnitude of a potential association between IBD and CKD is currently unknown. Method A comprehensive search was conducted in EMBASE, MEDLINE, Web of Science, the Cochrane database, and SCOPUS. Two separate reviewers were involved in the process of article selection and evaluation. In studies where a comparison between an IBD population and a non-IBD control population was available, odds ratios were calculated to quantify the association. To derive an overall estimate of the effect, the Mantel–Haenszel test was employed, utilising a random effect model. This systematic was registered with Prospero. Results Fifty-four articles were included in the review (Table 1). Eight articles compared an IBD population with a healthy control population and were included in the meta-analysis, although with high heterogeneity (I2 88%). An increased risk of CKD in the IBD population was observed, with an overall odds ratio of 1.59 (95% CI 1.31-1.93) (Fig. 1). Importantly, this effect was independent of 5-aminosalicylic acid use. Data on other therapies was sparse, except for known nephrotoxic medication like tacrolimus and cyclosporine. Interestingly, several studies reported a decreasing relative risk with increasing age, potentially reflecting an increased baseline risk in this ageing population. Conclusion The existing evidence on CKD in IBD was systematically reviewed, including all human epidemiological studies. An increased CKD prevalence in IBD was found; independent of medication. Hence, we suggest regular measurements of kidney function for all patients with IBD, irrespective of the therapeutic regimen. Measurements should include determination of the baseline creatinine, albumin/creatinine ratio and urinary sediment. For patients having a normal baseline function, we would recommend annual follow-up. A more systematic research approach regarding CKD and IBD is warranted

#2170 BET inhibitor nanotherapy reduces chronic kidney disease progression after ischemia-reperfusion injury

Abstract Background and Aims Targeting epigenetic mechanisms offer a potential breakthrough in addressing kidney diseases. Thus, inhibition of the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1, has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, clinical translation faces hurdles like poor pharmacokinetics and side effects. To tackle this, we engineered liposomes loaded with JQ1 to enhance kidney drug delivery and minimize side effects. By optimizing the encapsulation of JQ1, our aim was to improve its efficacy compared to freely delivered JQ1 and reduce its cytotoxicity. Moreover, the therapeutic potential of this new nanoformulation to halt the acute renal ischemic injury and its transition to CKD will be explored. Method JQ1 was encapsulated in different types of lipid nanoparticles (NPs), and its effect was analyzed in vitro on the HK2 tubular epithelial line. Additionally, in vivo administration was carried out 1 hour after the induction of 45 minutes bilateral renal ischemia, and its effect was further analyzed at 24 h in the AKI setting or at 21 days for CKD. Non-encapsulated JQ1 was used as a control, and in vivo biodistribution studies were conducted using IVIS and fluorescent Cy5.5-labelled NPs. Damage markers, inflammatory burden and fibrosis development were analyzed through conventional techniques (Western blot, RT-PCR, IHC, flow cytometry), and functional parameters (BUN, Creatinine) were assessed through colorimetric assays. Results Three types of liposomal NPs were assayed to optimize JQ1 encapsulation, being the combined encapsulation of JQ1 in both the core and lipid layer the ones proving the best effectiveness. With an average hydrodynamic diameter of 78 nm, these NPs effectively reached the kidneys as assayed by in vivo biodistribution assays. In vitro assays performed on the HK2 cell line reveal their potential to inhibit TNF-α-induced inflammation. Importantly, in vivo dose-dependent studies determined that a 40 mg/kg dose was effective in post-ischemic acute kidney injury, showing a reduction in inflammatory (Il6, Csf2, Ccl2, Ccl5) and renal damage markers (Ngal), as well as reduced immune cell infiltration into the renal tissue of monocytes (CD45+CD11b+Ly6G−Ly6ChiF4/80low) and neutrophils (CD45+CD11b+Ly6GhiLy6Clow) quantified by flow cytometry. In accordance, decreased levels of serum BUN and Creatinine were detected in the group of mice treated with NPs-JQ1, indicating a better renal function. Of note, none of these effects were observed when the same dose of the unencapsulated inhibitor was used, demonstrating no effectiveness whatsoever. In the long term, by establishing an AKI to CKD transition model analysing fibrosis at 21 days post-ischemia induction, mice receiving a single dose of NPs-JQ1 early after damage displayed reduced fibrosis markers expression (α-SMA and Fibronectin), decreased collagen deposits quantified by Masson's Trichrome tissue staining and a partial recovery of the renal function. Conclusion The encapsulation of JQ1 in lipid nanoparticles is an effective strategy that allows for a decrease in the minimum effective dose of the drug. Due to its higher concentration and stability in the kidney, this therapy could be relevant in the treatment of renal pathologies and represents a significant step forward in the development of an innovative therapy for AKI-to-CKD transition. Our pioneering liposomal formulation not only expands the therapeutic potential of JQ1 but also addresses a significant barrier to its clinical translation in the nephrology field.

#211 Low albumin levels are associated with intrarenal complement C4d deposits in critically ill patients with ANCA-associated renal vasculitides

Abstract Background and Aims Despite serum albumin levels being predictive for clinical outcome in ANCA-associated renal vasculitis, implications providing a direct link between low serum albumin levels and intrarenal lesions remain elusive. Therefore, we here aimed to systematically assess the clinical relevance of low albumin levels and scrutinize clinicopathological correlations to expand our current knowledge. Method We here retrospectively enrolled biopsy-proven cases of ANCA-associated renal vasculitis between 2015 till 2020 in a single-center observational study. Survival-curve analyses on long-term renal survival and short-term clinical recovery were performed. Correlative analyses between serum albumin levels, laboratory parameters, proteinuria levels, and histopathological lesions including tubulointerstitial immune cell infiltrates, lesions analogous to the Banff score and intrarenal complement deposition of C3c and C4d were performed. Results We here show that hypoalbuminemia below the median of 2.4 g/dL impairs long-term renal survival especially in MPO-positive ANCA-associated renal vasculitis (p = 0.006, HR: 5.0, 95% CI: 1.2-21.5), while short-term clinical recovery particularly in critically ill patients is negatively affected by low serum albumin levels (p = 0.0082, HR: 3.6, 95% CI: 1.1-11.7). Low albumin levels are associated with the urinary marker of tubular damage in the total cohort (β = −0.5, p = 0.01), PR3-ANCA (β = −0.5, p = 0.04) and MPO-ANCA (p = 0.002, β = −0.8), but not in critically ill patients (β = −0.5, p = 0.07) implying hypoalbuminemia to occur by extrarenal causation in the state of critical illness. We identified plasmacytic infiltrates to correlate with low albumin levels in the total cohort (β = −0.6, p = 0.004) and in the subgroup of critically ill patients (β = −0.7, p = 0.005). Banff-scored interstitial inflammation (i) was further observed to inversely correlate with albumin levels (β = −0.7, p = 0.02). Intrarenal C4d deposition showed a significant correlation with low serum albumin levels in the glomerular tuft (β = −0.4, p = 0.04) and in interstitial arteries (β = −0.7, p = 0.01). Conclusion In conclusion, long-term renal outcome is significantly affected by low serum albumin levels especially in the setting of MPO-ANCA seropositivity. By contrast, short-term recovery was predicted by low albumin levels in critically ill patients with ANCA-associated renal vasculitis. We here provide evidence that low levels of serum albumin might directly affect tubulointerstitial inflammation as reflected by plasmacytic immune-cell infiltration, and intrarenal C4d complement deposition in ANCA-associated renal vasculitis.

#2668 New treatments, new challenges: nephrotoxicity associated to naxitamab in patients with high-risk neuroblastoma

Abstract Background and Aims Neuroblastoma is the most common extracranial solid tumor in children, harboring a poor survival for those with high-risk (HR) tumors. Naxitamab (hu3F8) is a humanized monoclonal anti-disialoganglioside (GD2) antibody approved for the treatment of patients older than 1-year with refractory/relapsed HR-neuroblastoma limited to bone and/or bone marrow. First naxitamab (DanyelzaR) infusion was in 2017 in our institution and since then a large experience has been acquired with its use through several clinical trials as well as an expanded named patient access program. The objective is to describe the renal toxicity profile associated to naxitamab in patients with HR-neuroblastoma. Method Retrospective descriptive study including 244 patients treated with naxitamab (either on monotherapy or associated to chemotherapy) from June 2017 to December 2022 in whom renal involvement and/or hypertension (HT) was evaluated. Results Mean age of the cohort was 8 years (41% female -101- and 59% male -143-), presenting nephrotoxicity of some type up to 26.6% (n = 65): AHT (11.9%), acute renal damage (ARD 10.2%) and proteinuria (5.3%), developing all during the first 3 cycles. All of them were classified as mild adverse events (1-2) according to the Common Terminology Criteria for Adverse Events (CTCAE) scale, except for one patient with ARD grade 3 and two patients with HT grade 3. In the case of HT only in 6 patients an Ambulatory Blood Pressure Monitoring (ABPM) was performed, observing: 2 nocturnal-HT, 2 diurnal-HT without specific-pattern and 2 disautonomic-patterns. Among the ARD, all cases were tubular except for two patients who presented acute tubule-interstitial nephritis (AIN) (one with clinical-analytical pattern and another confirmed by biopsy). Eight of them (32%) had potential confounding factors (previous chemotherapy, ibuprofen or radiotherapy). Among patients with proteinuria (none in the nephrotic range): 38% tubular, 38% glomerular and 23% mixed. Two patients presented ARD + HT and 3 a combination of HT + ARD + proteinuria. All patients received prior chemotherapy and 2 developing chronic renal damage (CKD stage 2 and 3). Conclusion Short-and long-term follow-up, the systematic performance of ABPM, and the use of early markers of renal damage, could lead to a more efficient management of complications derived from this new treatment.

The ocrelizumab wearing-off phenomenon is associated with reduced immunomodulatory response and increased neuroaxonal damage in multiple sclerosis.

The wearing-off phenomenon is common in people with multiple sclerosis (MS) treated with ocrelizumab. We aim to evaluate the presence and severity of wearing-off to ocrelizumab in relation to demographic and MS clinical variables, immune profiling, and a marker of neuroaxonal damage (plasma neurofilament light chain (pNfl)). This cross-sectional study included MS patients treated with ocrelizumab from at least 1year. Wearing-off questionnaire and blood samples were collected between 21 and 23weeks after the previous ocrelizumab infusion. Lymphocyte subpopulations were evaluated on peripheral blood using flow cytometry. PNfl was evaluated using fully automated chemiluminescent enzyme immunoassay. We included 106 people with MS (age 49.5 ± 11.6years; females 42.3%; wearing-off 57.6%). On regression models, wearing-off was associated with higher pNfl, CD8, CD3, and CD3CD27 lymphocytes. Most frequent wearing-off symptoms were cognitive, sensory, and balance problems; wearing-off started < 1week (9.4%), 1-4weeks (10.7%) or > 4weeks (10.7%) before infusion; 44.8% of the complaints were moderate to severe. Severity of wearing-off was associated with higher pNfl and CD8 lymphocytes. Wearing-off is common in people with MS treated with ocrelizumab, and is associated with reduced immunomodulation (higher T lymphocytes) and increased neuroaxonal damage, suggesting reduced treatment response.

Early cognitive dysfunction development immunological predictors in the ischemic stroke acute period

The purpose of the study is activity identification CXC family cytokines in patients with cognitive impairments in the acute period of ischemic stroke.Material and methods. 78 patients with diagnosis “Ischemic stroke” were examined. Depending on cognitive impairment (Montreal Cognitive Assessment (MoCA)) patients were divided into two groups: the 1st group — 58 patients with cognitive decline (MoCA ≤ 25 points); the 2nd group — 20 patients without cognitive decline. Neuropsychologic testing was performed on the second day of hospitalisation and included episodic memory, executive function, speech, gnosis, praxis and IQCODE parameters examination. Laboratory diagnosis consisted of level assessing of CXC family chemokines (CXCL10, CXCL11, CXCL9, CXCL1, CXCL8) and TNF-α cytokine in patients’ plasma on the second day of hospitalisation. Statistical analysis was employed using the Python programing language and its libraries Pandas and SciPy.Results. Statistical analysis revealed the highest level of IP-10/CXCL10 chemokines (p = 0.002) and Gro-a/CXCL1 (p = 0.044) in patients of the 1st group, statistically significant correlations of MoCA and IQCODE with IP-10/CXCL10 and Gro-a/CXCL1 concentrations, correlations of IP-10/CXCL10 concentrations with semantic information processing functions (r = –0.512), subject gnosis (r = –0.211), memory (r = 0.275), speech (r = –0.400), and Gro-a/ CXCL1 level with semantic information processing (r = –0.418).Conclusion. The study of chemokines of the CXC cluster represents a relevant and promising direction in the diagnosis and assessment of progression of early post-stroke cognitive impairment of mixed genesis due to minimal invasiveness and high specificity. Further studies are needed to verify CXCL chemokines, particularly IP-10/CXCL10 and Gro-a/CXCL1 as potential molecular markers of neurological damage in neurodegenerative and inflammatory diseases of the central nervous system.

#1368 Podocytopathy associated with IgA nephropathy: is it really a prognostic factor?

Abstract Background and Aims Since 2017, the clinical value of podocyte injury in IgA nephropathy (IgAN) has been studied, the presence of podocyte hypertrophy and tip- lesions are markers of podocyte damage. It has been observed that these histological findings tend to be treated with immunosuppression, so they have a better kidney prognosis, but those who do not receive immunosuppression have a worse prognosis. In Latin America there are no cohorts that evaluate the clinical course of these lesions. Method Cases and control. Results A total of 37 patients with a diagnosis of IgAN were evaluated, of these 27% presented podocytopathy (IgAN-P) and 72.9% presented IgAN without histological data in the optical microscopy of podocytopathy (IgAN-NP), with an average follow-up of 41 ± 32 months. Clinically, the proteinuria in IgAN-P patients was higher with a mean of 3.9 ± 3.0 gr/gr vs 1.6 ± 1.5 gr/gr in the group with IgAN-NP, without being statistically significant p = 0.54. Kidney function in the group with podocytopathy was slightly lower with an average eGFR of 65.9 ± 45 ml/min/1.73 m2 vs 80.2 ± 36.4 ml/min/1.73 m2 p = 0.23, associated with a greater presence of granular casts in podocytopathy (92% vs 80% p = 0.02) describing probable associated acute tubular injury. Histologically, patients with podocytopathy presented 80% of podocyte hypertrophy and 2% of tip-type FSGS. The findings in the MEST-C score had no differences between the groups, except for mesangial proliferation that was present in 96.3% of subjects with podocytopathy compared to 70% of subjects without podocytopathy, p = 0.02. The prognosis based on the international score was not statistically significant between the groups p = 0.59. Patients with podocytopathy tended to be treated with immunosuppression prior to the biopsy in 50% vs 37% in the group without podocytopathy p = 0.01, however once the histological diagnosis was obtained it was more common to decide to continue with immunosuppressants in subjects with podocytopathy with 90% vs 63% (p = 0.11). Finally, long-term results reported no differences in the requirement for kidney replacement therapy. When evaluating MAKE outcomes, there were no differences between groups with respect to ESKD; 40% decrease in eGFR and need for kidney replacement therapy. Conclusion In previous reports the presence of podocytopathy reported as podocyte hypertrophy and tip-lesions were 16%. In our population this finding was more frequent (27%), with podocyte hypertrophy being more prevalent. Greater proteinuria and worse kidney function were observed compared to IgAN-NP, justifying a greater frequency of immunosuppressive therapy, impacting the kidney outcome being the same as that reported in patients without podocytopathy. Similar result to that observed in previous cohorts.

Understanding the impact of congenital infections and perinatal viral exposures on the developing brain using white matter magnetic resonance imaging: a scoping review

BackgroundMagnetic Resonance Imaging (MRI)-based imaging techniques are useful for assessing white matter (WM) structural and microstructural integrity in the context of infection and inflammation. The purpose of this scoping review was to assess the range of work on the use of WM neuroimaging approaches to understand the impact of congenital and perinatal viral infections or exposures on the developing brain.MethodsThis scoping review was conducted according to the Arksey and O’ Malley framework. A literature search was performed in Web of Science, Scopus and PubMed for primary research articles published from database conception up to January 2022. Studies evaluating the use of MRI-based WM imaging techniques in congenital and perinatal viral infections or exposures were included. Results were grouped by age and infection.ResultsA total of 826 articles were identified for screening and 28 final articles were included. Congenital and perinatal infections represented in the included studies were cytomegalovirus (CMV) infection (n = 12), human immunodeficiency virus (HIV) infection (n = 11) or exposure (n = 2) or combined (n = 2), and herpes simplex virus (HSV) infection (n = 1). The represented MRI-based WM imaging methods included structural MRI and diffusion-weighted and diffusion tensor MRI (DWI/ DTI). Regions with the most frequently reported diffusion metric group differences included the cerebellar region, corticospinal tract and association fibre WM tracts in both children with HIV infection and children who are HIV-exposed uninfected. In qualitative imaging studies, WM hyperintensities were the most frequently reported brain abnormality in children with CMV infection and children with HSV infection.ConclusionThere was evidence that WM imaging techniques can play a role as diagnostic and evaluation tools assessing the impact of congenital infections and perinatal viral exposures on the developing brain. The high sensitivity for identifying WM hyperintensities suggests structural brain MRI is a useful neurodiagnostic modality in assessing children with congenital CMV infection, while the DTI changes associated with HIV suggest metrics such as fractional anisotropy have the potential to be specific markers of subtle impairment or WM damage in neuroHIV.

#1193 Tracking kidney cancer initiation via somatic mutation analysis of pre-cancer cells from Von Hippel Lindau cancer predisposition syndrome

Abstract Background and Aims Somatic mutations promote the transition from a normal cell to a cancer clone. Little is known about the factors that promote mutation in normal tissues, but whole genome sequencing of pre-cancer cells, followed by mutational pattern analyses, is a revolutionary tool to understand somatic mutation mechanisms active during an individual's lifetime. In the kidney, we have shown that normal kidney tubule epithelial cells (TEC) derived from damaged PT display an excess of somatic mutations and a peculiar enrichment of mutations in exons and highly transcribed DNA compared to other cell types [1]. However, the direct contribution of this PT-specific mutational process to carcinogenesis needs further demonstration. Here, we have analyzed normal TEC that are genetically predisposed to evolve into clear cell renal cell carcinoma (ccRCC), as they harbor a germline heterozygous mutation in the tumor suppressor Von Hippel Lindau, VHL. Using this genetic model of tumor predisposition, we have tracked the PT-specific mutational process from normal cells to overt ccRCC. This study sheds light on the etiology of this mutational process, as well as its causative role in ccRCC. Method We detected somatic mutations in single genomes from normal kidney TEC from 8 control individuals and 7 VHL-disease patients in the age range 24-70. Different from our previous study [1], we isolated kidney tubule cells from the urine. Multiple TECs per individual were subjected to in vitro clonal expansion, followed by whole genome sequencing (WGS), a golden-standard method to obtain high confidence somatic mutation data. We analyzed concomitant somatic mutation- and gene expression-data from 58 clones, and expression data from 154 TEC clones. Results First, we compared our newly generated dataset (TECs obtained from urines) with our previous dataset (TECs obtained from kidney biopsies). QPCR measurements of marker gene expression and somatic mutational pattern analyses showed that the same two populations could be clonally expanded from both urines and kidney biopsies. One population expressed markers of proximal tubule (AQP1, SLC17A3) and tubule damage (VCAM1, KIM1), and showed an excess of somatic single base substitutions (SBSs). This TEC population was defined: VCAM1-PT cells. We characterized the types of SBSs that are more frequently found in VCAM1-PT genomes. The mutation spectrum was different from that induced by exposure to common mutagens, e.g. aristolochic acid, reactive oxygen species and tobacco. Normal cells, including TEC, fat and blood progenitors, showed mutation depletion in highly transcribed genes, in agreement with more efficient repair. Instead, VCAM1-PT cells showed an enrichment of specific SBS types, distinct from those induced by another transcription-coupled mutational process described in the liver. In vitro exposure of TECs to the alkylating agent ENU recapitulates some features of the spectrum of mutations observed in vivo, suggesting that VCAM1-PT cells might fail to repair damage induced by endogenous alkylating agents. The VCAM1-PT-specific mutational signature was observed in ccRCC, but not found in a variety of non-kidney cell-types and cancers. Importantly, the impact of the mutational signature was more evident in VCAM1-PT genomes from VHL-disease patients compared to similar samples from control individuals. Moreover, VCAM1-PT cells were more common (8-fold increase, chi squared test P = .026) in the urines of VHL-disease patients than in the urines of controls. Conclusion Our data show that TECs derived from damaged PTs are subjected to a specific somatic mutation process during adult life. The process is a distinctive feature of kidney cancer (ccRCC) genomes, and is more evident in patients affected by VHL-cancer predisposition syndrome. These data suggest that heterozygous loss of VHL facilitates a PT-specific, somatic mutation process involved in cancer initiation.

#302 Proteomic study reveals indoxyl sulfate induced changes in endothelial cells: role of extracellular vesicles in cardiovascular disease

Abstract Background and Aims Chronic kidney disease (CKD) lead to the accumulation of uremic toxins in the bloodstream, among which indoxyl sulfate (IS) is the most prominent. IS causes damage to endothelial cells lining blood vessels, ultimately leading to cardiovascular diseases in patients with CKD. Under physiological conditions, extracellular vesicles (EVs) are released by cells as intercellular communicators; however, after damage, the cargo of the EVs can be modified. This makes EV potential markers of damage, possibly carrying proteins of interest. This study aimed to investigate the impact of IS on endothelial cells and the release of EVs to identify potential therapeutic targets for preventing endothelial injury. Method Human umbilical vein endothelial cells (HUVEC) were cultured under control conditions and with IS exposure (250 μM IS, 24 h, n = 5). The released EVs were isolated from the supernatants by differential centrifugation and characterized by TEM and flow cytometry to confirm their identity as EVs (following the MISEV2018 guidelines). Proteome profiles of both cells and EVs were analyzed using a mass spectrometer (Orbitrap Exploris™ 240), followed by a Gene Set Enrichment Analysis (GSEA) which revealed altered functions. Results A total of 5871 proteins were identified in the proteomic analysis of cells, and 3614 proteins in EVs (with a False Discovery Rate (FDR) &amp;lt; 1%). In cells, 30 proteins were upregulated and 46 were found downregulated in response to IS, while in EVs, 51 proteins were found upregulated and 47 downregulated (IS vs C). GSEA revealed that IS potentiated adipogenesis (p-value &amp;lt; 0.001), TNF-α signaling via NFкB (p-value = 0.006), and xenobiotic metabolism in cells (p-value = 0.012). In contrast, EVs from cells treated with IS exhibited downregulation of extracellular matrix components (p-value = 0.003), myogenesis (p-value 0.009), genes downregulated in response to ultraviolet radiation (p-value = 0.014) and TNF-α signaling via NFкB (p-value = 0.041). Conclusion IS induces a change in protein expression in cells and their EVs, potentiating inflammation and atherosclerotic plaque formation with extracellular matrix loss. All these modifications contribute to endothelial dysfunction, which is the first step in the development of cardiovascular disease in patients with CKD. This study shed light on novel targets for intervention strategies to mitigate cardiovascular complications in patients with CKD. Funding Grants from the Instituto de Salud Carlos III (ISCIII) and cofounded by Fondos Europeo de Desarrollo Regional (FEDER): “PI19/00240” and “PI20/01321” and “FI20/00018” contract by the Instituto de Salud Carlos III and Ayuda de la Línea de Actuación “Excelencia para el Profesorado Universitario de la UAH” (EPU-INV-UAH/2022/001) from Alcala University.

#648 Relationship between renal tubular damage, interstitial fibrosis and semaphorin class 3C

Abstract Background and Aims The detailed mechanism of interstitial fibrosis in CKD has not been clarified. In this study, we focused on semaphorin class 3C (SEMA3C), a secreted protein expressed in the renal tubules, that is involved in embryonic kidney development [1], and investigated the relationship between SEMA3C and renal tubular damage and interstitial fibrosis. Methods &amp;lt; Clinical research&amp;gt; We investigated the correlation between serum SEMA3C concentration and the renal tubular damage marker urinary β2 microglobulin (β2MG) concentration in the 191 patients who underwent kidney biopsy from 2017 to 2021 at Yamagata University. &amp;lt; In vivo study&amp;gt; We generated proximal tubule-specific SEMA3C knockout mice (PT-SEMA3C KO) from male mice (C57BL/6) using the Cre/loxP system. The PT-SEMA3C KO group and control group were fed a 0.2% adenine diet for 5 weeks to induce CKD. The area of interstitial fibrosis/tubular atrophy (IFTA) region was assessed by Masson-Trichrome staining. Results &amp;lt; Clinical research&amp;gt; The median age of the patients was 54 (IQR 38-68) years, 94 men and 97 women. Median serum SEMA3C concentration was 0.14 (IQR 0.07-0.35) ng/ml. Median urinary β2MG levels was 0.22 (IQR 0-1.79) mg/gCr. Spearman's correlation analysis showed a significant negative correlation between serum SEMA3C and urinary β2MG levels (r=-0.208, P=0.005), but no correlation between eGFR and Urinary protein/ creatinine. &amp;lt; In vivo study&amp;gt; IFTA region was 5.6% in the control group, compared to 11.2% in the PT-SEMA3C KO group (p=0.031). Interstitial fibrosis was significantly suppressed in the PT-SEMA3C KO mice (Fig. 1). Conclusion Our clinical and in vivo results indicate that serum SEMA3C is associated with renal tubular damage and interstitial fibrosis.

#684 Safety and Tolerability of adipose-derived mesenchymal stem cell “ADR-001” in the treatment of immunoglobulin A nephropathy

Abstract Background and Aims Immunoglobulin A (IgA) nephropathy is one of the most frequent glomerular diseases and is sometimes refractory, requiring kidney replacement therapy. Although corticosteroids are mainly used for treatment, infection is a serious adverse event, and evidence for therapeutic efficacy is insufficient; therefore, new therapeutic options are highly desired. Mesenchymal stem cells (MSCs) contribute to the amelioration of inflammation and recovery of organ function. Due to these properties of MSCs, they have been clinically applied to various inflammatory diseases. Therefore, we decided to conduct the world's first investigator-initiated clinical trial to administer MSCs for refractory IgA nephropathy. Method This trial was a phase 1, open-label, multiple-center, dose-escalation study of adult patients with refractory IgA nephropathy resistant to or difficult to treat with existing therapies. This trial was registered and assigned the number, jRCT2043200002 and NCT04342325. ADR-001 was administered intravenously from three to six patients at a dose of 1 × 108 cells once in the first cohort and to six patients twice at two-week intervals in the second cohort (Fig. 1A). When MSCs were administered, heparin was also administered. The observational period continued until 52 weeks. The primary endpoint was the safety up to 6 weeks after ADR-001 administration. Secondary endpoints will be the respective percentages of patients with adverse events and efficacy, such as clinical remission, partial remission, remission of urine protein, remission of hematuria, time to remission, changes in urine protein, hematuria, and estimated glomerular filtration rate. Results First, regarding the primary endpoint, three patients in the first cohort received single dose of ADR-001, and the data safety monitoring board determined that the safety of the ADR-001 was well controlled because no adverse clinical events occurred. Therefore, the trial shifted the second cohort, and six patients received two doses of ADR-001. In the second cohort, the data safety monitoring board determined that safety was well controlled and that ADR-001 was safe and well tolerated for clinical use. Next, regarding the secondary endpoint, especially regarding to efficacy, kidney damage marker (Fig. 1B) and urinary protein were drastically reduced immediately after administration of ADR-001. Conclusion The safety and tolerability of ADR-001, an adipose-derived MSC, were confirmed. Furthermore, the efficacy of ADR-001 is strongly expected.

Markers of enterocyte damage, microbial translocation and systemic inflammation following 9 hours of heat exposure in young and older adults.

Heat stress induced damage to the gastrointestinal barrier can induce local and systemic inflammatory reactions implicated in heat-stroke. Gastrointestinal barrier damage has been shown to be greater in older relative to young adults following hyperthermia. However, comparisons between young and older adults have been limited to brief exposures (3-h), which may not reflect the duration of heat stress experienced during heat waves. We therefore evaluated markers of intestinal epithelial damage (Log transformed intestinal fatty acid binding protein, IFABPLOG), microbial translocation (soluble cluster of differentiation 14, sCD14LOG), and systemic inflammation (tumour necrosis factor alpha, TNF-αLOG; interleukin 6, IL-6LOG; C-reactive protein, CRP) in 19 young (interquartile range: 21-27 yr; 10 females) and 37 older (68-73 yr; 10 females) adults before and after 9-h of rest in 40°C (9% relative humidity). The magnitude of the increase in IFABPLOG was 0.38 log pg/mL (95% CI, 0.10, 0.65 log pg/mL) greater in the older relative to young cohort (P=0.049) after 9-h heat exposure. At baseline both IL-6LOG and CRP concentrations were higher in the older (IL-6: 2.67 (1.5) log pg/mL, CRP: 0.28 (1.5) mg/mL) relative to the young (IL-6: 1.59 log pg/mL, SD 1.2; CRP: 0.11 mg/mL, SD 1.7) group (both P<0.001). The change in IL-6 and CRP was similar between groups following 9-h heat exposure (IL-6: P=0.053; CRP: P=0.24). Neither sCD14LOG and TNF-αLOG were different between groups at baseline nor altered after 9-h heat exposure. Our data indicate that age may modify intestinal epithelial injury following 9 h of passive heat exposure.