Transcriptomic and behavioral analyses reveal unique target tissues and molecular pathways associated with embryonic exposure to low level glyphosate and metal mixtures in zebrafish.

Investigation of developmental molecular events following exposure to environmentally relevant agrochemical mixtures is critical to predicting their potential long-term ecological and human health risks. Here, we sought to uncover transcriptomic changes during zebrafish (Danio rerio) embryonic development following exposure to glyphosate and co-exposure to metals. Glyphosate is widely used globally with an allowable drinking water limit of 700 ppb. We examined effects of glyphosate (10 ppb) alone and when co-exposed to a metal mixture containing low levels of arsenic (4 ppb), lead (5 ppb), cadmium (2 ppb), and vanadium (15 ppb). This mixture was derived based on behavioral and morphological toxicity findings and environmentally relevant concentrations found in agricultural regions where glyphosate and metals are ubiquitously present. Gene expression patterns coupled to a single-cell transcriptomic dataset revealed that developmental exposure (28-72 h post fertilization) to glyphosate dysregulates expression of developmental genes specific to the central nervous system. Subsequent studies indicated significant suppression of larval zebrafish movement with 10 ppb glyphosate exposure. Studies with glyphosate + metals mixture and metals mixture alone showed unique developmental transcriptomic patterns and behavioral changes compared to glyphosate exposure alone. However, some outcomes (e.g., changes in expression of genes involved in epigenetic regulation and extracellular matrix patterning) were common across all three exposures compared to the control. Notably, glyphosate + metals co-exposure distinctly suppresses lysosomal transcripts and targets renal developmental genes. While further studies are required to uncover the precise nature of the interactions between glyphosate and metals, our study shows that glyphosate at very low levels is a behavioral and neurotoxicant that changes when metals are present. Given this herbicide affects distinctive physiological processes, including renal development and lysosomal dysregulation when co-exposed with metals, we conclude that environmental cation levels should be considered in glyphosate toxicity and risk assessment.

Fluoride exposure and pediatric kidney health in dry, wet and intermediate climatic zone communities in Sri Lanka: Implications from urinary Cystatin-C, and albumin-creatinine ratio.

BACKGROUND: High fluoride exposure is increasingly discussed attributing to kidney injury as a causative factor. Depending on geochemistry, differential fluoride levels in drinking water are identified in different regions in Sri Lanka. However, the levels of fluoride exposure, and associations with kidney health has not been adequately studied in Sri Lanka, particularly in pediatric communities. Hence, the present study aimed to assess fluoride exposure in selected pediatric communities in the dry, wet and intermediate climatic zones in Sri Lanka, along with an assessment of renal health using urinary Cystatin-C (uCys-C), and albumin-creatinine ratio (uACR). METHODS: We conducted a cross-sectional study with school students in selected education zones representing dry (N = 331), wet (N = 152), and intermediate (N = 292) climatic zones in Sri Lanka. Fluoride contents in urine and drinking water were assessed as measures of fluoride exposure. RESULTS: The median (interquartile distance) urinary fluoride levels of participants in the dry, wet and intermediate zones were 1.63(1.04-2.85), 1.29(0.85-2.21), and 1.07(0.61-1.98) mg/gCr while the fluoride contents of drinking water samples were 1.76(1.36-2.30), 0.25(0.18-0.37), and 0.43(0.26-0.63) ppm respectively with significant differences among the three groups. Median uCys-C level (ng/mgCr) of the participants in intermediate zone [30.26(8.49-71.44)] was significantly low (p < 0.05) compared to that of the participants in dry zone [56.19(7.08-211.8)], and wet zone [66.29(30.43-125.20)]. The incidences of elevated uCys-C levels above reference intervals in participants of dry zone (47.7%), and wet zone (50.0%) were significantly high (p < 0.001) compared to the intermediate zone (26.4%). CONCLUSION: Relatively high fluoride exposure is likely in dry and wet zone communities compared to the intermediate zone along with significantly higher incidence of uCys-C levels above reference intervals in study groups with higher fluoride exposure. However, to conclude a clear link between fluoride exposure and kidney health we need in-depth studies.

Environmental fluoride exposure and implications on potential pediatric kidney health risks: an approach with urinary biomarkers.

BACKGROUND: Environmental fluoride exposure at elevated levels is potentially linked to kidney injury, and may contribute to chronic kidney disease of uncertain etiology (CKDu) as a risk factor. However, this link remains unclear, and examining the risk of kidney damage from early life fluoride exposure may provide important insights. Hence, this study aimed to investigate associations of fluoride exposure with pediatric kidney health in CKDu impacted and unimpacted communities in Sri Lanka. METHODS: Considering the geographical variations in environmental fluoride, climate, and prevalence of CKDu, four study groups were established within selected education zones in CKDu-endemic dry zone regions (D-En), and CKDu-nonendemic regions within the dry (D-NE), wet (W-NE), and intermediate (I-NE) climatic zones. The study population included 922 school students (11-18 years of age). Participants in each group were divided into four subgroups based on quartiles of respective urinary fluoride (UF) distribution for comparison of urinary kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and albumin-creatinine ratio (ACR). RESULTS: UF levels in participants particularly in CKDu endemic dry zone regions were significantly high compared to the other regions. Significantly high median urinary NGAL (in D-NE) and ACR (in D-EN, and W-NE) levels were observed in subgroups of higher UF quartiles. Albuminuria was not particularly identified in subjects with high UF excretion. Urinary KIM-1 showed no significant variation across the UF quartile subgroups. Linear regression identified weak associations of UF with kidney injury biomarkers. CONCLUSIONS: Fluoride exposure is particularly high in CKDu-endemic dry zone communities. As implied by kidney injury biomarkers, a strong link between fluoride exposure and pediatric kidney health was not evident at the observed exposure levels in the study regions. A higher resolution version of the Graphical abstract is available as Supplementary information.

Bioenergetic Effects of Polycyclic Aromatic Hydrocarbon Resistance Manifest Later in Life in Offspring of Fundulus heteroclitus from the Elizabeth River.

Shifts in key physiological processes can confer resistance to chemical pollutants. However, these adaptations may come with certain trade-offs, such as altered energy metabolic processes, as evident in Atlantic killifish (Fundulus heteroclitus) in Virginia's Elizabeth River (ER) that have evolved resistance to polycyclic aromatic hydrocarbons (PAHs). We seek to understand the bioenergetic costs of PAH resistance among subpopulations of Atlantic killifish with differing contamination levels in order to examine how these changes manifest across multiple life stages and how these costs might be exacerbated by additional stressors. Bioenergetics data revealed differences in metabolic rates between offspring of PAH-resistant fish and reference fish were absent or minimal in both the embryo and larval stages but pronounced at the juvenile life stage, suggesting that bioenergetic changes in pollution-adapted killifish manifest later in life. We also provide evidence that killifish from remediated sites are more sensitive to PAH exposure than killifish from nonremediated sites, suggesting loss of PAH tolerance following relaxed selection. Collectively, our data suggest that the fitness consequences associated with evolved resistance to anthropogenic stressors may manifest differently over time and depend on the magnitude of the selection pressure. This information can be valuable in effective risk and remediation assessments as well as in broadening our understanding of species responses to environmental change.

Mitochondrial dysfunction and oxidative stress contribute to cross-generational toxicity of benzo(a)pyrene in Danio rerio.

The potential for polycyclic aromatic hydrocarbons (PAHs) to have adverse effects that persist across generations is an emerging concern for human and wildlife health. This study evaluated the role of mitochondria, which are maternally inherited, in the cross-generational toxicity of benzo(a)pyrene (BaP), a model PAH and known mitochondrial toxicant. Mature female zebrafish (F0) were fed diets containing 0, 12.5, 125, or 1250 µg BaP/g at a feed rate of 1% body weight twice/day for 21 days. These females were bred with unexposed males, and the embryos (F1) were collected for subsequent analyses. Maternally-exposed embryos exhibited altered mitochondrial function and metabolic partitioning (i.e. the portion of respiration attributable to different cellular processes), as evidenced by in vivo oxygen consumption rates (OCRs). F1 embryos had lower basal and mitochondrial respiration and ATP turnover-mediated OCR, and increased proton leak and reserve capacity. Reductions in mitochondrial DNA (mtDNA) copy number, increases in mtDNA damage, and alterations in biomarkers of oxidative stress were also found in maternally-exposed embryos. Notably, the mitochondrial effects in offspring occurred largely in the absence of effects in maternal ovaries, suggesting that PAH-induced mitochondrial dysfunction may manifest in subsequent generations. Maternally-exposed larvae also displayed swimming hypoactivity. The lowest observed effect level (LOEL) for maternal BaP exposure causing mitochondrial effects in offspring was 12.5 µg BaP/g diet (nominally equivalent to 250 ng BaP/g fish). It was concluded that maternal BaP exposure can cause significant mitochondrial impairments in offspring.

Prevalence of Chronic Kidney Disease of Uncertain Etiology Within Selected Farming Communities in Rural Sri Lanka.

Background: Chronic kidney disease of uncertain etiology (CKDu) is an emergent health concern, particularly in tropical farming communities in several global hotspots, including Sri Lanka. This particular nephropathy is characterized by a progressive decline in kidney function in the absence of conventional chronic kidney disease (CKD) risk factors such as diabetes mellitus, hypertension, and other identifiable kidney disorders. As it is almost asymptomatic at early stages, CKDu is mostly diagnosed at late stages with significant kidney damage. Hence, the identification of disease susceptibility and vulnerable communities at the earliest possible instances is highly important for the management of the disease. Objective: We aimed to compare kidney health across three different farming communities in Sri Lanka to identify CKDu susceptibilities. Methods: A cross-sectional study was performed with three selected farming communities: paddy farmers (PF; N = 581), sugarcane farmers (SF; N = 550), and vegetable farmers (VF; N = 244) in comparison with an age-matched control group of nonfarming (NF; N = 225) individuals. A medical examination was performed to investigate health status and medical history, whereas a urinalysis was performed to determine creatinine and albumin contents. Estimated glomerular filtration rate (eGFR) and urinary albumin-creatinine ratio (ACR) were used for assessment of kidney function. CKDu susceptibility was determined based on eGFR, and urinary ACR adhering to the clinical practice guidelines in Sri Lanka. Results: The median (interquartile range [IQR]) eGFR levels of PF (85 mL/min/1.73 m2 [72-97]) and SF (93 mL/min/1.73 m2 [73-112]) were significantly lower than that of the NF group (103 mL/min/1.73 m2 [87-125]) (P < .0001), whereas eGFR of VF (100 mL/min/1.73 m2 [80-111]) was not significantly different compared with NF. The median (IQR) urinary ACR levels of the study groups, PF, SF, VF, and NF, were 0.59 (0.26-1.45), 0.46 (0.28-0.88), 0.45 (0.34-0.90), and 0.44 (0.34-1.02) mg/mmol, respectively. However, urinary ACR did not differ significantly across the study groups (P > .05). The prevalence of CKDu within PF (13.60%), SF (12.54%), and VF (6.67%) communities was significantly higher (P < .05) compared with the NF (2.67%). Of the total CKD cases, CKDu susceptible cases represented 73%, 69%, 50%, and 25% in PF, SF, VF, and NF, respectively, indicating a high risk of CKDu susceptibility among farming communities. Moreover, a noteworthy association of CKDu was observed with agrochemical exposure (odds ratio [OR] = 3.11, 95% confidence interval [CI] = 1.36-7.09). Concerning the farming practices, sugarcane farming showed the highest association with CKDu prevalence (OR = 3.40, 95% CI = 1.49-7.78). Conclusions: Compared with the nonfarming group, a significant risk of CKDu was observed in the three farming communities, particularly among paddy and sugarcane farmers. Longitudinal epidemiological studies to identify vulnerable farming communities and associated risk factors are critically needed to develop effective management strategies against CKDu within farming communities.

Contexte: L'insuffisance rénale chronique d'étiologie incertaine (IRCi) est un problème de santé émergent, en particulier dans les collectivités agricoles de plusieurs régions tropicales dans le monde, dont le Sri Lanka. Cette néphropathie particulière se caractérise par un déclin progressif de la fonction rénale en absence de facteurs de risque classiques de l'IRC comme le diabète, l'hypertension et d'autres troubles rénaux identifiables. L'IRCi étant quasi asymptomatique dans les stades précoces, elle est souvent diagnostiquée à des stades avancés et en présence de lésions rénales importantes. Il serait donc très pertinent d'identifier les populations vulnérables et d'évaluer le plus tôt possible leur prédisposition à l'IRCi, afin de faciliter la prise en charge de la maladie. Objectif: Notre objectif était de comparer la santé rénale de trois différentes populations agricoles du Sri Lanka afin d'évaluer leur prédisposition à l'IRCi. Méthodologie: Une étude transversale a été réalisée auprès de trois populations agricoles, soit les cultivateurs de riz (CR; n=581), de canne à sucre (CCS; n=550) et de légumes (CL; n=244). Les groupes étudiés ont été comparés à un groupe témoin d'individus non-agriculteurs (NA; n=225) d'âge similaire. Les sujets se sont soumis à un examen de leur état de santé et leurs antécédents médicaux, ainsi qu'à une analyse d'urine pour déterminer leurs taux urinaires de créatinine et d'albumine. Le débit de filtration glomérulaire estimé (DFGe) et le rapport albumine-créatinine urinaire (RAC) ont servi à évaluer la fonction rénale. La prédisposition à l'IRCi a été établie sur la base du DFGe et du RAC urinaire, conformément aux recommandations de pratique clinique au Sri Lanka. Résultats: Le DFGe médian (ÉIQ) des CR (85 ml/min/1,73 m2 [72-97]) et des CCS (93 ml/min/1,73 m2 [73-112]) était significativement inférieur à celui du groupe témoin (103 ml/min/1,73 m2 [87-125]) (p < 0.0001); aucune différence significative n'a été observée pour le groupe des CL (100 ml/min/1,73 m2 [80-111]). Le RAC urinaire médian (ÉIQ) s'établissait à 0,59 mg/mmol (0,26-1,45) pour les CR, à 0,46 mg/mmol (0,28-0,88) pour les CCS, à 0,45 mg/mmol (0,34-0,90) pour les CL et à 0,44 mg/mmol (0,34-1,02) pour le groupe témoin; soit aucune différence significative pour le RAC urinaire entre les groupes à l'étude (p > 0.05). La prévalence de l'IRCi dans les populations de CR (13,60 %), de CCS (12,54 %) et de CL (6,67 %) était significativement plus élevée (p < 0.05) que dans le groupe témoin (2,67 %). Sur le total des cas d'IRC, ceux qui montraient une prédisposition à l'IRCi représentaient 73 % (CR), 69 % (CCS), 50 % (CL) et 25 % (NA) des cas d'IRC de chaque groupe, ce qui indique un risque élevé de prédisposition à l'IRCi dans les communautés agricoles. On a en outre observé une association notable entre l'IRCi et l'exposition aux agents agrochimiques (RC : 3,11 [IC 95 % : 1,36-7,09]). En ce qui concerne les différentes pratiques agricoles, la culture de la canne à sucre a montré la plus forte association avec la prévalence d'IRCi (RC : 3,40 [IC 95 % : 1,49-7,78]). Conclusion: Un risque significativement plus élevé d'IRCi a été observé dans les trois communautés agricoles par rapport au groupe des non-agriculteurs, en particulier chez les cultivateurs de riz et de canne à sucre. Il est essentiel de procéder à des études épidémiologiques longitudinales visant à identifier les communautés agricoles vulnérables et les facteurs de risque associés, afin d'élaborer des stratégies efficaces pour prévenir l'IRCi au sein de ces communautés.

River zebrafish combine behavioral plasticity and generalized morphology with specialized sensory and metabolic physiology to survive in a challenging environment.

Phenotypes that allow animals to detect, weather, and predict changes efficiently are essential for survival in fluctuating environments. Some phenotypes may remain specialized to suit an environment perfectly, while others become more plastic or generalized, shifting flexibly to match current context or adopting a form that can utilize a wide range of contexts. Here, we tested the differences in behavior, morphology, sensory and metabolic physiology between wild zebrafish (Danio rerio) in highly variable fast-flowing rivers and still-water sites. We found that river zebrafish moved at higher velocities than did still-water fish, had lower oxygen demands, and responded less vigorously to small changes in flow rate, as we might expect for fish that are well-suited to high-flow environments. River zebrafish also had less streamlined bodies and were more behaviorally plastic than were still-water zebrafish, both features that may make them better-suited to a transitional lifestyle. Our results suggest that zebrafish use distinct sensory mechanisms and metabolic physiology to reduce energetic costs of living in fast-flowing water while relying on morphology and behavior to create flexible solutions to a challenging habitat. Insights on animals' reliance on traits with different outcomes provide a framework to better understand their survival in future environmental fluctuations.

Environmental heat exposure and implications on renal health of pediatric communities in the dry climatic zone of Sri Lanka: An approach with urinary biomarkers.

Prolonged heat exposure during outdoor physical exertion can result in adverse renal health outcomes, and it is also supposed to be a driver of chronic kidney disease of uncertain etiology (CKDu) in tropical regions. School students are more likely to experience high heat exposure during outdoor sports practices, and the current knowledge on potential renal health outcomes associated with heat exposure carries many knowledge gaps. Hence, the present study aimed to perform biomarker-based assessment of the likelihood of pediatric renal injury focusing the communities in the dry climatic zone in Sri Lanka, where it prevails relatively harsh climate and high prevalence of CKDu. School students who engaged in regular outdoor sports practices (high-heat exposure), and an age-matched control of students who did not engage in sports practices (low-heat exposure) from four educational zones: Padavi Sripura (N = 159) and Medirigiriya (N = 171), Uhana (N = 165) and Thanamalwila (N = 169) participated in this cross-sectional study representing CKDu endemic and non-endemic regions. Effective temperature (ET), wet-bulb globe temperature (WBGT), heat index (HI) and humidex were used for comparison of thermal comfort in the environment. The intensity of environmental heat measured by thermal comfort indices showed no significant difference (p > 0.05) among the study regions. Urinary kidney injury molecule (KIM-1) and albumin-creatinine ratio (ACR) in participants with high heat exposure did not differ significantly from those in the control groups in the four study zones, where urinary neutrophil gelatinase-associated lipocalin showed substantial differences in some groups. Irrespective of heat exposure, increased KIM-1 excretion was observed (p < 0.01) in participants of CKDu endemic regions compared to those in non-endemic areas. Within the context of our findings, there is no plausibly strong evidence to establish potential association of heat exposure with the likelihood of developing renal injury or abnormal renal outcomes in dry zone school students in Sri Lanka.

The urinary proteome infers dysregulation of mitochondrial, lysosomal, and protein reabsorption processes in chronic kidney disease of unknown etiology (CKDu).

Chronic kidney disease (CKD) of uncertain etiology (CKDu) is a global health concern affecting tropical farming communities. CKDu is not associated with typical risk factors (e.g., diabetes) and strongly correlates with environmental drivers. To gain potential insights into disease etiology and diagnosis, here we report the first urinary proteome comparing patients with CKDu and non-CKDu controls from Sri Lanka. We found 944 differentially abundant proteins. In silico analyses identified 636 proteins of likely kidney and urogenital origin. As expected, renal tubular injury in patients with CKDu was evinced by increases in albumin, cystatin C, and ß2-microglobulin. However, several proteins typically elevated under CKD, including osteopontin and α-N-acetylglucosaminidase, were decreased in patients with CKDu. Furthermore, urinary excretion of aquaporins found higher in CKD was lower in CKDu. Comparisons with previous CKD urinary proteome datasets revealed a unique proteome for CKDu. Notably, the CKDu urinary proteome was relatively similar to that of patients with mitochondrial diseases. Furthermore, we report a decrease in endocytic receptor proteins responsible for protein reabsorption (megalin and cubilin) that correlated with an increase in abundance of 15 of their cognate ligands. Functional pathway analyses identified kidney-specific differentially abundant proteins in patients with CKDu denoted significant changes in the complement cascade and coagulation systems, cell death, lysosomal function, and metabolic pathways. Overall, our findings provide potential early detection markers to diagnose and distinguish CKDu and warrant further analyses on the role of lysosomal, mitochondrial, and protein reabsorption processes and their link to the complement system and lipid metabolism in CKDu onset and progression.NEW & NOTEWORTHY CKDu is a global health concern debilitating a number of tropical rural farming communities. In the absence of typical risk factors like diabetes and hypertension and the lack of molecular markers, it is crucial to identify potential early disease markers. Here, we detail the first urinary proteome profile to distinguish CKDu from CKD. Our data and in silico pathway analyses infer the roles of mitochondrial, lysosomal, and protein reabsorption processes in disease onset and progression.

Exploring the Utility of Urinary Creatinine Adjustment for KIM-1, NGAL, and Cystatin C for the Assessment of Kidney Function: Insights from the C-KidnEES Cohort.

Normalization of urinary biomarkers of kidney injury is a common practice in clinical and research settings to account for variations in urine concentration, and urinary creatinine is often used as a reference. However, to date, there is no consensus on the adjustment of urinary biomarkers with creatinine, and both absolute and creatinine-adjusted biomarker levels are adopted for making interpretations of kidney health. Hence, the present study aimed to investigate the associations of urinary creatinine with three widely used kidney injury biomarkers, KIM-1, NGAL, and cystatin C, to validate the applicability of urinary creatinine as a reference for normalization. A cross-sectional study was performed with 2100 students, 10-18 years of age in the Children's Kidney Environmental Exposure Study (C-KidnEES) cohort established in Sri Lanka. As identified in linear regression analyses, normalization of urinary KIM-1, NGAL, and Cys-C to urinary creatinine did not result in significant under-adjustment or over-adjustment to the absolute urinary concentrations, giving no specific rationale for creatinine adjustment. Hence, absolute urinary concentrations of the above biomarkers can be adopted for the characterization of subclinical kidney injury in adolescents in community studies where early morning urine sampling is practiced. However, for spot urine samples, consideration of both absolute and creatinine-adjusted biomarker levels would be a better approach.

Thermal physiology integrated species distribution model predicts profound habitat fragmentation for estuarine fish with ocean warming.

Species distribution models predict a poleward migration for marine ectotherms with ocean warming. However, a key limitation in current species distribution models (SDM) is that they do not account for population-specific heterogeneity in physiological responses to temperature change resulting from local adaptations and acclimatization. To address this gap, we developed a novel, Physiology Integrated BioClimate Model (PIBCM) that combines habitat-specific metabolic thermal physiological tolerance of a species into a bioclimate envelope model. Using a downscaling approach, we also established a fine-resolution coastal sea-surface temperature data set for 2050-2080, that showed a high degree of location-specific variability in future thermal regimes. Combining predicted temperature data with the PIBCM model, we estimated habitat distribution for a highly eurythermal intertidal minnow, the Atlantic killifish (Fundulus heteroclitus), a species that likely presents a best-case-scenario for coastal vertebrates. We show that the killifish northern boundary shifts southwards, while distinct habitat fragmentation occurs in the southern sub-population (due to migration of adjacent fish populations to the nearest metabolically optimal thermal habitat). When compared to current SDMs (e.g., AquaMaps), our results emphasize the need for thermal physiology integrated range shift models and indicate that habitat fragmentation for coastal fishes may reshape nursery habitats for many commercially and ecologically important species.

Nanoplastics in Aquatic Environments: Impacts on Aquatic Species and Interactions with Environmental Factors and Pollutants.

Plastic production began in the early 1900s and it has transformed our way of life. Despite the many advantages of plastics, a massive amount of plastic waste is generated each year, threatening the environment and human health. Because of their pervasiveness and potential for health consequences, small plastic residues produced by the breakdown of larger particles have recently received considerable attention. Plastic particles at the nanometer scale (nanoplastics) are more easily absorbed, ingested, or inhaled and translocated to other tissues and organs than larger particles. Nanoplastics can also be transferred through the food web and between generations, have an influence on cellular function and physiology, and increase infections and disease susceptibility. This review will focus on current research on the toxicity of nanoplastics to aquatic species, taking into account their interactive effects with complex environmental mixtures and multiple stressors. It intends to summarize the cellular and molecular effects of nanoplastics on aquatic species; discuss the carrier effect of nanoplastics in the presence of single or complex environmental pollutants, pathogens, and weathering/aging processes; and include environmental stressors, such as temperature, salinity, pH, organic matter, and food availability, as factors influencing nanoplastic toxicity. Microplastics studies were also included in the discussion when the data with NPs were limited. Finally, this review will address knowledge gaps and critical questions in plastics' ecotoxicity to contribute to future research in the field.

Urinary biomarkers indicate pediatric renal injury among rural farming communities in Sri Lanka.

Pediatric renal injury is an emerging health concern in communities affected by chronic kidney disease of uncertain etiology (CKDu). Early detection of susceptibilities through highly sensitive and specific biomarkers can lead to effective therapeutic and preventive interventions against renal diseases. Here, we aimed to investigate the utility of kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in early detection of renal abnormalities in selected pediatric communities in Sri Lanka. The study areas were stratified as CKDu endemic, emerging, and non-endemic based on the prevalence of CKDu, and a total of 804 school students (10-18 years of age) participated in the study. The median (IQR) urinary KIM-1 levels of the participants were 0.193 (0.026-0.338), 0.082 (0.001-0.220) and 0.040 (0.003-0.242) ng/mgCr for CKDu endemic, emerging and non-endemic regions respectively. Participants from CKDu endemic regions reported elevated (p < 0.0001) urinary KIM-1 expression compared to those from the other regions. The median (IQR) NGAL levels in participants from CKDu endemic (2.969; 1.833-5.641), emerging (3.374; 1.766-6.103), and non-endemic (3.345; 1.742-5.128 ng/mgCr) regions showed no significant difference. Also, urinary albumin-creatinine ratio (UACR) showed no significant differences across gender or residency. The prevalence of albuminuria was 1-2% in the locations irrespective of CKDu burden. Albuminuric participants reported higher (p < 0.05) urinary KIM-1 levels in comparison to normoalbuminuric participants. Significantly elevated urinary KIM-1 expression in a pediatric population from CKDu affected regions, especially in the presence of albuminuria, may indicate low-grade early renal damage supporting the utility of KIM-1 as a quantifiable biomarker.

Applicability of Novel Urinary Biomarkers for the Assessment of Renal Injury in Selected Occupational Groups in Sri Lanka: A Comparative Study with Conventional Markers.

Screening approaches with more robust biomarkers, are of the utmost importance in the characterization of renal injuries, particularly among communities with high burdens of chronic kidney disease of uncertain etiology (CKDu). The present study aimed to assess the utility of two emerging biomarkers: kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting renal injury in different occupational groups in Sri Lanka. A cross-sectional study was conducted with six occupational groups (n = 188): fisherfolk (FF), paddy farmers (PF), sugarcane farmers (SF), factory workers (FW) and plantation workers (PW) to assess the predictive performance of KIM-1 and NGAL against a CKDu patient (PT) group (n = 40). The median KIM-1 levels of the study groups; FF, PF, SF, FW, PW and PT were 0.67, 0.59, 0.49, 1.62, 0.67 and 5.24 ng/mgCr, respectively, while the median NGAL levels were 1.16, 2.52, 1.42, 1.71, 1.06 and 22.41 ng/mgCr respectively. In ROC analysis to predict CKDu susceptibility, the area under the curve for KIM-1 ranged from 0.88 to 0.99 for the study groups, and in overall analysis, the sensitivity and specificity were 100% and 96%, respectively, for a cutoff value of 2.76 ng/mgCr. Similarly, for NGAL the range of AUC was 0.78-0.94, and a cutoff value of 3.12 ng/mgCr produced 88% sensitivity and 82% specificity. Compared with conventional markers, KIM-1 was the best biomarker for the characterization of renal injury in the participants of the occupational groups. With further validations, KIM-1 may be adopted as a prognostic marker to identify early renal injury and CKDu susceptibilities in community screening.

A review of molecular mechanisms linked to potential renal injury agents in tropical rural farming communities.

The chronic kidney disease of unknown etiology (CKDu) is a global health concern primarily impacting tropical farming communities. Although the precise etiology is debated, CKDu is associated with environmental exposures including heat stress and chemical contaminants such as fluoride, heavy metals, and herbicide glyphosate. However, a comprehensive synthesis is lacking on molecular networks underpinning renal damage induced by these factors. Addressing this gap, here we present key molecular events associated with heat and chemical exposures. We identified that caspase activation and lipid peroxidation are common endpoints of glyphosate exposure, while vasopressin and polyol pathways are associated with heat stress and dehydration. Heavy metal exposure is shown to induce lipid peroxidation and endoplasmic reticulum stress from ROS activated MAPK, NFĸB, and caspase. Collectively, we identify that environmental exposure induced increased cellular oxidative stress as a common mechanism mediating renal cell inflammation, apoptosis, and necrosis, likely contributing to CKDu initiation and progression.

Urinary cystatin C: pediatric reference intervals and comparative assessment as a biomarker of renal injury among children in the regions with high burden of CKDu in Sri Lanka.

BACKGROUND: Cystatin C (Cys-C) is an emerging biomarker of renal diseases and its clinical use, particularly for screening the communities affected by chronic kidney disease of unknown etiology (CKDu), is hindered due to the lack of reference intervals (RIs) for diverse ethnic and age groups. The present study aimed to define RIs for urinary Cys-C (uCys-C) for a healthy pediatric population in Sri Lanka and in turn compare the renal function of the residential children in CKDu endemic and non-endemic regions in Sri Lanka. METHODS: A cross-sectional study was conducted with 850 healthy children (10-17 years) from selected locations for reference interval establishment, while a total of 892 children were recruited for the comparative study. Urine samples were collected and analyzed for Cys-C, creatinine (Cr) and albumin. Cr-adjusted uCys-C levels were partitioned by age, and RIs were determined with quantile regression (2.5th, 50th and 97.5th quantiles) at 90% confidence interval. RESULTS: The range of median RIs for uCys-C in healthy children was 45.94-64.44 ng/mg Cr for boys and 53.58-69.97 ng/mg Cr for girls. The median (interquartile range) uCys-C levels of children in the CKDu endemic and non-endemic regions were 58.18 (21.8-141.9) and 58.31 (23.9-155.3) ng/mg Cr with no significant difference (P = 0.781). A significant variation of uCys-C was noted in the children across age. CONCLUSIONS: Notably high uCys-C levels were observed in children with elevated proteinuria. Thus, uCys-C could be a potential biomarker in identifying communities at high risk of CKDu susceptibility.

Integrated physiological, transcriptome and metabolome analyses of the hepatopancreas of the female swimming crab Portunus trituberculatus under ammonia exposure.

Ammonia is a common environmental pollutant in aquatic ecosystem and is also a significant concern in closed aquaculture systems. The threat of ammonia has been increasing with rising anthropogenic activities including intensified aquaculture. In this study, we aimed to investigate ammonia toxicity and metabolism mechanisms in the hepatopancreas, a major organ for Vitellogenin (Vtg) synthesis and defending against ammonia stress, of female swimming crab Portunus trituberculatus which is an important fishery and aquaculture species, by integrating physiological, transcriptome and metabolome analyses. The results revealed that ammonia exposure (10 mg/L, an environmentally relevant concentration) resulted in a remarkable reduction in vtg expression and depression of multiple signaling pathways for reproductive regulators including methyl farnesoate, ecdysone and neuroparsin, demonstrating for the first time that ammonia impairs swimming crab female reproduction. In addition, a number of important genes and metabolites in glycolysis, the Krebs cycle, fatty acid ß-oxidation and synthesis were significantly downregulated, indicating that changes in ammonia levels lead to a general depression of energy metabolism in hepatopancreas. After ammonia exposure, an increased level of urea and a reduction of amino acid catabolism were observed in hepatopancreas, suggesting that urea cycle was utilized to biotransform ammonia, and amino acid catabolism was decreased to reduce endogenous ammonia generation. Furthermore, antioxidant systems were altered following ammonia exposure, which was accompanied by proteins and lipid oxidations, as well as cellular apoptosis. These results indicate that ammonia leads to metabolic suppression, oxidative stress and apoptosis in P. trituberculatus hepatopancreas. The findings improve the understanding for the mechanisms of ammonia toxicity and metabolism in P. trituberculatus, and provide valuable information for assessing potential ecological risk of environmental ammonia and improving aquaculture management.

Glucocorticoid-Responsive Transcription Factor Krüppel-Like Factor 9 Regulates fkbp5 and Metabolism.

Krüppel-like factor 9 (Klf9) is a feedforward regulator of glucocorticoid receptor (GR) signaling. Here we show that in zebrafish klf9 is expressed with GR-dependent oscillatory dynamics in synchrony with fkbp5, a GR target that encodes a negative feedback regulator of GR signaling. We found that fkbp5 transcript levels are elevated in klf9 -/- mutants and that Klf9 associates with chromatin at the fkbp5 promoter, which becomes hyperacetylated in klf9 -/ - mutants, suggesting that the GR regulates fkbp5 via an incoherent feedforward loop with klf9. As both the GR and Fkbp5 are known to regulate metabolism, we asked how loss of Klf9 affects metabolic rate and gene expression. We found that klf9 -/- mutants have a decreased oxygen consumption rate (OCR) and upregulate glycolytic genes, the promoter regions of which are enriched for potential Klf9 binding motifs. Our results suggest that Klf9 functions downstream of the GR to regulate cellular glucocorticoid responsivity and metabolic homeostasis.

Urinary Biomarkers of Renal Injury KIM-1 and NGAL: Reference Intervals for Healthy Pediatric Population in Sri Lanka.

Emerging renal biomarkers (e.g., kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)) are thought to be highly sensitive in diagnosing renal injury. However, global data on reference intervals for emerging biomarkers in younger populations are lacking. Here, we aimed to determine reference intervals for KIM-1 and NGAL across a pediatric population in Sri Lanka; a country significantly impacted by the emergence of chronic kidney disease of unexplained etiology (CKDu). Urine samples were collected from children (10-18 years) with no prior record of renal diseases from the dry climatic zone of Sri Lanka (N = 909). Urinary KIM-1 and NGAL concentrations were determined using the enzyme-linked immunosorbent assay (ELISA) and adjusted to urinary creatinine. Biomarker levels were stratified by age and gender, and reference intervals derived with quantile regression (2.5th, 50th, and 97.5th quantiles) were expressed at 95% CI. The range of median reference intervals for urinary KIM-1 and NGAL in children were 0.081-0.426 ng/mg Cr, 2.966-4.850 ng/mg Cr for males, and 0.0780-0.5076 ng/mg Cr, 2.0850-3.4960 ng/mg Cr for females, respectively. Renal biomarkers showed weak correlations with age, gender, ACR, and BMI. Our findings provide reference intervals to facilitate screening to detect early renal damage, especially in rural communities that are impacted by CKDu.