An Examination of Potential Renal Toxicity from Low Dose Glyphosate Exposure in Human Subjects

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Roberts, Nancy D. Denslow, Danielle S. Nelson, Juan G. Perez-Jimenez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7539235/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Widespread usage of glyphosate has resulted in contamination of water, air, and food products. The objective of this study was to examine the prevalence and magnitude of environmental exposure to glyphosate among subjects from the general population, as well as assess the potential risk of renal toxicity associated with these exposures. Methods Concentrations of glyphosate and its environmental breakdown product aminomethylphosphonic acid (AMPA) were measured in the urine of 168 random, de-identified subjects using liquid chromatograph-tandem mass spectrometry (LC-MS/MS) to determine the frequency of detection. Glyphosate doses producing the observed glyphosate in urine were estimated for comparison with regulatory safe exposure limits. Forty-eight of these samples were further screened for biomarkers of renal injury, with urinary extracellular vesicles prepared from a subset of 12 of these samples for miRNA analysis. Results Glyphosate was detected in the urine of 40% of all subjects with a maximum concentration of 25.3 µg/L, and AMPA was detected in 6.5% of all subjects with a maximum concentration of 0.86 µg/L. Estimated glyphosate doses corresponding to urinary levels were 1–2 orders of magnitude below U.S. and European exposure limits. There was no relationship between glyphosate or glyphosate plus AMPA concentrations in urine and any of 21 urinary biomarkers of renal injury, and no concentration related increase in urinary extracellular vesicular miRNAs previously associated with a high acute glyphosate exposure in humans. Conclusions Exposure to glyphosate, as reflected in urinary glyphosate levels, is common among members of the general public in the United States. Observed levels were below established exposure limits, and our data did not document adverse effects of glyphosate on the kidneys at doses at which the general population is exposed. However, more work is needed in subjects with known chronic glyphosate exposure before potential effects from prevalent low doses can be ruled out. Health sciences/Biomarkers Earth and environmental sciences/Environmental sciences Health sciences/Nephrology Health sciences/Risk factors Glyphosate Roundup aminomethylphosphonic acid (AMPA) urinary excretion human health risk renal biomarkers Figures Figure 1 Figure 2 Figure 3 BACKGROUND Glyphosate (N-[phosphonomethyl]-glycine; tradename Roundup®) is the most widely used herbicide in the world. It is applied before planting traditional crops, after planting genetically modified (GMO) glyphosate-resistant crops (60–80% of all GMO crops), and as a “harvest aid” on traditional grain crops (Van Bruggen et al., 2018 ). The widespread adoption of GMO herbicide-resistant crops since 1996 has resulted in a 15-fold increase in glyphosate use globally, and in 2014 enough glyphosate was sprayed to apply 0.53 kg on every hectare under cultivation worldwide (Benbrook, 2016 ). During that year, 113 million kg of glyphosate was used in agriculture in the United States (Benbrook, 2016 ). Not unexpectedly, extensive use of glyphosate has resulted in increased glyphosate resistance among weeds (Powles and Preston, 2006 ) leading to even greater application of glyphosate, often combined with other herbicides. With this widespread usage, glyphosate has been detected with increasing frequency in water, air, and food products (Munoz et al., 2023 ). As a result, interest in the potential risks from glyphosate exposure, once primarily focused on agricultural workers, is now also focused on the general population. A key component of the assessment of risk from glyphosate, as with any chemical, is the level of exposure. Attempts have been made to estimate glyphosate exposure among the general population through modeling as well as more direct estimation through biomonitoring (Gillezeau et al., 2019 ). Based on observations that absorbed glyphosate is eliminated almost completely in urine, measuring urinary glyphosate is an effective approach to estimate exposure levels (Niemann et al., 2015 ). Several studies have reported finding glyphosate in the urine of large percentages of subjects from the general population (see (Connolly et al., 2020 ; Gillezeau et al., 2019 ) for reviews). Among the largest studies, urine collected from individuals ages 6 and above during the 2013–2014 U.S. National Health and Nutrition Examination Survey (NHANES) had detectable glyphosate in 81.2% of samples (N = 2,130) (Ospina et al., 2022 ). Results from the 2014–2017 German Environmental Survey of children and adolescents ages 3–17 found that 52% had glyphosate in the urine above the level of quantitation (N = 2,144) (Lemke et al., 2021 ). Although the percentages of positive urines for glyphosate vary among studies due to differences in populations examined, analytical detection limits, and perhaps other factors, it is clear that glyphosate exposure is widespread in the general population. This raises the question of the toxicological significance of these exposures. Glyphosate effects on the kidney are of particular interest from a human health perspective. Glyphosate exposure has been proposed as a contributor to Chronic Kidney Disease of Unknown Origin (Gunatilake et al., 2019 ) and transcriptomic changes consistent with renal damage were observed in rats given very low doses of glyphosate chronically (Mesnage et al., 2015b ). In the study presented here, we initially obtained 120 random, deidentified urine samples from the UFHealth hospital clinical laboratory which were analyzed for glyphosate and its environmental decomposition product aminomethylphosphonic acid (AMPA) using liquid chromatography-mass spectrometry (LC-MS/MS). Forty-eight random, deidentified urine samples were subsequently obtained from among samples collected as part of routine clinical care at a UFHealth outpatient clinic, to assess whether a relationship existed between urinary glyphosate concentrations and 21 urinary biomarkers of renal injury. To evaluate the possibility of transcriptomic changes as precursors to toxicity in kidney tissue non-invasively, urinary extracellular vesicles were prepared from a subset of 12 of these 48 samples with and without elevated glyphosate concentrations and target miRNAs were compared. METHODS Subjects One-hundred-twenty de-identified urine samples were randomly selected from among routine urine samples submitted for hospitalized patients to the UFHealth hospital clinical pathology laboratory in Gainesville, FL. While the majority of hospital patients at UFHealth come from the city of Gainesville, the hospital is a tertiary referral center and does admit patients from surrounding rural areas in North Central Florida. In a second, follow-up study, focusing on renal biomarkers and miRNA analysis, random de-identified urine samples were obtained from among routine samples submitted by outpatients to the laboratory at a UFHealth family medicine/primary care clinic in the city of Gainesville. All methods were carried out in accordance with relevant guidelines and regulations. The study protocol was reviewed and approved as “exempt” by University of Florida IRB (protocol IRB201800432), contingent on the requirement that all samples be fully deidentified, that there be no contact with patients and that no personal health information (other than age and gender) be made available to investigators. Analysis of glyphosate and aminomethylphosphonic acid : Urine samples were stored frozen at -20°C until analysis. Analysis of glyphosate and AMPA in urine was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described by Jensen and coworkers (Jensen et al., 2016 ). Urine was diluted 1:1 with 0.1% formic acid and injected directly onto a Bio-Rad Cation-H guard column using a Shimadzu Prominence UHPLC-30AD coupled to the QTRAP 6500 (AB SCIEX, Framingham, MA). An isotopically labeled internal standard ( 13 C 2 , 15 N-glyphosate) was added to the samples and used for quantitation. The mobile phases were 0.1% formic acid in water for A and acetonitrile for B. The first minute of the chromatography separation was diverted to waste. Glyphosate and AMPA were analyzed in negative mode via multiple reaction monitoring (MRM) using the following transitions: glyphosate m/z 168/63 and 168/79 and AMPA m/z 110/63 and 110/79 for primary and secondary transitions, respectively. The primary transition was used for quantitation and the secondary transition for qualification. The urine samples were run in four batches, and the assay performance was evaluated for each run. The limits of detection for glyphosate ranged from 0.05–0.10 µg/L and from 0.1–0.26 µg/L for AMPA. Limits of quantitation were 0.1–0.36 µg/L for glyphosate and 0.5–0.86 µg/L for AMPA. Creatinine and osmolality measurements Urine creatinine concentrations were measured by the UFHealth Pathology Laboratories using a kinetic modification of the Jaffe procedure as developed by Cook (Cook, 1971 ). Creatinine concentration was determined by the rate of change in absorbance at 520/800 nm using a Beckman Coulter AU680 Chemistry Analyzer. Osmolality was measured using an osmometer (Advanced Instruments, model 3320). Protein biomarkers of renal injury Twenty-one biomarkers of renal injury were measured via the use of two Luminex panels (Human ProcartaPlexTM Panel 1 and Panel 2 from ThermoFisher) following the manufacturer’s recommendation. Aliquots of 50 ul urine were used for each of the panels. The Luminex panels were analyzed at the Proteomics Laboratory of the University of Florida Interdisciplinary Center for Biotechnology Research (UF/ICBR). The biomarkers analyzed were Interferon-Inducible Cytokine IP-10 (IP-10), Osteoactivin (GPNMB), retinol binding protein 4 (RBP4), Calbindin (CALB1), Glutathione S-Transferase Alpha 1 (GSTA1), Renin (REN), C-C Motif Chemokine Ligand 2 (MCP-1), Kidney Injury Marker − 1 (KIM-1), Epidermal Growth Factor (EGF), N-Acetyl-alpha glucosaminidase (NAG), interleukin 18 (IL-18), Clusterin (APO-J), Vascular Endothelial Growth Factor A (VEGF-A), Neutrophil Gelatinase-Associated Lipocalin (NGAL), Beta-2-microglobulin (B2M), TIMP Metallopeptidase Inhibitor 1 (TIMP-1), Trefoil Factor 3 (TFF3), Cystatin C (CST3). Uromodulin (UMOD), Osteopontin (OPN), and Alpha-1-microglobulin (AMBP). Each of these biomarkers was previously validated by Fisher for kidney disease induced by different mechanisms, including diabetes, hypertension, and drug-induce kidney disease, among others. Extracellular vesicle preparation Extracellular vesicles were prepared from 35 ml of urine using ultracentrifugation. Prior to ultracentrifugation, the urine samples were filtered through a 0.22 µm Nalgene filter. Ultracentrifugation was performed at 4 o C using a fixed angle rotor, Ti-70 rotor from Beckman. Samples were ultracentrifuged at 118,000 X g for 2 h. Pellets were resuspended in 1 X PBS and subjected to another round of ultracentrifugation at the same speed and time. The final pellets were resuspended in approximately 200 µl sterile and 0.22 µm filtered PBS solution (GIBCO). The extracellular vesicles were characterized for size and number by nanoparticle tracking analysis using a NanoSight NS300 instrument equipped with a high sensitivity Hamamatsu sCMOS camera, 20 x objective lens, and a 50 mW green 532 nm laser as described previously (Dang et al., 2017 ). Samples were processed by three 60 sec videos. miRNA preparation from extracellular vesicles and sequencing To isolate miRNAs from extracellular vesicles we used the Exosomal RNA Isolation Kits (Norgren Biotech Corp, Thorold, Ontario, Canada) following the manufacturer’s instructions. miRNA libraries were prepared by personnel in the Gene Expression and Genotyping Core of the Interdisciplinary Center for Biotechnology Research (ICBR) using SMARTer® smRNA-Seq Kit (Takara Bio, San Jose, CA), following the manufacturer’s protocol. Input was 1ng total small RNA. Illumina NovaSeq6000 Sequencing The Illumina NovaSeq 6000 was used to sequence the libraries for 2 x 150 cycles. Sequencing was performed at the UF/ICBR NextGen Sequencing Core. Briefly, normalized libraries were submitted to the “Free Adapter Blocking Reagent” protocol (FAB, Cat# 20024145) to minimize the presence of adaptor-dimers and index hopping rates. The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2x150 cycles) of the Illumina NovaSeq6000. The instrument’s computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated approximately 2.5 billion paired-end reads (~ 750Gb) with an average Q30%>= 92.5% and Cluster PF = 85.4%. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal. Bioinformatics Bioinformatics to identify the miRNAs and quantify their concentrations was performed in the Bioinformatics Core at UF/ICBR. Calculations for average daily glyphosate dose The average daily glyphosate dose (in mg glyphosate/kg body weight/day) corresponding to the glyphosate concentration in urine was estimated in a manner similar to the approach described by Curwin and coworkers (Curwin et al., 2007 ). Briefly, the absorbed daily glyphosate dose (ADD) corresponding to the urine concentration measured was estimated using the following equation where C is the concentration of glyphosate in urine (µg/g creatinine), Cn is the estimated creatinine excretion rate (g/day), and BW is body weight (kg). The creatinine excretion rate for each subject was estimated from age and body weight using the model of (Goldwasser et al., 1997 ). Because body weight information was not available for the subjects, a “standard” body weight of 70 kg was assumed. Glyphosate is excreted in the urine as parent compound rather than in the form of metabolites, and consequently no correction of the urinary concentration for metabolism is required. In the Curwin et al. study (Curwin et al., 2007 ), an oral bioavailability of 30% was assumed to derive an ingested glyphosate dose from the ADD. However, based on a more recent study of glyphosate urinary excretion in humans (Zoller et al., 2020 ), an oral bioavailability of 1% was assumed for ingested dose estimation, i.e. the ADD was multiplied by 100. RESULTS Glyphosate was detected in urine in 40% of subjects, with concentrations above the limit of quantitation (LOQ) in 15% of samples. Among samples from UFHealth hospital (n = 120) and a UFHealth outpatient clinic (n = 48), respectively, the percent positives were 38.3 and 52.25 and the percent above the LOQ were 15 and 12.5, differences which were not statistically significant. Glyphosate concentrations above the LOQ ranged from 0.21–25.3 µg/L with a geometric mean of 1.0 µg/L. The second highest glyphosate concentration was 5.5 µg/L. Ages of persons whose samples were analyzed ranged from 1 to 96 years (Fig. 1 ). For samples obtained from the hospital, average age of patients whose samples were tested was 52 years, significantly higher than the average age of 38 years for clinic patients whose samples were tested (p < 0.001, Mann Whitney U). However, the glyphosate concentrations in samples showed no significant relationship to subject age (r 2 = 0.003). Among the 138 subjects where gender was identified, 62% were female. Among samples with a glyphosate concentration above the LOQ and the gender of the subject identified, all but three were from female subjects (females, range 0.36–25.3 µg/L, N = 14; males, range 0.30–0.62 µg/L, N = 3). Environmental degradation of glyphosate to AMPA can lead to AMPA exposure as an indirect consequence of glyphosate use. AMPA was detected in the urine of 11 subjects (6.5%), and only two of these (0.75 and 0.86 µg/L) were above the LOQ. Risk-based criteria for glyphosate exposure based upon urine concentrations are not available. To gain perspective on potential health risks associated with glyphosate exposure as indicated by urinary excretion, daily dose rates were estimated corresponding to the urine concentrations. The estimated daily glyphosate dose in the subject with the highest glyphosate concentration in urine (25.3 µg/L in a 43 yo female) was 0.039 mg/kg body weight/day, while the estimated daily dose in the subject with the lowest concentration above the LOQ (0.21 µg/L in a 66 yo subject of unidentified gender) was 0.0005 mg/kg body weight/day. Protein biomarkers of kidney disease Two kidney-specific Luminex panels containing a total of 21 protein biomarkers related to kidney injury/disease were used to test the 48 urine samples from outpatient clinic patients. To evaluate potential nephrotoxicity of glyphosate in these subjects, correlations were sought between glyphosate dose, as indicated by glyphosate urine concentration, and the corresponding urine concentrations of kidney injury biomarkers. No significant correlations were observed (Fig. 2 and supplemental figure S1 ). As an alternative indicator of glyphosate exposure, the relationships between glyphosate plus AMPA urinary concentration and each of the biomarkers were also examined for this group of subjects. As with the concentration of glyphosate alone no significant relationship was observed for any of the kidney injury biomarkers (data not shown). Extracellular vesicle miRNA analysis Small extracellular vesicles were prepared from urine samples from 12 of the subjects based on urinary glyphosate concentrations: this included six samples with the highest glyphosate concentrations (ranging from 0.30 to 1.74 ng/ml) and six samples all having concentrations below the limit of detection (LOD) (< 0.1 ng/ml). miRNAs were isolated from the extracellular vesicles and quantified by RNA sequencing (RNAseq). The miRNAs chosen for study were selected from among a suite examined in a recent study of urinary miRNAs from subjects with acute kidney injury (AKI) associated with a variety of potential toxic exposures, including acute self-poisoning with glyphosate, as well as healthy controls (Shihana et al., 2021 ). The miRNAs included in the present study and their expression in urinary exosomes from the two glyphosate exposure groups are shown in Table 1 . Extracellular vesicles did not differ in size or concentration per ml urine (Fig. 3 ). Expression of miRNAs in extracellular vesicles from four of the six subjects with detectable glyphosate in urine was similar to, or less than, that in extracellular vesicles from urine without detectable glyphosate. This observation applies to both the miRNAs found to be of diagnostic value for AKI by Shihana et al. (Shihana et al., 2021 ) and the miRNAs overall. The greatest miRNA response was in extracellular vesicles from urine with the lowest measured glyphosate concentration, with a somewhat lesser response in extracellular vesicles from urine with the highest glyphosate concentration in the group. DISCUSSION Over the last decade several studies have examined glyphosate concentrations in urine of subjects to gain insight regarding the prevalence and magnitude of exposure to glyphosate in the general population (Buekers et al., 2022 ; Campbell et al., 2022 ; Connolly et al., 2020 ; Conrad et al., 2017 ; Gillezeau et al., 2019 ; Mills et al., 2017 ). These studies have been conducted primarily in the U.S. and Europe, although based upon patterns of use of glyphosate in agriculture, general population exposures can reasonably be assumed to occur worldwide. Although the cohorts studied vary widely in size and composition with respect to age, they have in common the detection of glyphosate in urine in a high percentage of subjects, typically with maximum concentrations between 1 and 10 µg/L. In this study, deidentified urine samples were randomly selected from among samples submitted to our hospital clinical laboratory and to the laboratory of an outpatient clinic. As with most previous studies, glyphosate in urine was measured using LC-MS-MS. As we were using de-identified samples we do not have data on possible exposures to glyphosate among the individuals whose samples were tested. Outpatient clinic patients whose samples were tested were significantly younger than patients whose samples were collected from the hospital laboratory; however, percent positivity did not differ significantly between samples collected in outpatient and inpatient settings, and age did not emerge as a significant predictor for presence of glyphosate. Overall, 40% of urine samples had detectable glyphosate, consistent with the literature. The highest concentration observed, 25.3 µg/L in a 43-year-old female subject, was outside the range for non-occupational glyphosate exposure reported in the literature, and, in the absence of exposure data, it is possible that she had occupational exposure to glyphosate. All other glyphosate concentrations above the LOD (range 0.21–5.5 µg/L) were consistent with previous studies of non-occupational acute exposures. One approach for placing these exposures in perspective in terms of potential adverse health effects is to compare them with regulatory safe exposure limits. The U.S. Environmental Protection Agency glyphosate reference dose (RfD) and the Agency for Toxic Substances and Disease Registry (ATSDR) Minimal Risk Level (MRL) are both 1 mg/kg body weight/day (ATSDR, 2020 ; USEPA, 2017). The European Food Safety Authority (EFSA) has an Allowable Daily Intake (ADI) for glyphosate of 0.5 mg/kg body weight/day, and Health Canada has an Acceptable Daily intake of 0.3 mg/kg body weight/day. Comparison with these limits requires estimation of the daily ingested dose of glyphosate corresponding to its concentration in urine for each of the subjects. The absence of glyphosate metabolism in humans and the presumed complete elimination of absorbed glyphosate in urine facilitates back extrapolation of a glyphosate dose; however assumptions such as body weight and urinary output are required (Connolly et al., 2020 ). Initial estimates of glyphosate doses from typical glyphosate concentrations in urine placed them orders of magnitude below regulatory criteria (e.g.,(Munoz et al., 2023 )). However, these estimates were based on assumed oral bioavailability of glyphosate derived from studies in rats. Recent data from human studies indicate that oral bioavailability of glyphosate is much lower (ca. 1%) resulting in higher estimated doses (Connolly et al., 2020 ). Using a 1% oral bioavailability assumption, the highest glyphosate concentration observed among our subjects (25.3 µg/L) corresponds to a dose of 0.039 mg/kg body weight/day, and the second highest concentration, more typical of results in non-occupationally exposed individuals, is estimated to result from a dose of 0.0074 mg/kg body weight/day. These dose estimates are 1–2 orders of magnitude below regulatory exposure limits. Using a somewhat different approach, Hays et al. (Hays et al., 2023 ) calculated a safe exposure limit for glyphosate concentration in urine extrapolating from the animal toxicity study on which the Health Canada acceptable daily intake criterion is based. The proposed Biomonitoring Equivalent, in effect a urine concentration resulting from a no-effect dose, was 5.4 mg/L glyphosate, roughly three orders of magnitude or more above concentrations reported in the literature for non-occupational exposures. Despite estimated glyphosate exposures well below regulatory limits, there remains concern that environmental glyphosate exposures may result in increased risk of disease, including kidney disease (Mesnage et al., 2015b ). For example, a recent study of chronic exposure of rats to glyphosate observed morphologic and transcriptomic changes suggestive of renal damage at a dose of 4 ng/kg body weight/day (Mesnage et al., 2015a ), well within the range of doses observed here and in other studies of glyphosate in the general population. Beyond standard clinical chemistry tests for renal function such as serum and urinary creatinine, blood urea nitrogen, etc., a number of studies have used urinary biomarkers as sensitive indicators of renal injury from a variety of causes (Van Nynatten et al., 2023 ), including glyphosate ingestion. Wunnapuk and coauthors (Wunnapuk et al., 2014 ) evaluated the diagnostic performance of 14 urinary biomarkers in predicting AKI in rats treated with sub-lethal doses of glyphosate. Urinary KIM-1 was the best early biomarker for kidney injury, although urinary clusterin and VEGF were also significantly elevated in animals given the highest dose of glyphosate. In a study of patients self-poisoned with glyphosate, 10 urinary biomarkers were used: KIM-1, clusterin, IL-18, cytochrome c (CytoC), cystatin c (CysC), albumin (Alb), trefoil factor 3 (TFF3), osteopontin (OstP), beta-2-microglobulin (B2M), and neutrophil gelatinase-associated lipocalin (NGAL) (Mohamed et al., 2016 ). None were elevated in patients without AKI or with mild AKI. Urinary IL-18, CytoC, NGAL, and CysC were increased in patients with moderate or severe AKI. We tested a large suite of 21 biomarkers, including all of the biomarkers observed to be elevated in glyphosate intoxicated humans or animals presented above, in urine samples from 48 subjects. No significant association was found between urinary glyphosate concentrations or glyphosate plus AMPA concentrations and any of these biomarkers. Medical histories for the subjects in this study were not available to us, and it is possible that the presence of other morbidities that produce elevations in kidney biomarkers (e.g., diabetes) could have interfered with glyphosate-biomarker comparisons. However, we note that Trasande et al. (Trasande et al., 2020 ) also found no evidence of kidney injury when urinary glyphosate concentrations ranging from 0.105–2.125 µg/L in children were compared with three biomarkers (viz., albuminuria, NGAL, and KIM-1) using multivariable regression. Transcriptomic changes often precede tissue injury and can provide an early and sensitive predictor of toxicity. Urinary extracellular vesicles, which are released from cells within the urinary tract, contain cellular constituents including proteins, RNA, and miRNA, and offer a means to assess the metabolic and transcriptomic status of the kidney non-invasively (Pisitkun et al., 2004 ; van Balkom et al., 2011 ). Results from analysis of extracellular vesicular miRNAs from 12 subjects with and without detectable glyphosate in urine were equivocal. Extracellular vesicles with the largest increase in targeted miRNAs were from urine samples with glyphosate, but the greatest response was from the sample with the lowest detectable glyphosate concentration. Extracellular vesicles from this and the sample with the highest glyphosate concentration had increases in miRNAs identified previously by Shihana (Shihana et al., 2021 ) as diagnostic for AKI in glyphosate poisoned subjects but also increases in several miRNAs that were not diagnostic. Many of the miRNAs increased in extracellular vesicles from two of the six urines with glyphosate above the LOD control pathways for cell proliferation, apoptosis, lipid metabolism, and mitochondrial energetics plausibly related to the higher order pathology observed in the chronic low dose study in rats by Mesnage et al. (Mesnage et al., 2015b ). However, inconsistency in these miRNA changes among subjects with detectable glyphosate and the absence of a clear relationship with glyphosate dose greatly limit any conclusions that can be drawn. Urinary extracellular vesicles are potentially a powerful tool for understanding kidney disease processes and efforts to understand potential glyphosate effects on the kidney need additional, much larger studies. Studies of patients with AKI from acute glyphosate poisoning will be valuable, but it is conceivable that chronic, lower dose exposure may produce renal pathology differently. Cohorts constructed from individuals with occupational and non-occupational exposure are especially important, but adequately addressing confounding variables from prevalent conditions such as diabetes, high blood pressure, infections, etc. will be challenging. An important limitation of this and most related studies is the difficulty in characterizing glyphosate exposure from the snapshot given by a single urinary sample. Longitudinal studies are essential to characterize whether glyphosate exposure is continuous or episodic and the extent to which it varies over time. This information is critical to developing meaningful dosimetry and dose-response relationships from which glyphosate risk of renal disease can be determined. The high level of chronic exposure to glyphosate in the general population, as reflected in the high rates of urinary glyphosate in random community populations reported in this and multiple other studies, underscores the critical importance of documenting long-term effects of low-level exposure. This, in turn, will require carefully conducted, multi-year cohort studies, which need to become a top priority for research funding. CONCLUSIONS This study is similar to others in terms of the prevalence and magnitude of glyphosate detected in urine among subjects from the general population. Measurement of biomarkers of kidney injury in subject urine, as well as target miRNAs in exosomes harvested from urine, showed no clear evidence that renal impairment results from acute glyphosate exposure at levels experienced by the general population. Abbreviations ADD, absorbed daily glyphosate dose AKI, acute kidney injury AMBP, Alpha-1-microglobulin AMPA, aminomethylphosphonic acid APO-J, Clusterin ATSDR, Agency for Toxic Substances and Disease Registry B2M, Beta-2-microglobulin CALB1, Calbindin CST3, Cystatin C EGF, Epidermal Growth Factor GMO, genetically modified GPNMB, Osteoactivin GSTA1, Glutathione S-Transferase Alpha 1 ICBR, Interdisciplinary Center for Biotechnology Research IL-18, interleukin 18 IP-10, Interferon-Inducible Cytokine IP-10 KIM-1, Kidney Injury Marker − 1 LC-MS/MS, liquid chromatograph-tandem mass spectrometry LOD, limit of detection LOQ, limit of quantitation MCP-1, C-C Motif Chemokine Ligand 2 MRL, Minimal Risk Level MRM, multiple reaction monitoring NAG, N-Acetyl-alpha glucosaminidase NGAL, Neutrophil Gelatinase-Associated Lipocalin NHANES, National Health and Nutrition Examination Survey OPN, Osteopontin RBP4, retinol binding protein 4 RfD, reference dose REN, Renin RNAseq, RNA sequencing TFF3, Trefoil Factor 3 TIMP-1, TIMP Metallopeptidase Inhibitor 1 UFHealth, University of Florida UMOD, Uromodulin VEGF-A, Vascular Endothelial Growth Factor A Declarations Ethics approval and consent to participate Study protocol reviewed and approved as “exempt” by University of Florida IRB (IRB protocol IRB201800432) Consent for publication: Not applicable Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests: The authors declare that they have no competing interests. Funding: This study had no external sponsors and was funded entirely with University of Florida internal funding sources. Authors’ contributions: SM , conception of project, analyzed and interpreted the patient data, performed risk assessment, writing of the initial version of the manuscript: ND , performed and analyzed primary data, writing of manuscript; DN, obtained deidentified patient data, reviewed manuscript; JP-J , performed miRNA separation from extracellular vesicles, reviewed manuscript; GM , conception of project, obtained IRB approved protocol, writing of manuscript. All authors read and approved the final manuscript. Acknowledgements: The authors acknowledge the excellent technical work of Ran Zheng and Jin Kor, in running the Luminex panels; YanPing Zhang, for preparing the miRNA libraries, David Moraga for processing RNAseq, and Fahong Yu, for bioinformatics support offered by UF/ICBR. 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Glyphosate's Synergistic Toxicity in Combination with Other Factors as a Cause of Chronic Kidney Disease of Unknown Origin. Int J Environ Res Public Health 16(15). (2019). Hays, S. M., Kirman, C. R., Flippin, J. & Lopez, T. Biomonitoring Equivalents for glyphosate. Regul. Toxicol. Pharmacol. 144 , 105481 (2023). Huang, Y., Tian, Y., Likhodii, S. & Randell, E. Baseline urinary KIM-1 concentration in detecting acute kidney injury should be interpreted with patient pre-existing nephropathy. Pract. Lab. Med. 15 , e00118. (2019). Jensen, P. K., Wujcik, C. E., McGuire, M. K. & McGuire, M. A. Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS. J. Environ. Sci. Health B . 51 (4), 254–259 (2016). Kim, N. H. et al. Vascular endothelial growth factor (VEGF) and soluble VEGF receptor FLT-1 in diabetic nephropathy. Kidney Int. 67 (1), 167–177 (2005). Krzeminska, E., Wyczalkowska-Tomasik, A., Korytowska, N. & Paczek, L. Comparison of Two Methods for Determination of NGAL Levels in Urine: ELISA and CMIA. J. Clin. Lab. Anal. 30 (6), 956–960 (2016). Lemke, N. et al. Glyphosate and aminomethylphosphonic acid (AMPA) in urine of children and adolescents in Germany - Human biomonitoring results of the German Environmental Survey 2014–2017 (GerES V). Environ. Int. 156 , 106769 (2021). Mesnage, R. et al. Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure. Environ. Health . 14 , 70 (2015a). Mesnage, R., Defarge, N., de Vendomois, S., Seralini, G. E. & J. and Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem. Toxicol. 84 , 133–153 (2015b). Mills, P. J. et al. Excretion of the Herbicide Glyphosate in Older Adults Between 1993 and 2016. JAMA 318 (16), 1610–1611 (2017). Mohamed, F. et al. Mechanism-specific injury biomarkers predict nephrotoxicity early following glyphosate surfactant herbicide (GPSH) poisoning. Toxicol. Lett. 258 , 1–10 (2016). Munoz, J. P., Silva-Pavez, E., Carrillo-Beltran, D. & Calaf, G. M. Occurrence and exposure assessment of glyphosate in the environment and its impact on human beings. Environ. Res. 231 (Pt 3), 116201 (2023). Munshi, R. et al. MCP-1 gene activation marks acute kidney injury. J. Am. Soc. Nephrol. 22 (1), 165–175 (2011). Musiał, K. et al. Clusterin as a New Marker of Kidney Injury in Children Undergoing Allogeneic Hematopoietic Stem Cell Transplantation-A Pilot Study. J. Clin. Med. 9 (8). (2020). Niemann, L., Sieke, C., Pfeil, R. & Solecki, R. A critical review of glyphosate findings in human urine samples and comparison with the exposure of operators and consumers. J. Verbrauch Lebensm . 10 (1), 3–12 (2015). Ospina, M. et al. Exposure to glyphosate in the United States: Data from the 2013–2014 National Health and Nutrition Examination Survey. Environ. Int. 170 , 107620 (2022). Pisitkun, T., Shen, R. F. & Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. U S A . 101 (36), 13368–13373 (2004). Powles, S. B. & Preston, C. Evolved glyphosate resistance in plants: Biochemical and genetic basis of resistance. Weed Technol. 20 (2), 282–289 (2006). Roy, R. et al. Urinary TIMP-1 and MMP-2 levels detect the presence of pancreatic malignancies. Br. J. Cancer . 111 (9), 1772–1779 (2014). Shihana, F. et al. Urinary microRNAs as non-invasive biomarkers for toxic acute kidney injury in humans. Sci. Rep. 11 (1), 9165 (2021). Tang, J. et al. Urinary Renin in Patients and Mice With Diabetic Kidney Disease. Hypertension 74 (1), 83–94 (2019). Trasande, L. et al. Glyphosate exposures and kidney injury biomarkers in infants and young children. Environ. Pollut . 256 , 113334 (2020). USEPA 2017 (United States Environmental Protection Agency). Glyphosate. Draft Human Health Risk Assessment in Support of Registration Review. Health Effects Division, O.o.P.P. (ed). van Balkom, B. W., Pisitkun, T., Verhaar, M. C. & Knepper, M. A. Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases. Kidney Int. 80 (11), 1138–1145 (2011). Van Bruggen, A. H. C. et al. Environmental and health effects of the herbicide glyphosate. Sci. Total Environ. 616–617 , 255–268 (2018). Van Nynatten, L. R. et al. A novel multiplex biomarker panel for profiling human acute and chronic kidney disease. Sci. Rep. 13 (1), 21210 (2023). Walshe, C. M., Odejayi, F., Ng, S. & Marsh, B. Urinary glutathione S-transferase as an early marker for renal dysfunction in patients admitted to intensive care with sepsis. Crit. Care Resusc. 11 (3), 204–209 (2009). Wunnapuk, K. et al. Use of a glyphosate-based herbicide-induced nephrotoxicity model to investigate a panel of kidney injury biomarkers. Toxicol. Lett. 225 (1), 192–200 (2014). Zoller, O., Rhyn, P., Zarn, J. A. & Dudler, V. Urine glyphosate level as a quantitative biomarker of oral exposure. Int. J. Hyg. Environ. Health . 228 , 113526 (2020). Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx GlyphosateSupplementaryinformation.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 20 May, 2026 Reviews received at journal 07 May, 2026 Reviews received at journal 01 May, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 25 Apr, 2026 Reviews received at journal 06 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor assigned by journal 23 Dec, 2025 Editor invited by journal 09 Sep, 2025 Submission checks completed at journal 05 Sep, 2025 First submitted to journal 05 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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14:18:25","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124110,"visible":true,"origin":"","legend":"","description":"","filename":"f83d7e3b3ac04a92a05065c0b7bc097d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/8ba594d02054487404e18a99.xml"},{"id":99317132,"identity":"93a00216-c1cb-41db-9c1f-33c126bada7a","added_by":"auto","created_at":"2025-12-31 16:29:41","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132501,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/51dae58b505fe144ee1beed0.html"},{"id":99317146,"identity":"4cb6153a-7449-4f64-8b82-a86888c5330e","added_by":"auto","created_at":"2025-12-31 16:29:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber of patients and glyphosate concentrations, by age\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/05cbd1ebb4980b1159e7c96b.png"},{"id":99165904,"identity":"112928e6-d7d8-4fa5-af6e-bfb4a7468d4b","added_by":"auto","created_at":"2025-12-29 14:18:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":206120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between urinary glyphosate and biomarker of kidney injury concentrations: \u003c/strong\u003eLuminex biomarkers that showed a small degree of correlation with glyphosate concentrations in urine of patients. \u0026nbsp;There are no thresholds reported for these biomarkers. To get a range equivalent to normal controls, we have plotted the average or geometric mean of normal human samples for each biomarker (dotted red line). Renin, geometric mean (range) of 1.64 pg/ml (0.14-17.2) among a group of 65 individuals (Tang et al., 2019); Kim-1, average (range) of controls of 0.6 (0.19-1.26 ng/ml) (Huang et al., 2019); MCP-1, range of controls, average 0.4 ng/ml (0-0.8 ng/ml) (Munshi et al., 2011); VEGF-A, median value of 220 pg/ml based on normal individuals with 27.8 pg/ mg creatinine and average creatinine values of 7.9 ug/ml (Kim et al., 2005); TIMP-1, average and range for controls of 0.33 ng/ml (0.12-2.98 ng/ml) (Roy et al., 2014); NGAL, average and range for controls of 5.5 ng/ml (0.2-28.6 ng/ml) (Krzeminska et al., 2016). Values for GSTA, range for controls 0-15 ng/ml) (Walshe et al., 2009); and Clusterin (APO-J), values for children (1.2-1.4 ng/ml) (Musiał et al., 2020) were higher than values reported in our study. Plots for the 12 additional biomarkers are in Supplementary Information.\u003c/p\u003e","description":"","filename":"2z.png","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/250530b316834f4cc666a1a0.png"},{"id":99165905,"identity":"37c4cbc6-4a59-43ec-b231-d0b81690596e","added_by":"auto","created_at":"2025-12-29 14:18:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140276,"visible":true,"origin":"","legend":"\u003cp\u003eExosome characterization by NanoSight analysis showing the diameter of small extracellular vesicles isolated from the urine of patients with different levels of urinary glyphosate. (a) a patient with no detectable glyphosate in their urine, (b) a patient with an intermediate level of 0.3 ng/ml glyphosate, (c) a patient with high levels of 1.15 ng/ml, and (d) average diameter of extracellular vesicles as a function of urinary glyphosate concentration.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/074af7b5fb3b8a6dffef2370.png"},{"id":99323652,"identity":"b011849b-cccc-4584-838c-40f101b3ce94","added_by":"auto","created_at":"2025-12-31 16:45:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1174013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/1f687757-084a-461c-b25e-033dfbcadd26.pdf"},{"id":99165900,"identity":"58cef856-8d13-4e48-81e1-95c92377f7ad","added_by":"auto","created_at":"2025-12-29 14:18:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23798,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/6b754c4ef3d24f259ecc7e7e.docx"},{"id":99315747,"identity":"6bec7fba-6e56-488a-a9fb-cac49cfeffb4","added_by":"auto","created_at":"2025-12-31 16:27:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":526765,"visible":true,"origin":"","legend":"","description":"","filename":"GlyphosateSupplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7539235/v1/d365594dd65c5ffa3683d0b7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"An Examination of Potential Renal Toxicity from Low Dose Glyphosate Exposure in Human Subjects","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eGlyphosate (N-[phosphonomethyl]-glycine; tradename Roundup\u0026reg;) is the most widely used herbicide in the world. It is applied before planting traditional crops, after planting genetically modified (GMO) glyphosate-resistant crops (60\u0026ndash;80% of all GMO crops), and as a \u0026ldquo;harvest aid\u0026rdquo; on traditional grain crops (Van Bruggen et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The widespread adoption of GMO herbicide-resistant crops since 1996 has resulted in a 15-fold increase in glyphosate use globally, and in 2014 enough glyphosate was sprayed to apply 0.53 kg on every hectare under cultivation worldwide (Benbrook, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During that year, 113\u0026nbsp;million kg of glyphosate was used in agriculture in the United States (Benbrook, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Not unexpectedly, extensive use of glyphosate has resulted in increased glyphosate resistance among weeds (Powles and Preston, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) leading to even greater application of glyphosate, often combined with other herbicides. With this widespread usage, glyphosate has been detected with increasing frequency in water, air, and food products (Munoz et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a result, interest in the potential risks from glyphosate exposure, once primarily focused on agricultural workers, is now also focused on the general population.\u003c/p\u003e \u003cp\u003eA key component of the assessment of risk from glyphosate, as with any chemical, is the level of exposure. Attempts have been made to estimate glyphosate exposure among the general population through modeling as well as more direct estimation through biomonitoring (Gillezeau et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on observations that absorbed glyphosate is eliminated almost completely in urine, measuring urinary glyphosate is an effective approach to estimate exposure levels (Niemann et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several studies have reported finding glyphosate in the urine of large percentages of subjects from the general population (see (Connolly et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gillezeau et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) for reviews). Among the largest studies, urine collected from individuals ages 6 and above during the 2013\u0026ndash;2014 U.S. National Health and Nutrition Examination Survey (NHANES) had detectable glyphosate in 81.2% of samples (N\u0026thinsp;=\u0026thinsp;2,130) (Ospina et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Results from the 2014\u0026ndash;2017 German Environmental Survey of children and adolescents ages 3\u0026ndash;17 found that 52% had glyphosate in the urine above the level of quantitation (N\u0026thinsp;=\u0026thinsp;2,144) (Lemke et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the percentages of positive urines for glyphosate vary among studies due to differences in populations examined, analytical detection limits, and perhaps other factors, it is clear that glyphosate exposure is widespread in the general population. This raises the question of the toxicological significance of these exposures.\u003c/p\u003e \u003cp\u003eGlyphosate effects on the kidney are of particular interest from a human health perspective. Glyphosate exposure has been proposed as a contributor to Chronic Kidney Disease of Unknown Origin (Gunatilake et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and transcriptomic changes consistent with renal damage were observed in rats given very low doses of glyphosate chronically (Mesnage et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). In the study presented here, we initially obtained 120 random, deidentified urine samples from the UFHealth hospital clinical laboratory which were analyzed for glyphosate and its environmental decomposition product aminomethylphosphonic acid (AMPA) using liquid chromatography-mass spectrometry (LC-MS/MS). Forty-eight random, deidentified urine samples were subsequently obtained from among samples collected as part of routine clinical care at a UFHealth outpatient clinic, to assess whether a relationship existed between urinary glyphosate concentrations and 21 urinary biomarkers of renal injury. To evaluate the possibility of transcriptomic changes as precursors to toxicity in kidney tissue non-invasively, urinary extracellular vesicles were prepared from a subset of 12 of these 48 samples with and without elevated glyphosate concentrations and target miRNAs were compared.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cstrong\u003eSubjects\u003c/strong\u003e \u003cp\u003eOne-hundred-twenty de-identified urine samples were randomly selected from among routine urine samples submitted for hospitalized patients to the UFHealth hospital clinical pathology laboratory in Gainesville, FL. While the majority of hospital patients at UFHealth come from the city of Gainesville, the hospital is a tertiary referral center and does admit patients from surrounding rural areas in North Central Florida. In a second, follow-up study, focusing on renal biomarkers and miRNA analysis, random de-identified urine samples were obtained from among routine samples submitted by outpatients to the laboratory at a UFHealth family medicine/primary care clinic in the city of Gainesville.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e All methods were carried out in accordance with relevant guidelines and regulations. The study protocol was reviewed and approved as \u0026ldquo;exempt\u0026rdquo; by University of Florida IRB (protocol IRB201800432), contingent on the requirement that all samples be fully deidentified, that there be no contact with patients and that no personal health information (other than age and gender) be made available to investigators.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of glyphosate and aminomethylphosphonic acid\u003c/b\u003e: Urine samples were stored frozen at -20\u0026deg;C until analysis. Analysis of glyphosate and AMPA in urine was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described by Jensen and coworkers (Jensen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Urine was diluted 1:1 with 0.1% formic acid and injected directly onto a Bio-Rad Cation-H guard column using a Shimadzu Prominence UHPLC-30AD coupled to the QTRAP 6500 (AB SCIEX, Framingham, MA). An isotopically labeled internal standard (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e15\u003c/sup\u003eN-glyphosate) was added to the samples and used for quantitation. The mobile phases were 0.1% formic acid in water for A and acetonitrile for B. The first minute of the chromatography separation was diverted to waste. Glyphosate and AMPA were analyzed in negative mode via multiple reaction monitoring (MRM) using the following transitions: glyphosate m/z 168/63 and 168/79 and AMPA m/z 110/63 and 110/79 for primary and secondary transitions, respectively. The primary transition was used for quantitation and the secondary transition for qualification. The urine samples were run in four batches, and the assay performance was evaluated for each run. The limits of detection for glyphosate ranged from 0.05\u0026ndash;0.10 \u0026micro;g/L and from 0.1\u0026ndash;0.26 \u0026micro;g/L for AMPA. Limits of quantitation were 0.1\u0026ndash;0.36 \u0026micro;g/L for glyphosate and 0.5\u0026ndash;0.86 \u0026micro;g/L for AMPA.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCreatinine and osmolality measurements\u003c/strong\u003e \u003cp\u003eUrine creatinine concentrations were measured by the UFHealth Pathology Laboratories using a kinetic modification of the Jaffe procedure as developed by Cook (Cook, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Creatinine concentration was determined by the rate of change in absorbance at 520/800 nm using a Beckman Coulter AU680 Chemistry Analyzer. Osmolality was measured using an osmometer (Advanced Instruments, model 3320).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProtein biomarkers of renal injury\u003c/strong\u003e \u003cp\u003eTwenty-one biomarkers of renal injury were measured via the use of two Luminex panels (Human ProcartaPlexTM Panel 1 and Panel 2 from ThermoFisher) following the manufacturer\u0026rsquo;s recommendation. Aliquots of 50 ul urine were used for each of the panels. The Luminex panels were analyzed at the Proteomics Laboratory of the University of Florida Interdisciplinary Center for Biotechnology Research (UF/ICBR). The biomarkers analyzed were Interferon-Inducible Cytokine IP-10 (IP-10), Osteoactivin (GPNMB), retinol binding protein 4 (RBP4), Calbindin (CALB1), Glutathione S-Transferase Alpha 1 (GSTA1), Renin (REN), C-C Motif Chemokine Ligand 2 (MCP-1), Kidney Injury Marker \u0026minus;\u0026thinsp;1 (KIM-1), Epidermal Growth Factor (EGF), N-Acetyl-alpha glucosaminidase (NAG), interleukin 18 (IL-18), Clusterin (APO-J), Vascular Endothelial Growth Factor A (VEGF-A), Neutrophil Gelatinase-Associated Lipocalin (NGAL), Beta-2-microglobulin (B2M), TIMP Metallopeptidase Inhibitor 1 (TIMP-1), Trefoil Factor 3 (TFF3), Cystatin C (CST3). Uromodulin (UMOD), Osteopontin (OPN), and Alpha-1-microglobulin (AMBP). Each of these biomarkers was previously validated by Fisher for kidney disease induced by different mechanisms, including diabetes, hypertension, and drug-induce kidney disease, among others.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExtracellular vesicle preparation\u003c/strong\u003e \u003cp\u003eExtracellular vesicles were prepared from 35 ml of urine using ultracentrifugation. Prior to ultracentrifugation, the urine samples were filtered through a 0.22 \u0026micro;m Nalgene filter. Ultracentrifugation was performed at 4 \u003csup\u003eo\u003c/sup\u003eC using a fixed angle rotor, Ti-70 rotor from Beckman. Samples were ultracentrifuged at 118,000 X g for 2 h. Pellets were resuspended in 1 X PBS and subjected to another round of ultracentrifugation at the same speed and time. The final pellets were resuspended in approximately 200 \u0026micro;l sterile and 0.22 \u0026micro;m filtered PBS solution (GIBCO).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe extracellular vesicles were characterized for size and number by nanoparticle tracking analysis using a NanoSight NS300 instrument equipped with a high sensitivity Hamamatsu sCMOS camera, 20 x objective lens, and a 50 mW green 532 nm laser as described previously (Dang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Samples were processed by three 60 sec videos.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003emiRNA preparation from extracellular vesicles and sequencing\u003c/strong\u003e \u003cp\u003eTo isolate miRNAs from extracellular vesicles we used the Exosomal RNA Isolation Kits (Norgren Biotech Corp, Thorold, Ontario, Canada) following the manufacturer\u0026rsquo;s instructions. miRNA libraries were prepared by personnel in the Gene Expression and Genotyping Core of the Interdisciplinary Center for Biotechnology Research (ICBR) using SMARTer\u0026reg; smRNA-Seq Kit (Takara Bio, San Jose, CA), following the manufacturer\u0026rsquo;s protocol. Input was 1ng total small RNA.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIllumina NovaSeq6000 Sequencing\u003c/strong\u003e \u003cp\u003eThe Illumina NovaSeq 6000 was used to sequence the libraries for 2 x 150 cycles. Sequencing was performed at the UF/ICBR NextGen Sequencing Core. Briefly, normalized libraries were submitted to the \u0026ldquo;Free Adapter Blocking Reagent\u0026rdquo; protocol (FAB, Cat# 20024145) to minimize the presence of adaptor-dimers and index hopping rates. The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2x150 cycles) of the Illumina NovaSeq6000. The instrument\u0026rsquo;s computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated approximately 2.5\u0026nbsp;billion paired-end reads (~\u0026thinsp;750Gb) with an average Q30%\u0026gt;= 92.5% and Cluster PF\u0026thinsp;=\u0026thinsp;85.4%. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBioinformatics\u003c/strong\u003e \u003cp\u003eBioinformatics to identify the miRNAs and quantify their concentrations was performed in the Bioinformatics Core at UF/ICBR.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCalculations for average daily glyphosate dose\u003c/strong\u003e \u003cp\u003eThe average daily glyphosate dose (in mg glyphosate/kg body weight/day) corresponding to the glyphosate concentration in urine was estimated in a manner similar to the approach described by Curwin and coworkers (Curwin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Briefly, the absorbed daily glyphosate dose (ADD) corresponding to the urine concentration measured was estimated using the following equation\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1767017643.png\" style=\"width: 193px;\"\u003e\u003c/p\u003e\u003cp\u003ewhere C is the concentration of glyphosate in urine (\u0026micro;g/g creatinine), Cn is the estimated creatinine excretion rate (g/day), and BW is body weight (kg). The creatinine excretion rate for each subject was estimated from age and body weight using the model of (Goldwasser et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Because body weight information was not available for the subjects, a \u0026ldquo;standard\u0026rdquo; body weight of 70 kg was assumed. Glyphosate is excreted in the urine as parent compound rather than in the form of metabolites, and consequently no correction of the urinary concentration for metabolism is required. In the Curwin et al. study (Curwin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), an oral bioavailability of 30% was assumed to derive an ingested glyphosate dose from the ADD. However, based on a more recent study of glyphosate urinary excretion in humans (Zoller et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), an oral bioavailability of 1% was assumed for ingested dose estimation, i.e. the ADD was multiplied by 100.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eGlyphosate was detected in urine in 40% of subjects, with concentrations above the limit of quantitation (LOQ) in 15% of samples. Among samples from UFHealth hospital (n\u0026thinsp;=\u0026thinsp;120) and a UFHealth outpatient clinic (n\u0026thinsp;=\u0026thinsp;48), respectively, the percent positives were 38.3 and 52.25 and the percent above the LOQ were 15 and 12.5, differences which were not statistically significant. Glyphosate concentrations above the LOQ ranged from 0.21\u0026ndash;25.3 \u0026micro;g/L with a geometric mean of 1.0 \u0026micro;g/L. The second highest glyphosate concentration was 5.5 \u0026micro;g/L.\u003c/p\u003e\n\u003cp\u003eAges of persons whose samples were analyzed ranged from 1 to 96 years (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). For samples obtained from the hospital, average age of patients whose samples were tested was 52 years, significantly higher than the average age of 38 years for clinic patients whose samples were tested (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann Whitney U). However, the glyphosate concentrations in samples showed no significant relationship to subject age (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.003). Among the 138 subjects where gender was identified, 62% were female. Among samples with a glyphosate concentration above the LOQ and the gender of the subject identified, all but three were from female subjects (females, range 0.36\u0026ndash;25.3 \u0026micro;g/L, N\u0026thinsp;=\u0026thinsp;14; males, range 0.30\u0026ndash;0.62 \u0026micro;g/L, N\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n\u003cp\u003eEnvironmental degradation of glyphosate to AMPA can lead to AMPA exposure as an indirect consequence of glyphosate use. AMPA was detected in the urine of 11 subjects (6.5%), and only two of these (0.75 and 0.86 \u0026micro;g/L) were above the LOQ.\u003c/p\u003e\n\u003cp\u003eRisk-based criteria for glyphosate exposure based upon urine concentrations are not available. To gain perspective on potential health risks associated with glyphosate exposure as indicated by urinary excretion, daily dose rates were estimated corresponding to the urine concentrations. The estimated daily glyphosate dose in the subject with the highest glyphosate concentration in urine (25.3 \u0026micro;g/L in a 43 yo female) was 0.039 mg/kg body weight/day, while the estimated daily dose in the subject with the lowest concentration above the LOQ (0.21 \u0026micro;g/L in a 66 yo subject of unidentified gender) was 0.0005 mg/kg body weight/day.\u003c/p\u003e\n\u003ch3\u003eProtein biomarkers of kidney disease\u003c/h3\u003e\n\u003cp\u003eTwo kidney-specific Luminex panels containing a total of 21 protein biomarkers related to kidney injury/disease were used to test the 48 urine samples from outpatient clinic patients. To evaluate potential nephrotoxicity of glyphosate in these subjects, correlations were sought between glyphosate dose, as indicated by glyphosate urine concentration, and the corresponding urine concentrations of kidney injury biomarkers. No significant correlations were observed (Fig. 2 \u003cstrong\u003eand supplemental figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). As an alternative indicator of glyphosate exposure, the relationships between glyphosate plus AMPA urinary concentration and each of the biomarkers were also examined for this group of subjects. As with the concentration of glyphosate alone no significant relationship was observed for any of the kidney injury biomarkers (data not shown).\u003c/p\u003e\n\u003ch3\u003eExtracellular vesicle miRNA analysis\u003c/h3\u003e\n\u003cp\u003eSmall extracellular vesicles were prepared from urine samples from 12 of the subjects based on urinary glyphosate concentrations: this included six samples with the highest glyphosate concentrations (ranging from 0.30 to 1.74 ng/ml) and six samples all having concentrations below the limit of detection (LOD) (\u0026lt;\u0026thinsp;0.1 ng/ml). miRNAs were isolated from the extracellular vesicles and quantified by RNA sequencing (RNAseq). The miRNAs chosen for study were selected from among a suite examined in a recent study of urinary miRNAs from subjects with acute kidney injury (AKI) associated with a variety of potential toxic exposures, including acute self-poisoning with glyphosate, as well as healthy controls (Shihana et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The miRNAs included in the present study and their expression in urinary exosomes from the two glyphosate exposure groups are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles did not differ in size or concentration per ml urine (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Expression of miRNAs in extracellular vesicles from four of the six subjects with detectable glyphosate in urine was similar to, or less than, that in extracellular vesicles from urine without detectable glyphosate. This observation applies to both the miRNAs found to be of diagnostic value for AKI by Shihana et al. (Shihana et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) and the miRNAs overall. The greatest miRNA response was in extracellular vesicles from urine with the lowest measured glyphosate concentration, with a somewhat lesser response in extracellular vesicles from urine with the highest glyphosate concentration in the group.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOver the last decade several studies have examined glyphosate concentrations in urine of subjects to gain insight regarding the prevalence and magnitude of exposure to glyphosate in the general population (Buekers et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Campbell et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Connolly et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Conrad et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gillezeau et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mills et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These studies have been conducted primarily in the U.S. and Europe, although based upon patterns of use of glyphosate in agriculture, general population exposures can reasonably be assumed to occur worldwide. Although the cohorts studied vary widely in size and composition with respect to age, they have in common the detection of glyphosate in urine in a high percentage of subjects, typically with maximum concentrations between 1 and 10 \u0026micro;g/L. In this study, deidentified urine samples were randomly selected from among samples submitted to our hospital clinical laboratory and to the laboratory of an outpatient clinic. As with most previous studies, glyphosate in urine was measured using LC-MS-MS. As we were using de-identified samples we do not have data on possible exposures to glyphosate among the individuals whose samples were tested. Outpatient clinic patients whose samples were tested were significantly younger than patients whose samples were collected from the hospital laboratory; however, percent positivity did not differ significantly between samples collected in outpatient and inpatient settings, and age did not emerge as a significant predictor for presence of glyphosate. Overall, 40% of urine samples had detectable glyphosate, consistent with the literature. The highest concentration observed, 25.3 \u0026micro;g/L in a 43-year-old female subject, was outside the range for non-occupational glyphosate exposure reported in the literature, and, in the absence of exposure data, it is possible that she had occupational exposure to glyphosate. All other glyphosate concentrations above the LOD (range 0.21\u0026ndash;5.5 \u0026micro;g/L) were consistent with previous studies of non-occupational acute exposures.\u003c/p\u003e \u003cp\u003eOne approach for placing these exposures in perspective in terms of potential adverse health effects is to compare them with regulatory safe exposure limits. The U.S. Environmental Protection Agency glyphosate reference dose (RfD) and the Agency for Toxic Substances and Disease Registry (ATSDR) Minimal Risk Level (MRL) are both 1 mg/kg body weight/day (ATSDR, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; USEPA, 2017). The European Food Safety Authority (EFSA) has an Allowable Daily Intake (ADI) for glyphosate of 0.5 mg/kg body weight/day, and Health Canada has an Acceptable Daily intake of 0.3 mg/kg body weight/day. Comparison with these limits requires estimation of the daily ingested dose of glyphosate corresponding to its concentration in urine for each of the subjects. The absence of glyphosate metabolism in humans and the presumed complete elimination of absorbed glyphosate in urine facilitates back extrapolation of a glyphosate dose; however assumptions such as body weight and urinary output are required (Connolly et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Initial estimates of glyphosate doses from typical glyphosate concentrations in urine placed them orders of magnitude below regulatory criteria (e.g.,(Munoz et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)). However, these estimates were based on assumed oral bioavailability of glyphosate derived from studies in rats. Recent data from human studies indicate that oral bioavailability of glyphosate is much lower (ca. 1%) resulting in higher estimated doses (Connolly et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Using a 1% oral bioavailability assumption, the highest glyphosate concentration observed among our subjects (25.3 \u0026micro;g/L) corresponds to a dose of 0.039 mg/kg body weight/day, and the second highest concentration, more typical of results in non-occupationally exposed individuals, is estimated to result from a dose of 0.0074 mg/kg body weight/day. These dose estimates are 1\u0026ndash;2 orders of magnitude below regulatory exposure limits. Using a somewhat different approach, Hays et al. (Hays et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) calculated a safe exposure limit for glyphosate concentration in urine extrapolating from the animal toxicity study on which the Health Canada acceptable daily intake criterion is based. The proposed Biomonitoring Equivalent, in effect a urine concentration resulting from a no-effect dose, was 5.4 mg/L glyphosate, roughly three orders of magnitude or more above concentrations reported in the literature for non-occupational exposures.\u003c/p\u003e \u003cp\u003eDespite estimated glyphosate exposures well below regulatory limits, there remains concern that environmental glyphosate exposures may result in increased risk of disease, including kidney disease (Mesnage et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). For example, a recent study of chronic exposure of rats to glyphosate observed morphologic and transcriptomic changes suggestive of renal damage at a dose of 4 ng/kg body weight/day (Mesnage et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e), well within the range of doses observed here and in other studies of glyphosate in the general population. Beyond standard clinical chemistry tests for renal function such as serum and urinary creatinine, blood urea nitrogen, etc., a number of studies have used urinary biomarkers as sensitive indicators of renal injury from a variety of causes (Van Nynatten et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), including glyphosate ingestion. Wunnapuk and coauthors (Wunnapuk et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) evaluated the diagnostic performance of 14 urinary biomarkers in predicting AKI in rats treated with sub-lethal doses of glyphosate. Urinary KIM-1 was the best early biomarker for kidney injury, although urinary clusterin and VEGF were also significantly elevated in animals given the highest dose of glyphosate. In a study of patients self-poisoned with glyphosate, 10 urinary biomarkers were used: KIM-1, clusterin, IL-18, cytochrome c (CytoC), cystatin c (CysC), albumin (Alb), trefoil factor 3 (TFF3), osteopontin (OstP), beta-2-microglobulin (B2M), and neutrophil gelatinase-associated lipocalin (NGAL) (Mohamed et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). None were elevated in patients without AKI or with mild AKI. Urinary IL-18, CytoC, NGAL, and CysC were increased in patients with moderate or severe AKI.\u003c/p\u003e \u003cp\u003eWe tested a large suite of 21 biomarkers, including all of the biomarkers observed to be elevated in glyphosate intoxicated humans or animals presented above, in urine samples from 48 subjects. No significant association was found between urinary glyphosate concentrations or glyphosate plus AMPA concentrations and any of these biomarkers. Medical histories for the subjects in this study were not available to us, and it is possible that the presence of other morbidities that produce elevations in kidney biomarkers (e.g., diabetes) could have interfered with glyphosate-biomarker comparisons. However, we note that Trasande et al. (Trasande et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also found no evidence of kidney injury when urinary glyphosate concentrations ranging from 0.105\u0026ndash;2.125 \u0026micro;g/L in children were compared with three biomarkers (viz., albuminuria, NGAL, and KIM-1) using multivariable regression.\u003c/p\u003e \u003cp\u003eTranscriptomic changes often precede tissue injury and can provide an early and sensitive predictor of toxicity. Urinary extracellular vesicles, which are released from cells within the urinary tract, contain cellular constituents including proteins, RNA, and miRNA, and offer a means to assess the metabolic and transcriptomic status of the kidney non-invasively (Pisitkun et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; van Balkom et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Results from analysis of extracellular vesicular miRNAs from 12 subjects with and without detectable glyphosate in urine were equivocal. Extracellular vesicles with the largest increase in targeted miRNAs were from urine samples with glyphosate, but the greatest response was from the sample with the lowest detectable glyphosate concentration. Extracellular vesicles from this and the sample with the highest glyphosate concentration had increases in miRNAs identified previously by Shihana (Shihana et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) as diagnostic for AKI in glyphosate poisoned subjects but also increases in several miRNAs that were not diagnostic. Many of the miRNAs increased in extracellular vesicles from two of the six urines with glyphosate above the LOD control pathways for cell proliferation, apoptosis, lipid metabolism, and mitochondrial energetics plausibly related to the higher order pathology observed in the chronic low dose study in rats by Mesnage et al. (Mesnage et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). However, inconsistency in these miRNA changes among subjects with detectable glyphosate and the absence of a clear relationship with glyphosate dose greatly limit any conclusions that can be drawn.\u003c/p\u003e \u003cp\u003eUrinary extracellular vesicles are potentially a powerful tool for understanding kidney disease processes and efforts to understand potential glyphosate effects on the kidney need additional, much larger studies. Studies of patients with AKI from acute glyphosate poisoning will be valuable, but it is conceivable that chronic, lower dose exposure may produce renal pathology differently. Cohorts constructed from individuals with occupational and non-occupational exposure are especially important, but adequately addressing confounding variables from prevalent conditions such as diabetes, high blood pressure, infections, etc. will be challenging. An important limitation of this and most related studies is the difficulty in characterizing glyphosate exposure from the snapshot given by a single urinary sample. Longitudinal studies are essential to characterize whether glyphosate exposure is continuous or episodic and the extent to which it varies over time. This information is critical to developing meaningful dosimetry and dose-response relationships from which glyphosate risk of renal disease can be determined. The high level of chronic exposure to glyphosate in the general population, as reflected in the high rates of urinary glyphosate in random community populations reported in this and multiple other studies, underscores the critical importance of documenting long-term effects of low-level exposure. This, in turn, will require carefully conducted, multi-year cohort studies, which need to become a top priority for research funding.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study is similar to others in terms of the prevalence and magnitude of glyphosate detected in urine among subjects from the general population. Measurement of biomarkers of kidney injury in subject urine, as well as target miRNAs in exosomes harvested from urine, showed no clear evidence that renal impairment results from acute glyphosate exposure at levels experienced by the general population.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cp\u003eADD, absorbed daily glyphosate dose\u003c/p\u003e \u003cp\u003eAKI, acute kidney injury\u003c/p\u003e \u003cp\u003eAMBP, Alpha-1-microglobulin\u003c/p\u003e \u003cp\u003eAMPA, aminomethylphosphonic acid\u003c/p\u003e \u003cp\u003eAPO-J, Clusterin\u003c/p\u003e \u003cp\u003eATSDR, Agency for Toxic Substances and Disease Registry\u003c/p\u003e \u003cp\u003eB2M, Beta-2-microglobulin\u003c/p\u003e \u003cp\u003eCALB1, Calbindin\u003c/p\u003e \u003cp\u003eCST3, Cystatin C\u003c/p\u003e \u003cp\u003eEGF, Epidermal Growth Factor\u003c/p\u003e \u003cp\u003eGMO, genetically modified\u003c/p\u003e \u003cp\u003eGPNMB, Osteoactivin\u003c/p\u003e \u003cp\u003eGSTA1, Glutathione S-Transferase Alpha 1\u003c/p\u003e \u003cp\u003eICBR, Interdisciplinary Center for Biotechnology Research\u003c/p\u003e \u003cp\u003eIL-18, interleukin 18\u003c/p\u003e \u003cp\u003eIP-10, Interferon-Inducible Cytokine IP-10\u003c/p\u003e \u003cp\u003eKIM-1, Kidney Injury Marker \u0026minus;\u0026thinsp;1\u003c/p\u003e \u003cp\u003eLC-MS/MS, liquid chromatograph-tandem mass spectrometry\u003c/p\u003e \u003cp\u003eLOD, limit of detection\u003c/p\u003e \u003cp\u003eLOQ, limit of quantitation\u003c/p\u003e \u003cp\u003eMCP-1, C-C Motif Chemokine Ligand 2\u003c/p\u003e \u003cp\u003eMRL, Minimal Risk Level\u003c/p\u003e \u003cp\u003eMRM, multiple reaction monitoring\u003c/p\u003e \u003cp\u003eNAG, N-Acetyl-alpha glucosaminidase\u003c/p\u003e \u003cp\u003eNGAL, Neutrophil Gelatinase-Associated Lipocalin\u003c/p\u003e \u003cp\u003eNHANES, National Health and Nutrition Examination Survey\u003c/p\u003e \u003cp\u003eOPN, Osteopontin\u003c/p\u003e \u003cp\u003eRBP4, retinol binding protein 4\u003c/p\u003e \u003cp\u003eRfD, reference dose\u003c/p\u003e \u003cp\u003eREN, Renin\u003c/p\u003e \u003cp\u003eRNAseq, RNA sequencing\u003c/p\u003e \u003cp\u003eTFF3, Trefoil Factor 3\u003c/p\u003e \u003cp\u003eTIMP-1, TIMP Metallopeptidase Inhibitor 1\u003c/p\u003e \u003cp\u003eUFHealth, University of Florida\u003c/p\u003e \u003cp\u003eUMOD, Uromodulin\u003c/p\u003e \u003cp\u003eVEGF-A, Vascular Endothelial Growth Factor A\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy protocol reviewed and approved as \u0026ldquo;exempt\u0026rdquo; by University of Florida IRB (IRB protocol IRB201800432)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study had no external sponsors and was funded entirely with University of Florida internal funding sources.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions: SM\u003c/strong\u003e, conception of project, analyzed and interpreted the patient data, performed risk assessment, writing of the initial version of the manuscript:\u0026nbsp;\u003cstrong\u003eND\u003c/strong\u003e, performed and analyzed primary data, writing of manuscript;\u0026nbsp;\u003cstrong\u003eDN,\u003c/strong\u003e obtained deidentified patient data, reviewed manuscript;\u0026nbsp;\u003cstrong\u003eJP-J\u003c/strong\u003e, performed miRNA separation from extracellular vesicles, reviewed manuscript;\u0026nbsp;\u003cstrong\u003eGM\u003c/strong\u003e, conception of project, obtained IRB approved protocol, writing of manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors acknowledge the excellent technical work of Ran Zheng and Jin Kor, in running the Luminex panels; YanPing Zhang, for preparing the miRNA libraries, \u0026nbsp;David Moraga for processing RNAseq, and Fahong Yu, for bioinformatics support offered by UF/ICBR.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e(Agency for Toxic Substances and Disease Registry) Toxicological Profile for Glyphosate. ATSDR \u0026amp; Prevention C.f.D.C.a. (ed). (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenbrook, C. M. Trends in glyphosate herbicide use in the United States and globally. \u003cem\u003eEnviron. Sci. Eur.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (1), 3 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuekers, J. et al. 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Care Resusc.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (3), 204\u0026ndash;209 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWunnapuk, K. et al. Use of a glyphosate-based herbicide-induced nephrotoxicity model to investigate a panel of kidney injury biomarkers. \u003cem\u003eToxicol. Lett.\u003c/em\u003e \u003cb\u003e225\u003c/b\u003e (1), 192\u0026ndash;200 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZoller, O., Rhyn, P., Zarn, J. A. \u0026amp; Dudler, V. Urine glyphosate level as a quantitative biomarker of oral exposure. \u003cem\u003eInt. J. Hyg. Environ. Health\u003c/em\u003e. \u003cb\u003e228\u003c/b\u003e, 113526 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Glyphosate, Roundup, aminomethylphosphonic acid (AMPA), urinary excretion, human health risk, renal biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-7539235/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7539235/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWidespread usage of glyphosate has resulted in contamination of water, air, and food products. The objective of this study was to examine the prevalence and magnitude of environmental exposure to glyphosate among subjects from the general population, as well as assess the potential risk of renal toxicity associated with these exposures.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eConcentrations of glyphosate and its environmental breakdown product aminomethylphosphonic acid (AMPA) were measured in the urine of 168 random, de-identified subjects using liquid chromatograph-tandem mass spectrometry (LC-MS/MS) to determine the frequency of detection. Glyphosate doses producing the observed glyphosate in urine were estimated for comparison with regulatory safe exposure limits. Forty-eight of these samples were further screened for biomarkers of renal injury, with urinary extracellular vesicles prepared from a subset of 12 of these samples for miRNA analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGlyphosate was detected in the urine of 40% of all subjects with a maximum concentration of 25.3 \u0026micro;g/L, and AMPA was detected in 6.5% of all subjects with a maximum concentration of 0.86 \u0026micro;g/L. Estimated glyphosate doses corresponding to urinary levels were 1\u0026ndash;2 orders of magnitude below U.S. and European exposure limits. There was no relationship between glyphosate or glyphosate plus AMPA concentrations in urine and any of 21 urinary biomarkers of renal injury, and no concentration related increase in urinary extracellular vesicular miRNAs previously associated with a high acute glyphosate exposure in humans.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eExposure to glyphosate, as reflected in urinary glyphosate levels, is common among members of the general public in the United States. Observed levels were below established exposure limits, and our data did not document adverse effects of glyphosate on the kidneys at doses at which the general population is exposed. However, more work is needed in subjects with known chronic glyphosate exposure before potential effects from prevalent low doses can be ruled out.\u003c/p\u003e","manuscriptTitle":"An Examination of Potential Renal Toxicity from Low Dose Glyphosate Exposure in Human Subjects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-29 14:18:20","doi":"10.21203/rs.3.rs-7539235/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-20T06:27:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T14:13:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T08:39:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152914468722365517835216421966141474497","date":"2026-04-29T11:12:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182236465389880643679728428988990308893","date":"2026-04-25T04:49:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T14:15:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309697884072330551882625342397352609748","date":"2025-12-29T11:56:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T13:15:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T12:15:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-09T20:42:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T15:43:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-05T15:39:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"79d67916-933b-4256-ab0c-886ef312fd1c","owner":[],"postedDate":"December 29th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-20T06:27:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T14:13:19+00:00","index":109,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":60253840,"name":"Health sciences/Biomarkers"},{"id":60253841,"name":"Earth and environmental sciences/Environmental sciences"},{"id":60253842,"name":"Health sciences/Nephrology"},{"id":60253843,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2026-05-20T06:39:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-29 14:18:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7539235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7539235","identity":"rs-7539235","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}