Nephrotoxic Effects of Glyphosate and Metals: From Mechanisms of Chronic Renal Dysfunction to Complex Mixture Interactions

Abstract

Chronic kidney disease (CKD) is a high prevalence, high burden disease which affects over 840 million people globally. Protecting the kidney from exogenous injuries represents an important priority for public health. Nephrotoxic injury, such as from environmental contaminants, can accelerate the accumulation of damage over a lifetime and increase likelihood of developing CKD. Environmental drivers of kidney health remain poorly understood despite the kidney's inherent vulnerabilities to exogenous stressors. The kidney is in continuous contact with the contents of the blood throughout the lifetime, including circulating contaminants. Renal function demands intense mitochondrial activity which renders kidney tissue particularly vulnerable to mitochondrial toxicants. Finally, even mild, subcytotoxic injury can initiate progressive cycles of damage that advance the pathogenesis underlying CKD. There is a critical need to understand how environmental hazards target subcellular processes and how chronic, low-level injury deteriorates renal health over time.Furthermore, the emergence of chronic kidney disease of unknown etiology (CKDu) in agricultural communities worldwide underscores the urgency of understanding the impact of environmental hazards on kidney health outcomes. CKDu presents as rapidly progressive, early-onset kidney failure and is diagnosed in the absence of traditional risk factors, such as hypertension and diabetes. CKDu is associated with agrochemical contamination of drinking water and occupational hazards to kidney health, but most evidence to support the role of suspect contaminants is from epidemiological approaches, including survey and analytical-based exposure assessment. Experimental investigation of the nephrotoxic potential of environmental hazards is needed, particularly at low concentrations and in exposure-relevant mixtures that reflect contamination of drinking water. This dissertation work investigated the nephrotoxic potential of glyphosate and the metals Cd, As, Pb, and V to gain fundamental insight about environmental drivers of kidney health and health risks from exposure to these near ubiquitous pollutants. We identified Cd, As, Pb, V, and glyphosate as contaminants of concern because of widespread exposure, association with CKDu incidence in Sri Lanka, and existing evidence supporting their toxicity to kidneys and mitochondria at high concentrations. We investigated how co-exposure to environmentally-relevant mixtures affect biological outcomes and how subcellular processes targeted by environmental nephrotoxicants contribute to renal dysfunction over time. We tested two central hypotheses: first, that interactions between glyphosate and the metals Cd, As, Pb, and V increase nephrotoxic potential even at concentrations typically found in drinking water, and second, that chronic exposure to glyphosate and metals disrupts mitochondrial function in the kidney and degrades kidney health. We used zebrafish (Danio rerio) across developmental and adult life stages as a robust in vivo model to address these questions. In Chapter 2, we systematically evaluated how co-exposure to multiple nephrotoxic stressors affect developmental outcomes to understand potential interactions between key environmental stressors, as exposure to nephrotoxic chemicals rarely occurs in isolation. We compared the developmental toxicity of individual metals and population-relevant mixtures based on drinking water from CKDu-endemic areas and the urinary metal content of women in the US. We investigated whether chelation of metals by glyphosate represents a significant interaction for toxicity and also evaluated whether heat stress, an orthogonal nephrotoxic stressor of concern for CKDu-affected populations, exacerbates chemical toxicity during development. Using larval zebrafish, we exposed embryos to individual metals, metal mixtures, glyphosate, glyphosate and metals, and combined with elevated temperature across a range of doses. We assessed whole-embryo bioenergetics, larval behavior, and gross developmental toxicity. We identified synergistic interactions between metals in mixtures to impair larval kidney function. In addition, co-exposure to glyphosate and metals impaired mitochondrial function, larval behavior, and larval kidney function more severely than individual exposures alone. Our functional and molecular findings provided evidence that synergistic interactions between glyphosate and metals arise due to cumulative impacts on shared mechanisms of toxicity, such as mitochondrial dysfunction. We demonstrate that heat stress amplified these effects. Glyphosate, the metals mixture, and heat stress also produced unique impacts on developing tissues beyond their overlapping mechanisms. These findings indicated greater nephrotoxic potential of glyphosate and metals co-exposure and underscored the importance of evaluating toxicity of environmental stressors in exposure-relevant contexts. Chapter 3 addressed the multidimensional complexity of assessing toxicity from environmental exposures and developed methods to identify specific components driving toxicity in complex mixtures, such as in drinking water. Direct testing of interactive effects remains limited by the vast number of chemical combinations present in environmental samples. We aimed to capture systematic differences between drinking water in CKDu-endemic and non-endemic regions of Sri Lanka and to develop statistical approaches for identifying drivers of observed toxicity from exposure studies using complex mixtures. We screened 180 drinking water samples from Sri Lanka using zebrafish to assess developmental toxicity across multiple endpoints throughout embryogenesis and larval stages. We developed a hierarchical linear mixed modeling framework to enhance the sensitivity and accuracy of analyzing functional endpoints and to identify components driving observed toxicity across all water samples. Water samples from CKDu-endemic areas consistently impaired whole-embryo respiration and larval behavior more strongly than samples from non-endemic areas. The differential toxicity between regions supported the hypothesis that specific drinking water contaminants present in endemic areas interfere with mitochondrial processes and other essential cellular functions, which may contribute to CKDu pathogenesis. Our modeling approach successfully estimated the impact of individual metal elements on behavioral outcomes and mitochondrial function in a statistically robust manner and established an approach to capture "bad actors" and key chemical interactions in complex mixtures. This work supports the emerging focus on new approach methodologies to understand potential health impacts from exposure to the myriad of environmental exposures. In Chapter 4, we investigated mechanisms of renal dysfunction induced by chronic exposure to glyphosate and metals to link environmental exposures to kidney health outcomes. Previous chapters demonstrated synergistic toxicity of co-exposures and differential impacts of drinking water from endemic versus non-endemic regions, but the consequences of chronic, low-level exposure to these contaminants for kidney health remained a central gap. We tested the hypothesis that long-term, low-level exposure to glyphosate, metals, and their combination impairs kidney function and structure, with co-exposures worsening kidney toxicity. We exposed adult zebrafish for 10 or 60 days to glyphosate, metals, or glyphosate and metals at concentrations at or below drinking water regulatory thresholds. We assessed kidney outcomes through low-molecular weight proteinuria, histopathology, metabolomics, mitochondrial function, mitochondrial copy number, and mitophagy. Chronic co-exposure to glyphosate and metals compromised kidney structure and function. We documented the onset of low-molecular weight proteinuria which indicates proximal tubule dysfunction, cytosolic vacuolation, disruption of mitochondrial metabolism, and a near ablation of mitophagy in kidney tissue. Co-exposure induced greater nephrotoxicity than individual chemicals, with effects most apparent after 60 days. Mechanistic analyses revealed the importance of mitochondrial disruption from exposure for renal dysfunction. This work is the first to demonstrate that glyphosate can alter the distribution of metals during co-exposure, which supports the role of glyphosate-metal chelation in the biological consequences of co-exposure. This work experimentally demonstrated that chronic exposure to contaminants of concern in agricultural communities affected kidney health even at drinking water concentrations and provided mechanistic insight into how toxicant-induced dysregulation of mitochondrial health impairs renal health and may reduce resilience to other stressors. Overall, this work developed zebrafish as an experimental tool for advancing nephrotoxicity research and demonstrated how environmental stressors synergistically threaten kidney health. We showed that the widespread pollutants glyphosate, Cd, As, Pb, and V exhibit nephrotoxic potential in exposure-relevant contexts, such as at concentrations at or below regulatory thresholds and in population-relevant mixtures. Further, this work experimentally contributed to a mechanistic framework for understanding how chronic mitochondrial dysfunction induced by environmental toxicants can degrade kidney health over time. These findings reveal that co-exposures produce greater nephrotoxicity than individual chemicals and converge on disruption of mitochondrial function in the proximal tubule as a priority target of these environmental stressors. This research addresses critical knowledge gaps regarding environmental drivers of CKD and provides biological evidence connecting suspect environmental exposures in CKDu-endemic regions to mechanisms of kidney disease pathogenesis.