Kidney Toxicity of Roundup, AMPA, and Cadmium in Zebrafish

Abstract

First marketed in the 1970s, glyphosate is now the most widely used herbicide in the world, with usage increasing consistently in recent decades. Emerging toxicological data link exposure to glyphosate with several deleterious health endpoints, including some evidence suggesting a role in chronic kidney disease of unknown etiology (CDKu). Chronic kidney disease (CKD) remains a leading cause of death, with incidence of CKDu growing at an alarming rate in certain agricultural regions. The compound glyphosate, as well as its degradation product aminomethyl phosphonic acid (AMPA), are capable of strongly chelating environmentally and agriculturally relevant nephrotoxic metals such as cadmium, potentially leading to unique environmental fate and toxicological effects upon co-exposure and is posited be a mechanism by which this pesticide contributes to CKDu. This study aimed to assess kidney toxicity by characterizing damage to the proximal tubule. I used a transgenic proteinuria reporter in larval zebrafish, a standard medical model organism with great morphological and functional homology to the adult human nephron. In this study, I assessed the impacts of cadmium, glyphosate in a standard commercial Roundup® formulation, and its lesser-studied degradation product AMPA in isolation to establish baseline kidney toxicological data. Alongside these, I conducted co-exposure experiments with cadmium and Roundup/AMPA to assess how these compounds might modify cadmium nephrotoxicity within environmentally relevant concentration ranges. In a healthy kidney, low molecular weight proteins pass through the glomerular filtration and into the proximal tubule, where they are eventually reabsorbed and recirculated back into the bloodstream. If the proximal tubule is damaged, such as through exposure to a nephrotoxic chemical, the low molecular weight proteins will not be reabsorbed and instead pass into the urine, termed proteinuria. Presence of elevated proteinuria is a clinical marker of kidney damage and is the health endpoint of interest in this study. In order to quantify proteinuria in zebrafish samples, we followed a modified proteinuria reporter assay using a transgenic line of zebrafish (Danio rerio), termed “NL-D3”. This line of zebrafish expresses a luciferase-modified low molecular weight protein that, upon contact with its natural substrate, emits measurable luminescence. As such, we use the luminescence measurements from water samples individually collected from each larval zebrafish over the same overnight period as a proxy for proteinuria content, and thus for kidney damage in the proximal tubule. The conclusions of this study rely on five primary experiments: a cadmium range-finding experiment, a Roundup range-finding experiment, an AMPA range-finding experiment, a Roundup and cadmium co-exposure experiment, and an AMPA and cadmium co-exposure experiment. The range-finding experiments provide baseline toxicity data for the pure compounds or, in the case of Roundup, chemical mixture. The co-exposure experiments aim to replicate environmental exposures in agricultural communities and to test the hypothesis that the presence of glyphosate or AMPA alters the nephrotoxic effect of cadmium. The experimental data support four main conclusions: (i) cadmium is nephrotoxic, (ii) neither Roundup nor AMPA increase proteinuria in isolation, (iii) both Roundup and AMPA decrease proteinuria in a non-monotonic manner, and (iv) both Roundup and AMPA modulate cadmium nephrotoxicity in a non-monotonic manner. The term “non-monotonic” refers to a dose-response curve in which an increasing chemical exposure dose does not correspond to an increase in the measured response. Roundup specifically may increase the nephrotoxicity of cadmium at environmentally relevant levels, resulting in statistically higher proteinuria with respect to control. While certain single-doses and pairwise combinations with cadmium decreased proteinuria, this should be interpreted as a sign of some genuine biological effect, likely outside the kidney, that is unable to be aptly assessed in this assay and not construed as an absence of toxicity nor as a protective effect. In sum, the results of this study demonstrate that Roundup and AMPA affect kidney function to some degree, though likely through secondary mechanisms and not through direct damage to the proximal tubule, given the lower proteinuria counts in several instances. Roundup, however, was found to increase the nephrotoxicity of cadmium at some environmentally relevant concentrations. As such, while the commercial formulation Roundup alone may not damage the proximal tubule, it can increase the deleterious effects of cadmium already present in the environment. When paired with chronic exposure, it is possible that this co-exposure, which is particularly present in agricultural communities, may accelerate some of the effects associated with chronic kidney disease. Taken together, these results may provide further clarity into the link between environmental exposures and CKDu in certain agricultural regions while also providing some of the first kidney toxicological data for the lesser-studied AMPA.