Multiple epidemics of chronic kidney disease of an unknown etiology (CKDu), primarily in young healthy agricultural workers, have emerged in agricultural communities around the world. It is proposed that heat stress, dehydration and/or toxicant exposures may be a cause of this emerging disease. We have hypothesized that the harvest and burning of sugarcane leading to inhalation of sugarcane ash may contribute to development of CKDu. Sugarcane stalks consist of ~80% amorphous silica and we have demonstrated that following burning of sugarcane, nano-sized silica particles (~200 nm) are generated.

1. Sugarcane Ash and Sugarcane Ash-Derived Silica Nanoparticles
Alter Cellular Metabolism and Mitochondrial Function
in Human Proximal Tubular Kidney Cells
Arthur D. Stem, Keegan L. Rogers, and Jared M. Brown
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of Colorado, Anschutz Medical Campus, Aurora, CO, 80045
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BACKGROUND
METHODS
Multiple epidemics of chronic kidney disease of an unknown etiology
(CKDu), primarily in young healthy agricultural workers, have
emerged in agricultural communities around the world. It is proposed
that heat stress, dehydration and/or toxicant exposures may be a
cause of this emerging disease. We have hypothesized that the
harvest and burning of sugarcane leading to inhalation of sugarcane
ash may contribute to development of CKDu. Sugarcane stalks
consist of ~80% amorphous silica and we have demonstrated that
following burning of sugarcane, nano-sized silica particles (~200 nm)
are generated.
1. An immortalized human proximal convoluted tubule cell line
(HK-2), were seeded in 96 well plates at a density of 20,000
in standard/heat stress (37/39ºC) conditions.
2. Cells were exposed to media containing sugarcane ash (Ash)
sourced from Nicaragua, desilicated ash (D/S Ash), silica ash
derived silica nanoparticles (SAD Particles), and pristine
manufactured 200nm silica nanoparticles.
3. Viability was determined with a membrane integrity/esterase
activity fluorescence assay and compared to mitochondrial
activity (colorimetric tetrazolium MTS assay). Mitochondrial
membrane permeability changes were investigated with a JC-
1 fluorescence-based assay.
4. A Seahorse XFe96 Analyzer was used to detect changes to
oxygen concentration and pH, allowing for quantification of
mitochondrial respiration and glycolytic function parameters.
5. Metabolomics (UHPLC-MS with Absolute quant) were
performed on cells following 24 hours of exposure in order to
detect perturbations to glycolysis and TCA cycle. GSH-Glo (a
luminescence-based assay) was used to quantify glutathione
levels in cell cultures.
• Exposure to sugarcane ash alters
cellular metabolism and
mitochondrial membrane potential.
• SAD particle exposure reduces
mitochondrial respiration/ATP
production within 6 hours.
• SAD particles promote a short-
term shift to a glycolytic energy
phenotype, increasing glycolytic
activity and prolonging cell survival.
• Overreliance on glycolysis reduces
energetic flexibility and glycolytic
capacity, leading to a subsequent
reduction of glycolysis associated
metabolites and an increase in
fatty acid metabolism associated
metabolites.
• SAD particle exposure perturbs cell
energy metabolism, resulting in a
stress response, changes to
glutathione homeostasis, and
eventual cell death.
CONCLUSIONS
FUTURE DIRECTIONS
R01DK125351
REFERENCES & FUNDING
Analyze generation of reactive oxygen
species following treatment.
Measure extent of cellular
nanoparticle uptake and correlate to
degree of metabolic effects.
Perform metabolomics on agricultural
worker biomatrices to determine if
similar metabolic shifts occur and
establish physiologic relevance.
Correa-Rotter, R., Wesseling, C. & Johnson, R. J. CKD
of Unknown Origin in Central America: The Case for a
Mesoamerican Nephropathy. Am. J. Kidney Dis. 63,
506–520 (2014).
Jayasekara, K. B. et al. Relevance of heat stress and
dehydration to chronic kidney disease (CKDu) in Sri
Lanka. Prev. Med. Rep. 15, 100928 (2019).
Rovani, S., Santos, J. J., Corio, P. & Fungaro, D. A.
Highly Pure Silica Nanoparticles with High Adsorption
Capacity Obtained from Sugarcane Waste Ash. ACS
Omega 3, 2618–2627 (2018).
Please Direct Any Questions To
Arthur.Stem@cuanschutz.edu
Figure 3: All treatments cause a slight, but not statistically significant increase in glycolysis at 6 hours of exposure. SAD particle treatment maximized glycolytic
activity (as indicated by reduced glycolytic reserve) by 6 hours. This is followed by reduced glycolysis after 24 hours of exposure, suggesting a shift to a
glycolytic energy phenotype following mitochondrial shutdown. Data is presented as mean and SEM (N=3). Data was analyzed with Welch’s ANOVA (P value
<0.0001) and Dunnett’s T3 multiple comparison test for group-to-control comparisons (P value <0.05).
Figure 2: Ash and D/S treatments cause a significant increase in mitochondrial respiration suggestive of a stress response, pristine 200nm particles do not
cause a significant change relative to control, SAD particles result in a major reduction to mitochondrial respiration and ATP generation as early as 6 hours of
exposure. Data is presented as mean and SEM (N=3). Data was analyzed with Welch’s ANOVA (P value <0.0001) and Dunnett’s T3 multiple comparison test
for group-to-control comparisons (P value <0.05).
RESULTS
6
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100
****
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Mitochondrial Activity
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****
Percent
Relative
toControl
Viability
0 20 40 60 80
0
20
40
60
80
Time (minutes)
Glucose Oligomycin 2-DG
Ash 25µg/mL
D/S 25µg/mL
SAD 2.5µg/mL
200 25µg/mL
Control
24hr Glycolysis Stress Test
Extra
Cellular
Acidification
Rate
(mpH/min/10,000
cells)
0 20 40 60 80
0
20
40
60
80
100
120
Time (minutes)
Ash 25µg/mL
D/S 25µg/mL
SAD 2.5µg/mL
200 25µg/mL
Control
Oligomycin FCCP Rot/AA
24hr Mito Stress Test
Oxygen
Consumption
Rate
(pmol/min/10,000
cells)
Figure 1: Viability (A) indicates significant cytotoxicity after 48 hours of SAD particle exposure, yet mitochondrial activity (B) only indicates a significant decline
at 24 hours, suggesting mitochondrial shutdown proceeds cell death. Changes to mitochondrial membrane potential (C) are indicative of a shift to a more
depolarized membrane following exposure to ash containing treatments. Data is presented as mean and standard deviation (N=3). Data was analyzed with
one-way ANOVA (P value <0.0001) and Tukey’s multiple comparison test for group-to-group comparisons (P value <0.0001).
A. B. C.
1.
2.
3.
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
50
100
150
ATP Generation
Percent
Relative
to
Control
***
**
****
****
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
50
100
150
Maximal Respiration
****
****
*
*
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
25
50
75
100
125
Glycolysis
Percent
Relative
to
Control
*
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
25
50
75
100
125
Glycolytic Reserve
****
****
0
h
r
1
h
r
2
h
r
3
h
r
4
h
r
5
h
r
6
h
r
7
h
r
8
h
r
40
60
80
100
120
2
4
h
r
3
0
h
r
3
6
h
r
4
2
h
r
4
8
h
r
Percent
Relative
to
Control
Ash 25µg/mL
D/S 25µg/mL
SAD 2.5µg/mL
200 25µg/mL
Mitochondrial Membrane Potential
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
50
100
150
Basal Respiration
*
**
****
****
6hr 24hr 6hr 24hr 6hr 24hr 6hr 24hr
0
25
50
75
100
125
Glycolytic Capacity
** *
***
Ash D/S SAD 200
0.0
0.5
1.0
1.5
D-Fructose 1-6-bisphosphate
Fold
Change
Relative
to
Control
*
4.
Ash D/S SAD 200
0.0
0.5
1.0
4.0
4.5
5.0
5.5
6.0
Acetyl-Carnitine
Fold
Change
Relative
to
Control
**
Ash D/S SAD 200
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Creatine
Fold
Change
Relative
to
Control
**
Ash D/S SAD 200
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
5-Oxoproline
Fold
Change
Relative
to
Control **
Ash D/S SAD 200
0.0
0.5
1.0
1.5
Fold
Change
Relative
to
Control
Glutathione
**
*
*
Figure 4: Metabolomic analyses reveal a statistically significant reduction to glycolysis associated metabolites (A) and statistically significant increase in those
associated with fatty acid metabolism (B) following 24 hours of exposure to SAD particles, indicating increased fatty acid metabolism activity following
glycolytic shutdown. This energy metabolism perturbation occurs alongside indications of cell stress response (C) and alterations to glutathione homeostasis
(D). This is confirmed by direct measurement of glutathione levels in cultures at 24 hours of exposure (E). Data is presented as mean and SEM (N=4). Data
was analyzed with Welch’s ANOVA (P value <0.005) and Dunnett’s T3 multiple comparison test for group-to-control comparisons (P value <0.05).
A. B. C. D. E.