Chronic kidney disease of an unknown etiology (CKDu) presents a prominent and rapidly growing risk to coastal agricultural workers in tropical regions in Central America and Asia. CKDu presents as chronic tubulointerstitial nephritis that leads to end stage kidney disease. So far tens of thousands have died from the disease. Due to a lack of clinical symptoms, the disease is only diagnosed histologically. Risk factors for CKDu have been identified as heat stress, exercise, and dehydration. However, if these are rescued, CKDu still progresses. Workers in these regions are exposed to ash from burning crops, like sugarcane. Sugarcane Ash (SA) has been shown to contain a large amount of amorphous silica nanoparticles (SiNP). We hypothesize that exposure to SiNPs from SA drive pathogenesis of CKDu.

1. Characterization of Nano-Sized Silica in Agricultural Exposures and its
Role in CKDu Pathogenesis
Keegan L. Rogers1, Arthur Stem1, Carlos A. Roncal2, Richard J. Johnson2, and Jared M. Brown1
1Skaggs School of Pharmacy and Pharmaceutical Sciences, Anschutz Medical Campus, University of Colorado
2Division of Renal Diseases and Hypertension, Anschutz Medical Campus, University of Colorado
BACKGROUND
METHODS
RESULTS SUMMARY
FUNDING SOURCES
R01DK125351
n=3
II. RENAL TOXICITY OF SUGARCANE ASH AND NANO-SIZED SILICA
I. NANO-SILICA ANALYSIS BY SP-ICP-MS IN ASH AND KIDNEY BIOPSIES
Control CKDu
PAS
Darkfield
Figure 1. PAS-stained
histological sections of control
and CKDu patients viewed under
light and darkfield microscopy
REFERENCES
Chronic kidney disease of an unknown etiology (CKDu)
presents a prominent and rapidly growing risk to coastal
agricultural workers in tropical regions in Central America
and Asia. CKDu presents as chronic tubulointerstitial
nephritis that leads to end stage kidney disease. So far tens
of thousands have died from the disease. Due to a lack of
clinical symptoms, the disease is only diagnosed
histologically. However, Figure 1 shows a darkfield image of
a CKDu positive patient containing unknown deposits of
nanomaterial in the tubular region of the kidney.
Risk factors for CKDu
have been identified as
heat stress, exercise, and
dehydration. However, if
these are rescued, CKDu
still progresses. Workers
in these regions are
exposed to ash from
burning crops, like
sugarcane. Sugarcane
Ash (SA) has been shown
to contain a large amount
of amorphous silica
nanoparticles (SiNP). We
hypothesize that exposure
to SiNPs from SA drive
pathogenesis of CKDu
Single particle (SP) inductively coupled plasma- mass
spectrometry (ICP-MS), was used to analyze and characterize
the presence of SiNPs in different matrices.
Human kidney proximal convoluted tubule cells (HK-2) were
cultured for functional assays.
Sodium silicate (Na2SiO3) precipitation on sugarcane ash
followed by a biphasic sol-gel emulsion was used to generate
SAD SiNPs and silica-free ash.
Vimentin staining was used to determine EMT state.
FUTURE DIRECTIONS
A single particle ICP-MS approach was utilized to
characterize silica nanoparticle (SiNP) content in
sugarcane ash (SA).
SiNP content in SA seen with SP-ICP-MS were
corroborated by using a TEM and DLS approach.
SiNP content in a CKDu positive patient kidney biopsy
matched the representative SiNP content seen in SA. The
SiNP content was not present in the CKDu negative
patient.
There was a trend toward greater total SiNP content
present in CKDu patients against non-CKDu patients.
There was a significant increase in the odds that a patient
was diagnosed with CKDu given an elevated amount of
SiNP was found in their kidney biopsy.
SiNPs can be precipitated out of SA leaving purified SAD
SiNPS and DS ash.
SiNPs induce ROS and mitochondrial superoxide
generation in HK-2 cells. Following this, HK-2 cells undergo
epithelial-mesenchymal transition and cell death in a SiNP
dependent manner.
• Verify a correlation between SiNP
deposition in kidney biopsies with CKDu
in patients from alternative geographical
regions.
• Examine the mechanism by which EMT
contributes to CKDu pathogenesis in
vivo.
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).
0 200 400 600 800
0
2
4
6
8
10
Diameter (nm)
Frequency
Representative SiNP Population
in Sugarcane Ash
0 200 400 600 800
0
1
2
3
4
5
Diameter (nm)
Frequency
CKDu Positive Tissue Sample
0.054 Particles/μm3
Most Frequent Size:
193 nm
0 200 400 600 800
0
1
2
3
4
5
Diameter (nm)
Frequency
CKDu Negative Tissue Sample
0.006 Particles/μm3
Most Frequent Size:
Below LLODd
Average Size: 212 nm
CKD CKDu
0
5
10
15
20
Diagnosis Against
Particle Threshold
Number
of
Cases
Not Reached
Reached
OR: 7.8 (1.079 - 43.75)
p = 0.0384
CKD CKDu
0.00
0.01
0.02
0.03
0.04
0.05
190-230nm
SiNPs/
mm
3
Tissue
Diagnosis Against
SiNP Content
p = 0.054
A.
B. C.
A. B.
Figure 3. (Left) A)
Frequency histogram of the
size distribution of SiNP
content in sugarcane ash. B)
SiNP content in a CKDu
biopsy patient C) SiNP
contentbiopsy in a CKDu
negative patient. Black lines
represent individual detected
nanoparticle sizes and red
line represents a gaussian
non-linear distribution of the
frequency histogram.
Figure 4. (Above) A) Amount of detected SiNPs between 190
and 230 nm in CKD patients (n=18) and CKDu patients (n=8).
Error bars represent one standard deviation on either side of
the mean. B) Contingency graph displaying proportion of CKD
and CKDu patients reaching an elevated SiNP threshold in their
kidney biopsy where red represents patients reaching the
elevated SiNP threshold and black represents patients who do
not reach the elevated SiNP threshold. OR represents the odds
ratio of a patient being diagnosed with CKDu as a function of
reaching the elevated SiNP threshold in their kidney biopsy.
Values in parenthesis are the 95% CI. p < 0.05 represents a
significant result.
CellRox staining was used to determine ROS generation.
Sugarcane
ash
Silica-free
ash
Sugarcane ash
derived silica
nanoparticles
200nm silica
nanoparticles
CellROX MitoSOX Vimentin TUNEL
Figure 5. (Left) Microscopic analyses
to examine toxicological effects of
different SiNP and ash treatments on
human kidney proximal convoluted
tubule cells (HK-2 cells). Assays from
left to right are a CellROX assay for
ROS, a MitoSOX assay for
mitochondrial superoxide generation, a
vimentin stain to determine epithelial
mesenchymal transition state and a
TUNEL assay to determine cell death.
Cells were fixed with 4%
paraformaldehyde in PBS and
permeabilized in 0.2% Triton X-100 in
PBS and counterstained with 1μg/mL
DAPI.
T32ES029074
MitoSOX staining was used to determine generation of
mitochondrial superoxide.
The TUNEL assay was used to determine DNA strand
damage and cell death.
Sugarcane ash was collected in Nicaragua.
Pristinely synthesized 200nm silica nanoparticles were
purchased from NanoComposix.