Chemosphere 257 (2020) 127186

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

The Influence of fluoride on chronic kidney disease of uncertain

aetiology (CKDu) in Sri Lanka
Shanika Nanayakkara a, b, *, S.T.M.L.D. Senevirathna c, Kouji H. Harada d,
Rohana Chandrajith e, Nishantha Nanayakkara f, Akio Koizumi g
a
School of Dentistry, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia
b
Institute of Dental Research, Westmead Centre for Oral Health, Westmead Hospital, Westmead, Australia
c
Faculty of Business, Justice and Behavioural Sciences, School of Engineering, Charles Sturt University, Bathurst, Australia
d
Department of Health and Environmental Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan
e
Department of Geology, Faculty of Science, University of Peradeniya, Sri Lanka
f
Kandy Teaching Hospital and Center for Research and Training on Kidney Diseases, University of Peradeniya, Kandy, Sri Lanka
g
Institute of Public Health and Welfare Research, Nakagyoku, Kyoto, Japan

h i g h l i g h t s

Fluoride in drinking water and serum of CKDu patients and healthy controls were evaluated.

Drinking water fluoride concentration in more than 95% of the samples were below the WHO guideline of 1.5 ppm.

The serum fluoride concentration of healthy controls was comparable with the levels reported from other healthy populations.

Data suggests that when renal function decreases fluoride tends to accumulate in the body.

A health-based target guideline for fluoride in drinking water for the CKDu affected region in Sri Lanka is recommended.

a r t i c l e i n f o a b s t r a c t

Article history: Fluoride is an element that is widely distributed in the environment. The involvement of fluoride in
Received 14 April 2020 pathogenesis of Chronic Kidney Disease of uncertain aetiology (CKDu) in Sri Lanka is a much-debated
Received in revised form topic. This study aimed to investigate the fluoride concentration in drinking water in CKDu affected
18 May 2020
areas in Sri Lanka and to evaluate the possible effect of renal impairment on serum fluoride levels in
Accepted 21 May 2020
Available online 27 May 2020
CKDu patients.
Drinking water (n ¼ 60) from the common water sources from two CKDu prevalent areas and serum
Handling editor: Veeriah (Jega) Jegatheesan samples of CKDu patients (n ¼ 311) and healthy controls (n ¼ 276) were collected. Both environmental
and biological samples were analysed for the concentration of fluoride. The fluoride concentration in
Keywords: over 95% of drinking water samples was below the WHO guideline of 1.5 mg/L. Serum fluoride con-
Chronic kidney disease of uncertain centrations in majority of the unaffected and early-stage CKDu patients (stages 1 and 2, eGFR >60 ml/
aetiology min/1.73m2) were below the normal upper concentration of 50 mg/l and significantly higher levels were
Fluoride observed in patients in late stages of CKDu compared to the healthy controls. The available guidelines for
Fluoride toxicity
drinking water are solely based on healthy populations with normal renal function. But, it is evident that
Fluoride bioaccumulation
once the kidney function is impaired, patients enter a vicious cycle as fluoride gradually accumulates in
Drinking water
Fluoride guidelines the body, further damaging the kidney tissue. Thus, close monitoring of serum fluoride levels in CKDu
patients and establishing health-based target guidelines for fluoride in drinking water for the CKDu
patients are recommended to impede the progression to end stage renal disease.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction

Fluorine is a ubiquitous element in the environment and natu-

* Corresponding author. School of Dentistry, Faculty of Medicine and Health, The
rally occurs in varying amounts in different geographical locations
University of Sydney, Sydney, NSW, 2006, Australia.
E-mail address: shanika.nanayakkara@sydney.edu.au (S. Nanayakkara). in its anionic form, fluoride. Fluoride enters the human body mostly

https://doi.org/10.1016/j.chemosphere.2020.127186
0045-6535/© 2020 Elsevier Ltd. All rights reserved.
2 S. Nanayakkara et al. / Chemosphere 257 (2020) 127186
through drinking water and the diet. After fluoride enters the
gastrointestinal tract, it is rapidly absorbed into the body by a
process of diffusion and distributed to the tissues through the
systemic circulation (Rashid et al., 2013). Calcified tissues such as
bone and teeth rapidly uptake about 50% of fluoride from the cir-
culation and the rest is primarily excreted in urine to maintain a
serum fluoride concentration of 10-50 mg/L (Joshi et al., 2011). In the
kidneys, fluoride is freely filtered through glomerular capillaries
and as it passes through the tubular system, a variable degree of re-
absorption occurs (Whitford, 1994). Renal clearance of fluoride is
around 30-40 mL/min in healthy adults and is directly related to the
glomerular filtration rate (Spak et al., 1985; Ludlow et al., 2007).
Therefore, the kidneys play a pivotal role in regulating the con-
centration of fluoride in serum and preventing the accumulation of
fluoride to toxic levels.
Fluoride is considered beneficial to human health in low doses.
Thus, water fluoridation has been recognised as one of the major
public health achievements during the 20th century. Extensive
literature on the benefits of fluoride suggests protective effects of
water fluoridation on reducing dental caries. International health
and dental organizations, including the World Health Organization
(WHO) and the International Association for Dental Research have
strongly supported this initiative as a safe and effective way of
preventing tooth decay (National Health and Medical Research
Council, 1999; McDonagh et al., 2000; Council, 2007; Ludlow
et al., 2007).
However, existing evidence suggests that at toxic concentrations
fluoride can exert pathological effects on different organs and body
systems with the skeletal system, teeth, kidneys, liver and brain
being the main targets (Hodge, 1968; Ludlow et al., 2007; Xiong
et al., 2007; Yang and Liang, 2011). Previous studies, mostly on
animals have suggested different pathophysiological mechanisms
for fluoride induced organ damage such as oxidative stress, cell
cycle arrest, altering gene expressions and cell apoptosis (Barbier
et al., 2010; Ozbek, 2012; Rashid et al., 2013). Some research sug-
gests that there is a possibility for systemic fluorosis in patients
with diminished renal function due to impaired excretion of fluo-
ride (Juncos and Donadio, 1972). Thus, these patients have rela-
tively lower margin of safety than a healthy person when it comes
to the adverse effects of fluoride.
Chronic kidney disease (CKD), a condition characterised by the
gradual loss of kidney function is one of the most prevalent non-
communicable diseases in both developed and developing coun-
tries causing a significant global health and economic burden (Hill
et al., 2016). In most of the countries, the high prevalence of CKD is
due to an ageing population and an increasing incidence of hy-
pertension and diabetes (Gansevoort et al., 2013; Hill et al., 2016).
However, epidemiological evidence demonstrates that in some
geographical regions of a number of tropical/subtropical countries
such as Sri Lanka, India and some countries in Central America, a
substantial number of CKD patients in low socioeconomic, agri-
cultural communities do not have such identifiable aetiology for
the disease (Jayatilake et al., 2013; O’Callaghan-Gordo et al., 2019).
Thus, this disease entity is named as chronic kidney disease of
uncertain aetiology (CKDu). In Sri Lanka, the rapid progressions of
kidney damage in these CKDu patients, leading to end stage renal
disease (ESRD) is a major problem due to lack of sufficient resources
for renal replacement therapy. Hence, impeding disease progres-
sion is crucial to minimize CKDu related deaths in this community.
The possible involvement of fluoride in the pathogenesis of CKDu in
Sri Lanka has been a much debated topic for several years (Wasana
et al., 2016; Fernando et al., 2019), but evidence on the role of
fluoride on CKDu is inconsistent. Thus, the objective of this study is
to assess the fluoride content in drinking water collected from
CKDu affected regions in Sri Lanka and to investigate the effect of
renal impairment on the serum fluoride levels in CKDu patients at
different stages of the disease. This information will open an
avenue to consider the requirements to reset the guidelines for
total fluoride intake for the CKDu affected regions.
2. Materials and methods
2.1. Dataset
The data of serum fluoride analysis has been originally pub-
lished as a supplementary figure in one of our previous publications
on aetiological factors of CKDu in Sri Lanka (Nanayakkara et al.,
2013). This paper presents a secondary analysis of the data with a
focus on disease progression.
This study was approved by the human research ethics com-
mittees at the University of Peradeniya (Sri Lanka) and Kyoto Uni-
versity (Japan). All human samples and information were obtained
following informed written consent and the study was performed
in accordance with the Declaration of Helsinki.
2.2. Sample collections
Drinking water samples (n ¼ 60) were collected from common
water sources in two CKDu prevalent areas (Girandurukotte and
Medawachchiya) into polypropylene tubes that were rinsed with
nitric acid and then with purified water before sampling. Sample
locations were randomly selected to represent different water
sources such as dug wells, tube wells, surface water sources and
treated water which provide drinking water to the affected
communities.
Venous blood samples were collected into K-EDTA tubes from
male patients diagnosed with CKDu (biopsy proven renal tubu-
lointerstitial disease, uncontrolled hypertension or diabetes at the
time of initial diagnosis, negative immunofluorescence for IgG, IgM,
IgA, and C3, serum creatinine >1.2 mg/dL and/or A1M > 15.5 mg/L,
HbA1C<6.5%) (n ¼ 311) and healthy controls (no history of hyper-
tension, diabetes or renal impairment, blood pressure not more
than 140/90 mmHg, no proteinuria or glycosuria based on the
dipstick urine test, HbA1C<6.5%, serum creatinine <1.2 mg/dL and/
or A1M < 15.5 mg/L) (n ¼ 276) following informed written consent.
Samples were immediately centrifuged at 3000 rpm for 10 min to
separate serum. All the collected samples were shipped to Japan at
-20  C and stored at -30  C until analysis.
2.3. Sample analysis
Flow injection analysis with a fluoride ion-selective electrode
was used to analyse ionic fluoride concentrations (Itai and Tsunoda,
2001). The system comprised two double-plunger backflow pumps
(Uniflo se, Tokyo, Japan), an injector, a flow cell, a fluoride electrode
(Orion 94-09, Orion Research, Cambridge, MA) and a reference
electrode (Model 4400, DKK, Tokyo, Japan). Purified water prepared
by Milli-Q systems (Millipore, Tokyo, Japan) was used as the carrier.
Each sample (0.2 ml) was mixed with 1.2 ml of acetate buffer (pH
adjusted to 5.4) and centrifuged supernatant was injected into the
system.
2.4. Statistical analysis
IBM SPSS Statistics (version 23, IBM SPSS Inc., Chicago, IL) was
used to perform statistical analysis of the collected data. Descriptive
data was presented using mean and standard deviations. Means
were compared using the analysis of variance (ANOVA) test and
p  0.05 was considered statistically significant.
 S. Nanayakkara et al. / Chemosphere 257 (2020) 127186
3. Results and discussion
3.1. Fluoride concentration in drinking water
Groundwater serves as the primary drinking water source in the
CKDu affected regions studied. Previous reports suggest high levels
of naturally occurring fluoride in groundwater sources in some
parts of the dry zone in Sri Lanka (Chandrajith et al., 2012). The
distribution of fluoride concentration in drinking water samples is
demonstrated in Fig. 1. The mean fluoride concentration in drinking
water samples analysed in this study was 0.68 mg/L (SD ¼ 0.48),
with a minimum of 0.007 mg/L and a maximum of 2.03 mg/L. Only
two out of 60 samples had a fluoride concentration more than the
World Health Organization (WHO) guideline of 1.5 mg/L (3.33%)
(Organization, 1993; Fawell et al., 2006; Edition, 2011) but, 18
samples exceeded the Sri Lanka drinking water standards guideline
which is 1 mg/L (30%) (Sri Lanka Standards Institution, 2013).
The World Health Organization has emphasized that fluoride is
an essential element for all living organisms and prevents com-
plications related to teeth pathophysiology in low doses, but it can
have adverse health outcomes, if exposed to higher doses (World
Health Organization, 1984). The total fluoride intake of an indi-
vidual mainly depends on drinking water but other sources of
fluoride such as diet, oral health products and air also contribute
(Jime
nez-Co
rdova et al., 2018). The WHO has established a general
standard for fluoride in drinking water (1.5 mg/L) but, recommends
a maximum intake of 6 mg/day when setting national and local
guidelines (Organization, 1993; Fawell et al., 2006; Edition, 2011).
Water consumption depends on temperature, humidity, exercise
and the state of health of an individual. For example, the water
consumption of residents living in countries close to the equator is
relatively higher than the other parts of the world (Murray and
Organization, 1986). Thus, it is important to note that even if the
fluoride concentration in drinking water is below 1.5 mg/L, there is
a possibility for consumption of over the maximum allowable daily
intake in people living in hot climates and exposed to sun for longer
hours.
Considering both the beneficial and adverse effects of fluoride,
drinking water standards are set to allow sufficient fluoride intake
to prevent dental caries and to avoid possible adverse health out-
comes such as dental and skeletal fluorosis in otherwise healthy
Fig. 1. Fluoride concentration in drinking water.
3
populations. In addition to WHO standards, different countries
have established guidelines for fluoride content in drinking water
considering the variations in the above mentioned factors. For
example, the National Health and Medical Research Council
(NHMRC) of Australia recommends fluoride content in drinking
water within a range of 0.6e1.1 mg/L to ensure the population re-
ceives the beneficial effects of fluoride while avoiding the adverse
effects (National Health and Medical Research Council, 2017;
National Health and Medical Research Council, 2017).
3.2. Fluoride in serum
Table 1 summarizes the demographic and clinical characteristics
of CKDu patients and the healthy controls who participated in this
study. When compared with the healthy controls, the CKDu pa-
tients were relatively older. Variations in blood pressure observed
are likely due to the antihypertensive medications prescribed for
CKDu patients. a1-Microglobulin, which is a known marker for
renal tubular damage (Nanayakkara et al., 2012), showed a signif-
icantly higher excretion pattern in CKDu stages 3e5 when
compared with the healthy controls, indicating progressive damage
to the renal tubular system (see Table 1).
Serum sample analysis for fluoride demonstrated that the
serum fluoride concentrations in majority of the unaffected and
early-stage CKDu patients (stages 1 and 2, eGFR >60 ml/min/
1.73m2) were below the reported normal upper concentration of
50 mg/L (Joshi et al., 2011) (Table 2, Fig. 2). When compared with the
healthy controls, except stage 1 patients, all the other stages had
significantly higher mean serum fluoride concentrations (Table 2).
This observation supports the existing evidence that when renal
function declines, fluoride tends to accumulate in the body due to
impaired excretion (Spencer et al., 1980; Torra et al., 1998; Ludlow
et al., 2007). Fig. 1 also clearly demonstrates the trend of the
gradual retention of fluoride in the body with the deterioration of
renal function.
The available evidence, mostly based on animal studies reports
that the detrimental effects of fluorosis are not limited to the
skeletal system and teeth but also affect organs such as the brain,
liver and kidneys (Dote et al., 2000; Shashi and Thapar, 2001; Xiong
et al., 2007). Approximately 60% of the total daily fluoride absorbed
to the human body is filtered and excreted in urine and kidneys
have the ability concentrate fluoride in urine up to 50-fold as in
plasma. Hence, kidneys become a key target organ in fluorosis
(Jime
nez-Co
rdova et al., 2018). The oxidative stress mediated
mechanism of renal tissue injury in excess fluoride exposure is well
documented based on in-vitro studies and animal studies
(Anuradha et al., 2001; Barbier et al., 2010; Jime
nez-Co

rdova et al.,
2018). Some studies have also indicated that fluoride can alter gene
expression and induce cell apoptosis leading to organ damage
(Zhang et al., 2008; Barbier et al., 2010). It is noteworthy that these
adverse effects of fluoride on human organs are closely related to
the dose and concentration of exposure.
As discussed, it is evident that once the renal function is
impaired, it can cause an accumulation of fluoride in the human
body causing further damage to the renal tissue and accelerating
the disease progression. This suggests that CKDu patients are more
sensitive to fluoride toxicity and have a lower margin of safety for
fluoride induced adverse effects than a healthy person with normal
renal function. Recommendation of non-fluoridated water to pa-
tients with CKD was a much-debated topic from the time the
artificial fluoridation came into action. However, the United Sates
National Kidney Foundation (NKF) or Kidney Health Australia
(KHA) have not issued specific recommendations regarding fluo-
ride intake and kidney disease due to the limited available research
evidence on this topic. In a report issued by NKF in 2008, it was
4 S. Nanayakkara et al. / Chemosphere 257 (2020) 127186

Table 1
Demographic and clinical characteristics of the participants.

CKD Stage 0 1 2 3 4 5
(n ¼ 276) (n ¼ 10) (n ¼ 60) (n ¼ 160) (n ¼ 72) (n ¼ 9)

Age (yrs) 41.1 (8.2) 42.1 (14.4) 43.4 (9.5) 46.9 (8.4)* 48.7 (8.6)* 49.1 (7.7)*
Duration of stay in the study area (Years) 32.77 (12.26) 27.11 (12.49) 36.07 (10.76) 38.47 (12.32)* 44.23 (11.23)* 32.44 (13.27)
Blood Pressure eSystolic (mmHg) 129.15 (12.26) 122.00 (11.03) 122.74 (15.80)* 127.12 (21.30) 119.53 (15.32)* 127.50 (11.94)
Blood Pressure-Diastolic (mmHg) 74.80 (9.62) 67.78 (8.90) 69.22 (10.44)* 73.24 (13.54) 67.51 (12.64)* 74.00 (8.62)
a1-microglobulin (mg/gCr) 1.61 (2.61) 12.62 (14.81) 5.71 (9.06) 15.74 (19.24)* 37.63 (36.18)* 63.44 (45.89)*

CKD stage classification is based on the National Kidney Foundation Kidney Disease Quality Outcome Initiative (K/DOQI) guidelines (Levey et al., 2005); *p < 0.05 when
compared with the healthy controls.

Table 2
Estimated glomerular filtration rates and Serum fluoride concentrations of the CKDu cases and the controls.

CKD Stage 0 1 2 3 4 5
(n ¼ 276) (n ¼ 10) (n ¼ 60) (n ¼ 160) (n ¼ 72) (n ¼ 9)

eGFR (mL/min/1.73m2) 104.2 (17.2) 102.4 (8.9) 70.7 (8.8) 43.7 (8.4) 23.1 (4.1) 11.0 (3.0)
Serum fluoride Concentration (mg/L) Mean (SD) 35.5 (16.3) 38.1 (18.1) 53.9 (34.2)* 82.8 (41.9)* 123.4 (59.9)* 123.9 (52.6)*
95% CI for mean 33.6e37.5 24.2e52.0 45.0e62.7 76.3e89.4 109.3e137.6 94.2e172.8
Min-Max 9.5e99.0 21.0e72.5 12.7e175.1 24.3e256.7 43.6e367.0 39.9e178.4
Percentage with >50 mg/l 17.4 20.0 36.7 78.8 97.2 88.9

CKD stage classification is based on the National Kidney Foundation Kidney Disease Quality Outcome Initiative (K/DOQI) guidelines (Levey et al., 2005); *p < 0.05 when
compared with the healthy controls.

the initiation of renal tissue damage leading to CKD, there is evi-

dence that once renal function is impaired, fluoride retention can
exert a potential risk of further damage to the renal tissue. In Sri
Lanka, parallel with the efforts to identify the aetiology for CKDu,
there is an urgent need to identify the potential risk factors
involved in the rapid progression of CKDu to ESRD. Controlling the
disease progression and reducing the rate of CKDu patients pro-
gressing to a stage requiring costly and resource intensive man-
agement will help to reduce the overall burden on the healthcare
system and also CKDu related deaths which is one of the most
common causes of hospital mortality in CKDu affected regions in Sri
Lanka (http://www.health.gov.lk/moh_final/english/). This study
suggests that monitoring and controlling the serum fluoride con-
centration in CKDu patients is important to consider in patient
management. Further research is required to identify the threshold
of tolerance for fluoride exposure to establish a safe drinking water
fluoride concentration for CKD patients.

Fig. 2. Variation of serum fluoride concentration with eGFR at different CKD stages. 4. Conclusion
CKD stage classification is based on the National Kidney Foundation Kidney Disease
Quality Outcome Initiative (K/DOQI) guidelines (Levey et al., 2005). Serum samples from CKDu patients and healthy controls and
water samples from their common drinking water sources were
analysed for fluoride content. Over 95% of water samples met the
stated that "there is insufficient evidence at this time to recom-
WHO guideline of 1.5 mg/L. CKDu patients showed significantly
mend the use of fluoride-free drinking water for all patients with
higher serum fluoride concentrations than the healthy controls.
renal diseases" (United States National Kidney Foundation, 2008)
The estimated glomerular filtration level was inversely propor-
and in the latest position statement of KHA, it states that the
tional to the serum fluoride concentration, indicating the accu-
NHMRC and KHA have not found any evidence to confirm a link
mulation of fluoride in the body with the progression of CKDu,
between fluoride in water at optimal levels and chronic kidney
which can further aggravate renal tissue damage. Thus, this study
disease (Kidney Health Australia, 2018). KHA further states that
highlights the importance of monitoring and controlling the serum
there is no evidence to support that the consumption of optimally
fluoride concentration in CKDu patients to prevent further renal
fluoridated drinking water increases the risk of developing CKD.
damage. It also recommends the development of new health-based
However, the report admits that there is consistent evidence that
target guidelines for fluoride in drinking water for CKDu affected
renal function impairment can affect fluoride metabolism, resulting
regions, to replace the current guideline values which are solely
in an increased burden of fluoride in the body and recommends
based on healthy populations with normal renal function. Further
monitoring of fluoride intake for patients with stages 4 and 5 CKD
research is required to identify the threshold of tolerance for
(Kidney Health Australia, 2018). Thus, in summary, even though
fluoride exposure to establish the safe drinking water fluoride
optimally fluoridated water cannot be considered a risk factor for
concentration for CKD patients.
 S. Nanayakkara et al. / Chemosphere 257 (2020) 127186
Declaration of competing interest
The authors declare that they have no conflict of interest.
CRediT authorship contribution statement
Shanika Nanayakkara: Conceptualization, Investigation,
Formal analysis, Writing - original draft. S.T.M.L.D. Senevirathna:
Conceptualization, Investigation, Formal analysis, Writing - review
& editing. Kouji H. Harada: Funding acquisition, Investigation,
Project administration, Writing - review & editing. Rohana Chan-
drajith: Writing - review & editing. Nishantha Nanayakkara:
Writing - review & editing. Akio Koizumi: Funding acquisition,
Supervision.
Acknowledgement
The authors would like to acknowledge the Special Coordination
Funds for Promoting Science and Technology from the Ministry of
Education, Culture, Sports, Science and Technology in Japan for the
funding support.
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