Clinical Characteristics of Chronic Kidney Disease of Nontraditional Causes in Salvadoran Farming Communities

INTRODUCTION In Central America, beginning in the 1990s, chronic kidney disease cases were reported unassociated with traditional risk factors (CKDnt), primarily affecting farming communities and male agricultural workers.[1] In 2000, it became apparent in Sri Lanka that a CKD of unknown etiology was affecting male rice farm-ers[2] and that its characteristics were similar to Balkan endemic nephropathy.[3] Recent reports of similar CKD cases in rural areas have come from Egypt[4] and India, where it is known as Udhanam endemic nephropathy.[5] In Central America, a number of publications have chronicled the disease: • García-Trabanino's 2002 study of dialysis patients in El Salva-dor (1999–2000), found that cause of renal failure could not be identifi ed in 67% of cases. This led to suspicion of a relationship with occupational exposure to insecticides or pesticides.[6] • In 2003, Domínguez analyzed CKD prevalence and risk factors on the Pacifi c coast of southern Mexico, Guatemala, El Salvador and Honduras, fi nding an inverse association between protein-uria prevalence and municipal altitude. Among men with protein-uria living on the coast (altitude ≤200 m), 71% had no signs of hypertension (HT) or diabetes. Agricultural work and contact with pesticides were common to persons with CKD at all altitudes.[7] • Research by Sanoff in León and Chinandega, Nicaragua (2003), reported endemic levels of CKD in young farmers, unrelated to diabetes or HT and associated with environmental and occupational exposures, working conditions, consumption of homemade liquor (lija) and drinking >5 L of water per day.[8] • Prevalence of renal replacement therapy was 12.5 cases/100,000 population in a study of dialysis and renal transplant patients in eight Salvadoran hospitals from August through November, 2003.[9] • A 2005 study by García-Trabanino in Salvadoran farming communities detected proteinuria in 45.7% of coastal residents versus 12.9% of those living at high altitudes. Elevated blood glucose levels were also more common in coastal areas than in areas ≥500 m above sea level (25% vs. 8%, respectively). Proteinuria was not signifi cantly related to agricultural work, pesticides or alcohol use.[10] • Torres' 2007 cross-sectional community study of CKD of unknown etiology in Nicaragua described prevalence of above-ABSTRACT INTRODUCTION Chronic kidney disease is a serious health problem in El Salvador. Since the 1990s, there has been an increase in cases unassociated with traditional risk factors. It is the second leading cause of death in men aged >18 years. In 2009, it was the fi rst cause of …


INTRODUCTION
In Central America, beginning in the 1990s, chronic kidney disease cases were reported unassociated with traditional risk factors (CKDnt), primarily affecting farming communities and male agricultural workers.[1] In 2000, it became apparent in Sri Lanka that a CKD of unknown etiology was affecting male rice farmers [2] and that its characteristics were similar to Balkan endemic nephropathy.[3] Recent reports of similar CKD cases in rural areas have come from Egypt [4] and India, where it is known as Udhanam endemic nephropathy.[5] In Central America, a number of publications have chronicled the disease: • García-Trabanino's 2002 study of dialysis patients in El Salvador (1999Salvador ( -2000)), found that cause of renal failure could not be identifi ed in 67% of cases.This led to suspicion of a relationship with occupational exposure to insecticides or pesticides.[6] • In 2003, Domínguez analyzed CKD prevalence and risk factors on the Pacifi c coast of southern Mexico, Guatemala, El Salvador and Honduras, fi nding an inverse association between proteinuria prevalence and municipal altitude.Among men with protein-uria living on the coast (altitude ≤200 m), 71% had no signs of hypertension (HT) or diabetes.Agricultural work and contact with pesticides were common to persons with CKD at all altitudes.[7] • Research by Sanoff in León and Chinandega, Nicaragua (2003), reported endemic levels of CKD in young farmers, unrelated to diabetes or HT and associated with environmental and occupational exposures, working conditions, consumption of homemade liquor (lija) and drinking >5 L of water per day.[8] • Prevalence of renal replacement therapy was 12.5 cases/100,000 population in a study of dialysis and renal transplant patients in eight Salvadoran hospitals from August through November, 2003.[9] • A 2005 study by García-Trabanino in Salvadoran farming communities detected proteinuria in 45.7% of coastal residents versus 12.9% of those living at high altitudes.Elevated blood glucose levels were also more common in coastal areas than in areas ≥500 m above sea level (25% vs. 8%, respectively).Proteinuria was not signifi cantly related to agricultural work, pesticides or alcohol use.[10] • Torres' 2007 cross-sectional community study of CKD of unknown etiology in Nicaragua described prevalence of above-

Original Research
normal serum creatinine levels (defi nition: >1.2 mg/dL in men and >0.9 mg/dL in wo men) of 31% in male and 24% in female agricultural workers in a community at 100-300 m altitude.[11] • According to the Latin American Society of Nephrology and Hypertension, in 2008, El Salvador reported 531 patients receiving renal replacement therapy per million population (pmp).Of these, 347 were on peritoneal dialysis, 121 on hemodialysis and 63 had received kidney transplants, fi gures above the mean for Central American countries of similar economic development levels.[12] CKD in El Salvador is a serious public health problem and its epidemiology is not completely understood.It is the fi fth leading cause of death nationwide in persons aged >18 years and the second cause of death in men.In 2009, prevalence of renal replacement therapy was 566 pmp.[13] According to the Ministry of Health's 2011-2012 Annual Report, end-stage renal disease (CKD stages 3-5) was the third leading cause of hospital deaths in adults of both sexes (fi rst for men and fi fth for women), with an in-hospital case fatality rate of 12.6%.[14] A study of farmers in fi ve Salvadoran communities-two on the coast devoted to growing sugarcane, three at 500 m altitude with economies focused on services and non-sugarcane crops-found prevalence of chronic renal failure (CRF) (glomerular fi ltration rate, GFR <60 mL/min/1.73m 2 body surface area) of 18% on the coast, compared to 1% in communities at >500 m.Proteinuria was infrequent, or low grade, with no differences among communities.The study concluded that sugarcane cultivation in coastal areas was associated with decreased kidney function in patients studied, possibly related to strenuous work in hot environments with repeated fl uid depletion.[15] The Nefrolempa Study (2009) in rural communities of the Bajo Lempa region reported an all-stage CKD point prevalence in adults of 17.9%, higher in men (25.7% vs. 11.8% in women).CRF (stages 3-5) prevalence was 9.8%, higher in men (17% vs. 4.1% in women).Neither diabetes nor HT, nor any other primary renal disease accounted for the majority (54.7%) of cases.[16] Other population-based research in El Salvador found a CKD prevalence in adults of 15.4% (men 22.8%, women 9.5%) and CRF prevalence of 8.8% (men 15.9%, women 3.2%).CKD point prevalence observed varied from 13.3% to 21.1% (men 13.1%-29%, women 13.4%-21.5%).CRF point prevalence was 13.3%, higher in men (22.4% vs. 3% in women).[17] Published overviews of CKDnt in the Central American region conclude that the disease has not been completely studied clinically or histopathologically. [1,18] This is also the case for El Salvador, where knowledge of its etiology, frequency and distribution in both the general population and in agricultural communities is incomplete, as is knowledge of its epidemiology, toxicoepidemiology, etiology and pathophysiology, anatomical pathology and clinical manifestations.This study's objective was to characterize CKDnt's clinical manifestations (including extrarenal) and pathophysiology in Salvadoran farming communities.

METHODS
A

Psychological assessment
From in-depth patient interview: general personality traits (extraversion, self control stability, independence); behaviors Emotional state (including reaction to disease): [20]

Chronic kidney disease[21]
Abnormalities of kidney structure or function, present for >3 months, with implications for health Criteria: markers of kidney damage or GFR <60 mL/min/ Ethics The study was conducted at the request of the Ministry of Health of El Salvador (MINSAL, the Spanish acronym) and the research protocol was approved by the Salvadoran National Committee on Clinical Research of the Higher Council on Public Health.Written informed consent was obtained from all participants.The consent form described study objectives, benefi ts and risks, and provisions for confi dentiality of the information obtained; and assured patients that they could withdraw at any time with no consequences for their medical care.Study participants received medical treatment as indicated by study fi ndings.

Analysis
LimeSurvey (an open-source online survey application installed on MINSAL's server) was used to input and store information, and to design clinical history and data forms for access to all data-individual and aggregate-obtained.Data were fi ltered with the platform's built-in tool and other software needed for specifi c purposes.Design included procedures to validate data before entry into the database, inclusion of imaging and other binary elements, and geocoding of each participant's home and workplace.Information was entered into a database and exported to SPSS for calculation of frequencies (point estimates and 95% confi dence intervals) of study variables.Of the 46 patients studied, 36 (78.3%) were men and 10 (21.7%) were women (Table 2).

Social determinants
The sociological study found that patients' community habitat is characterized by poverty, homes in poor condition, poor quality drinking water, low educational level, poor diet, inadequate health services, and inadequate domestic electricity.In addition, farmers' working conditions are characterized by indiscriminate use of agrochemicals (combining several at once, some prohibited, without protection for the farmer and with consequent environmental contamination), as well as long hours of intense outdoor physical activity, and profuse sweating in the absence of adequate hydration.

Psychological characterization
The assessment of personality traits and mental health indicators showed that the psychological sphere and behavior patterns of these patients were characterized by anxiety, depression, bargaining phase subsequent to denial of their illness, domestic violence, alcohol consumption and fear of dying.
Renal symptoms Disorders of micturition were the most common symptoms: nycturia in 30 patients (65.2%); dysuria in 18 (39.1%);post-void dribbling in 15 (32.6%); urinary hesitancy in 9 (19.6%); and foamy urine in 29 (63%).All symptoms were seen as early as stage 2 and tended to increase as the disease advanced.Some, including thin stream, urinary hesitancy, and dysuria were more evident from the start.

Genitourinary system
The most common risk factors reported were contact with agrochemicals in 44 patients (95.7%), farming 41 (89.1%), male sex 36 (78.3%) and profuse sweating during the workday 35 (76%).Only two reported being diabetic.See Table 2 for prevalence of other risk factors.
Visual acuity showed typical age-related changes; fundoscopic, intraocular pressure and visual fi elds tests were normal.
Hemopoietic system Patients' mean hemoglobin was 14 g/dL (95% CI 13.5-14.4).Only 6 had below-normal hemoglobin values.No coagulation abnormalities were found.Respiratory system Spirometry and chest x-ray were normal in all patients.

Original Research
Skin No dermatological lesions were detected specifi c to damage from metal exposures.

DISCUSSION
The fact that patients with CKDnt come from farming communities, and that more are male farmers, with fewer women and adolescents, requires analysis of contextual factors related to participants' place of work and residence.Three general conditions that affect these patients were identifi ed: poverty, with all its repercussions; unhealthy working conditions; and a contaminated environment.These elements link the disease to deep social roots.These families' living conditions, coupled with the impotence of watching the disease's progression towards death of loved ones, with no solution in sight, have plunged them into a state of grief.
The relatively high prevalence of NSAID use could be related to the high proportion of patients reporting joint pain.This in turn could be due to their strenuous work, and more research is needed to determine whether a toxic component may also contribute to pain.The cramping and fainting described could be the result of hyponatremia, seen in almost half the patients.
Lower urinary tract symptoms reported by patients, evident from early stages of the disease, mimic lower urinary tract obstructive syndrome, but most patients had neither obstruction nor urinary infection.Similar symptoms, without urinary infection, have been reported in Nicaraguan farmers, a condition called chistata.[18] Neurotoxic bladder irritation is one possible cause.Foamy urine is the manifestation of macroalbuminuria, detected in most study participants.
The fact that few patients had a history of traditional CKD risk factors-diabetes, HT and obesity-makes it unlikely that CKDnt is caused by the same factors that drive the global CKD epidemic.
The main risk factors identifi ed were nontraditional ones: contact with agrochemicals, agricultural work, male sex, family history of CKDnt, history of malaria, profuse sweating and use of NSAIDs.The predominance of male farmers suggests work-related risk factors are important, but the appearance of the disease in women and adolescents implies that there are additional risk factors to which the general population is also exposed.This raises the possibility of chronic low-level exposure to environmental toxins from proximity to agricultural activities.
Normal kidney function increases risk of renal damage from environmental toxins due to the high volume of renal blood fl ow, since large quantities of toxic substances can pass through the kidneys.The kidney's capacity to concentrate substances through fi ltration, reabsorption and secretion can increase the toxicity of agents that in low concentrations would not lead to renal damage.CKD may be manifested in chronic tubular defects, as has been seen in chronic poisoning from cadmium, lead and other agents.Furthermore, patients with previous renal damage are vulnerable to toxicity of substances that normally are excreted in the urine.
[24] Thus, further studies are needed of environmental contamination and measurement of toxins in biological fl uids.
Research in Sri Lanka found elevated urinary arsenic concentrations in patients with CKD of unknown etiology and detected high cadmium concentrations in well water where patients lived.The authors considered pesticides a possible source of environmental contamination by these metals.[25,26] Moderately high levels of urinary and blood cadmium were found to be associated with a higher proportion of albuminuria and CKD in NHANES study participants in the USA.[27] Almost all our cases had contact with pesticides.
The markers of renal damage observed made clear-because of the absence of proteinuria >1g-that this is not a proteinuric glomerular disease.Rather, tubulointerstitial damage is suggested by biomarkers of tubular damage, such as elevated urinary β2 microglobulin levels.It has been posited that elevated β2 microglobulin, NGAL and NAG may be associated with damaged proximal renal tubules.[28,29] A study of eight Salvadoran farmers with CKDnt found elevated β2 microglobulin and NAG, and tubulointerstitial damage was corroborated by renal biopsy.[30] It has long been known that chronic exposure to high doses of cadmium is associated with decreased renal tubular reabsorption of β2 microglobulin, as shown in an early study of work-related renal disease.[31] Renal ultrasound and Doppler ultrasound ruled out other traditional causes of CKD such as polycystic disease, vascular nephropathy and obstructive nephropathy.In most cases, no lower urinary obstruction was detected in bladder, prostate and gynecological ultrasounds.
Electrolyte loss in urine-primarily magnesium, phosphorus, sodium and potassium-beginning in early stages of the disease, signifi es primarily tubular damage as the initial site of renal damage, which once more points to chronic tubulointerstitial nephropathy.The proximal tubule reabsorbs 60% of electrolytes, which is why this must be the segment most involved.This electrolyte loss explains the electrolyte polyuria and low concentrations of some electrolytes in blood, as well as the symptoms of cramping and fainting.The absence of acidosis could indicate a relative conservation of the distal segment of the nephron, with bicarbonate reabsorption and hydrogen ion excretion.[32] López-Marín's histopathological characterization of renal biopsies from these same patients corroborated that chronic tubulointerstitial nephropathy was the initial damage.[33] Histopathology studies in Sri Lanka had similar results.[34] Wijkström's study of eight Salvadoran patients identifi ed damage to both glomerular and tubulointerstitial compartments, but nearly all were in advanced stages of CKD, [30] when all tissue compartments are typically compromised.[19] However, this form of chronic tubulointerstitial nephropathy in Salvadoran farming communities has extrarenal manifestations not attributable to renal disease progression, which suggests factors that could damage the kidney and other organs at the same time.
The main complications of traditional CKD from its early stages are cardiovascular.[35,36] However, in our patients, only a small percentage were hypertensive, they had relatively low heart rates, and most had normal fi ndings on EKG, stress test and cardiac Doppler ultrasound.The fact that the study patients were relatively young and members of populations with very low prevalence of HT, diabetes, obesity and smoking, suggests that vascular damage from these factors is minimal and that the vascular damage observed is more likely caused by their CKD rather than the converse.The protective effect of exercise in physically demanding jobs is evident in the almost athletic performance of the patients on the treadmill test.These facts could make CKDnt an interesting clinical model for studying the cardiovascular impact of CKD isolated from other vascular risk factors.
Traditionally, CKD patients have a high prevalence of peripheral vascular disease from the confl uence of two pathological abnormalities-atherosclerosis and arteriosclerosis-that are more frequent in patients with obesity, diabetes and HT, the peripheral vascular damage progressing with CKD evolution.It has been reported that frequency of atherosclerotic plaques in carotid arteries is four time greater in CKD patients than in controls.[37,38] In contrast, our patients' carotids were relatively unharmed, with the main vascular damage occurring in the tibial arteries.Atherosclerosis in all upper arteries was rare, becoming more evident in the lower body and peaking in the tibial arteries.One hypothesis for this selective damage to tibial arteries could be their greater contact with toxic substances on the job.Farmers' legs, sometimes bare, are the parts most exposed to agrochemicals from spraying, which is done using backpack applicators at high ambient temperatures, with consequent vasodilation and opening of skin pores.A Japanese study of patients with chronic arsenic exposure, showed that this metal produced endothelial dysfunction from inhibition of endothelial nitric oxide synthase enzyme and decreased nitric oxide production, associated with overproduction of reactive oxygen species, both inductive mechanisms for vascular damage.[39] From early stages of the disease, symptoms of anterior motor neuron damage-refl ex disorders-were detected, as well as Babinski sign and myoclonus.The sensorineural hearing loss found did not correspond to deafness related to hereditary nephropathy (which shows predominantly glomerular damage and is accompanied by proteinuria).Uremic neurotoxicity does not explain the neurological symptoms we detected in early CKD stages.Heavy metal and pesticide exposure have been associated with nervous system diseases (Parkinson, Alzheimer, amyotrophic lateral sclerosis), dopaminergic system impairment, impaired nerve conduction velocity, diminished refl exes, irritability, memory loss and other diseases.[40,41] Almost all patients had fatty liver with normal enzymes; this was associated with the risk factors of dyslipidemia and/or alcohol consumption.It must be kept in mind that the liver is the main organ for metabolism and removal of toxins.Dyslipidemia was present in the majority of cases, possibly infl uenced by diets high in fats and calories, associated with metabolic disorders typical of CKD.[42] Paradoxically, HDL was normal or high in most patients, which could refl ect the protective effect of their physical activity.
People in farming communities are subjected to the same traditional CKD risk factors as the rest of the world's population.However, the minimal presence of traditional risk factors in study patients points to environmental and occupational factors that could act synergistically to exacerbate a predominant one.Agricultural workers are also exposed to many toxic substances contained in dozens of agrochemicals, many of them prohibited, yet used in large quantities and mixed together without protection.In addition, these farmers carry out intense physical activity during long hours in high temperatures, without adequate hydration.[15,43] It is noteworthy that, although the disease occurs primarily in male farmers, it also affects women and adolescents, who do not necessarily work in the fi elds.
The clinical picture of CKDnt in this study is consistent with the hypothesis of environmental toxic agents (heavy metals and chemicals) from natural sources or from human activity as the pathogenetic trigger.Such toxins could be present in air, soil, water and food; subject to transformation by weather, topography and land use; and transported by air, water, clothing and food.Occupation, behaviors and drinking water quality could facilitate chronic exposure through inhalation, ingestion and/or skin contact.
Different levels of exposure are possible: a consistently high level over time from multiple acute exposures becoming chronic, primarily affecting farmers; and chronic low-level exposure affecting the general population, as well as farmers.In both cases, there could be interaction with genetic susceptibility.
In addition to chronic circulation in the blood of toxins eliminated through the kidneys, in agricultural fi elds with high temperatures, these toxins also concentrate in the renal medulla under the effects of dehydration from profuse sweating and low fl uid intake.[15,18,24,44,45] Besides this cascade of events, other infl uences are undoubtedly at work, such as social conditions-poverty paramount among them-that increase the likelihood of renal damage from low birth weight due to maternal malnutrition, infectious diseases (such as malaria), diabetes, HT, alcohol consumption, NSAID use and other factors.[21] Chief among the study's limitations is its small sample size, insuffi cient for estimating extent and signifi cance of associations among such a large number of variables.Furthermore, we were unable to measure toxins in biological fl uids.On the other hand, this is the largest clinical study of CKDnt in the Americas to date, with the greatest multidisciplinary involvement and the most thorough treatment of clinical and pathophysiological aspects, and including study of women and adolescents.Also, since over half of study patients were in CKD stages 2 and 3a, it permitted analysis of disease course from early stages.

CONCLUSIONS
CKDnt in Salvadoran farming communities is associated with social and working conditions and behaves like a chronic tubulointerstitial nephropathy.It has extrarenal manifestations not attributable to the progression of renal disease, suggesting that the kidney damage is a component of a more systemic process.This is compatible with the hypothesis of multifactorial etiopathogenesis with environmental nephrotoxic agents at its core.Environmental and biological toxicology studies should further explore the working conditions of farmers and the behavior of this disease in women, children and adolescents in these communities.

7 a
one patient unable to complete testing b patients could have more than one abnormality Peer Reviewed [19]4 were aged <60 and in stages 2, 3a and 3b of the disease.[19]Ofthese patients, 60 gave informed consent to participate in the study and 46 met inclusion criteria.
descriptive clinical study was conducted, involving 46 participants identifi ed through population screening for CKD in 5018 persons in 11 agricultural communities in 4 regions of El Salvador; of them, 2388 were aged ≥18 years, of whom 431 had CKD, and of those

Table 1 : Variables* Variable Defi nition Imaging patterns on renal, bladder, prostate and gynecological ultrasound and on renal Doppler ultrasound
Normal or abnormal images and measurements based on international reference patterns for normal 1.73 m 2 Chronic kidney disease of nontraditional causes CKD not attributable to diabetes mellitus, hypertension, urological causes, primary or secondary kidney disease, or other systemic diseases General symptoms By human body organs and systems, reported in order of appearance Renal symptoms By human body organs and systems, reported in order of appearance Signs Present or absent by human body organs and systems, detected by physical examination at time of study or by specifi c tests Body mass index (kg/m 2 )[22] Serum creatinine (mg/dL) Normal: 0.53-1.2CKD stages[21] 1: Presence of damage markers, GFR ≥90 mL/min 2: Presence of damage markers.GFR 89-60 mL/min 3a: GFR 59-45 mL/min/1.73m 2 3b: GFR 44-30 mL/min/1.73m 2 4: GFR 29-15 mL/min/1.73m 2 5: GFR <15 mL/min/1.73m 2 Chronic renal failure (CRF): Stages 3-5

patterns on echocardiogram and cardiovascular Doppler ultrasound Normal
or abnormal images and measurements based on international reference patterns for normal

Nervous system function Fundus
[21]ye, visual fi elds, intraocular pressure, visual acuity Audiometry Liver function Liver enzymes (ALT, AST, GGT, ALP), bilirubin: normal or abnormal Liver ultrasound: normal or abnormal, based on imaging results *Except as noted, all reference values from: Manual of Clinical Laboratory Standards, Procedures and Reference Values.San Juan de Dios National Hospital, San Miguel, El Salvador[19] CH 2 O: free water clearance Cosm: osmolal clearance E-Cosm: electrolyte osmolal clearance EF-Cosm: electrolyte-free osmolal clearance GFR: glomerular fi ltration rate (per Modifi cation of Diet in Renal Disease formula)[21]