Chronic interstitial nephritis in agricultural communities is a toxin-induced proximal tubular nephropathy
Authors: Benjamin A. Vervaet, Cynthia C. Nast, Channa Jayasumana, Gerd Schreurs, Frank Roels, Chula Herath, Nika Kojc, Vahid Samaee, Sonali Rodrigo, Swarnalata Gowrishankar, Christiane Mousson, Rajeewa Dassanayake, Carlos M. Orantes, Vincent Vuiblet, Claire Rigothier, Patrick C. D’Haese, Marc E. De Broe
Journal: Kidney International (2019)

Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity. Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity. In the last decade of the 20th century, alert clinicians in Central America and Sri Lanka described a large number of patients in their rural agricultural communities with relatively rapidly progressive chronic kidney disease (CKD) without hypertension or diabetes. Males were predominantly affected, including sugar cane workers in Central America and paddy farmers in Sri Lanka.1Weaver V.M. Fadrowski J.J. Jaar B.G. Global dimensions of chronic kidney disease of unknown etiology (CKDu): a modern era environmental and/or occupational nephropathy?.BMC Nephrol. 2015; 16: 2-8Crossref PubMed Scopus (79) Google Scholar In Central America the disease was named "Meso-American Nephropathy" (MeN), whereas in Sri Lanka, "CKD of unknown etiology" (CKDu) was the prefered terminology. In light of the comparable clinical, pathological, and epidemiological expression of the disease, the term Chronic Interstitial Nephritis in Agricultural Communities (CINAC) was proposed to describe both entities.2Jayasumana C. Orantes C. Herrera R. et al.Chronic interstitial nephritis in agricultural communities: a worldwide epidemic with social, occupational and environmental determinants.Nephrol Dial Transplant. 2017; 32: 234-241PubMed Google Scholar Classic presentations of CINAC are young men, primarily agricultural workers, with common socioeconomic and occupational determinants including a hot tropical climate, poverty, and exposure to potentially toxic agrochemicals through ingestion of contaminated food, drinking water from shallow contaminated wells, inhalation, and (in)direct skin contact. CINAC cases also have been identified among less exposed individuals including nonagricultural workers, women, and children who live in the same tropical environment.3Ordunez P. Nieto F.J. Martinez R. et al.Chronic kidney disease mortality trends in selected Central America countries, 1997–2013: clues to an epidemic of chronic interstitial nephritis of agricultural communities.J Epidemiol Community Health. 2018; 72: 280-286Crossref PubMed Scopus (27) Google Scholar Recent reports suggest that similar patterns of CKD may occur in the Uddanam region of Andhra Pradesh in India, Egypt, Tunisia, Senegal, and very recently in Peru.4Caplin B. Jakobsson K. Glaser J. et al.International collaboration for the epidemiology of eGFR in low and middle income populations—rationale and core protocol for the Disadvantaged Populations eGFR Epidemiology Study (DEGREE).BMC Nephrol. 2017; 18: 1Crossref PubMed Scopus (39) Google Scholar Clinically, CINAC is characterized by absent or low tubular proteinuria, CKD stages 3 to 4, small kidneys with irregular contours on imaging, and without traditional causes of CKD, such as glomerulonephritis, diabetes, and hypertension. Renal biopsies show varying degrees of tubular atrophy, interstitial fibrosis, interstitial inflammation, and glomerulosclerosis.5Athuraliya N. Abeysekera T. Amerasinghe P. et al.Uncertain etiologies of proteinuric-chronic kidney disaese in rural Sri Lanka.Kidney Int. 2011; 80: 1212-1221Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar,6Wijkström J. Jayasumana C. Dassanayake R. et al.Morphological and clinical findings in Sri Lankan patients with chronic kidney disease of unknown cause (CKDu): similarities and differences with Mesoamerican nephropathy.PLoS One. 2018; 13e0193056Crossref PubMed Scopus (37) Google Scholar The progression of renal disease is relatively rapid, with end-stage renal failure occurring over 4 to 10 years. However, no specific/sensitive "signature" lesions have been found to date to define CINAC and differentiate it from other chronic renal diseases. Two primary triggers have been proposed: toxic exposures in the agricultural communities and heat stress with repeated episodes of dehydration causing recurrent acute kidney injury leading to CKD. The dehydration hypothesis, however, cannot explain why the incidence of CINAC has increased in association with the increased use of agrochemicals in paddy farming in the 1990s, why CINAC is absent in hotter northern Sri Lanka, Cuba, and Myanmar where agrochemicals are rarely used, why there is a mosaic geographical pattern in CINAC endemic areas, why CINAC occurs in women, children, and adolescents who are not agricultural workers, and why there are extrarenal manifestations of CINAC.2Jayasumana C. Orantes C. Herrera R. et al.Chronic interstitial nephritis in agricultural communities: a worldwide epidemic with social, occupational and environmental determinants.Nephrol Dial Transplant. 2017; 32: 234-241PubMed Google Scholar,7Herath C. Jayasumana C. Mangala P. et al.Kidney diseases in agricultural communities: a case against heat-stress nephropathy.Kidney Int Rep. 2018; 3: 271-280Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Heat stress and dehydration may be contributory risk factors to disease development but cannot be assigned an isolated causal role in the development of CINAC. Here we present compelling evidence that CINAC is a lysosomal tubulopathy likely caused by a toxic substance or substances. All patients with CINAC were selected based on current clinical criteria.2Jayasumana C. Orantes C. Herrera R. et al.Chronic interstitial nephritis in agricultural communities: a worldwide epidemic with social, occupational and environmental determinants.Nephrol Dial Transplant. 2017; 32: 234-241PubMed Google Scholar Periodic acid–Schiff and Jones stained renal biospy sections demonstrated the classic histopathologic features associated with CINAC at all 4 locations2Jayasumana C. Orantes C. Herrera R. et al.Chronic interstitial nephritis in agricultural communities: a worldwide epidemic with social, occupational and environmental determinants.Nephrol Dial Transplant. 2017; 32: 234-241PubMed Google Scholar,6Wijkström J. Jayasumana C. Dassanayake R. et al.Morphological and clinical findings in Sri Lankan patients with chronic kidney disease of unknown cause (CKDu): similarities and differences with Mesoamerican nephropathy.PLoS One. 2018; 13e0193056Crossref PubMed Scopus (37) Google Scholar,8Wijkström J. Leiva R. Elinder C.-G. et al.Clinical and pathological characterization of Mesoamerican nephropathy: a new kidney disease in Central America.Am J Kidney Dis. 2013; 62: 908-918Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,9López-Marín L. Chávez Y. García X.A. et al.Histopathology of chronic kidney disease of unknown etiology in Salvadoran agricultural communities.MEDICC Rev. 2014; 16: 49-54Crossref PubMed Google Scholar: varying extents of tubular atrophy, tubulointerstitial fibrosis (sometimes striped), inflamed fibrosis, glomerulosclerosis, glomerulomegaly, and vascular hyalinosis/sclerosis (Supplementary Figure S1 and Supplementary Table S1). None of the biopsies showed evidence of uric acid deposition on hematoxylin and eosin stain or electron microscopy (EM). Jones staining revealed accumulations of silver positive light brown to black cytoplasmic granules in cortical tubular cells (Figure 1). These granules, optimally observed at magnifications ≥400×, varied in size from finely granular to prominent (≥ one-third of nuclear size), with discrete borders and a round to irregular/dysmorphic shape (Figure 1b, d, and f; Supplementary Figure S2). Tubular profiles ranged from unaffected to heavily involved, and affected tubular cells had few to several dozen granules observed on 2- to 4-μm sections (Figure 1). Serial sections stained with Jones, periodic acid–Schiff, and gamma-glutamyltranspeptidase 1, respectively, showed that the intracellular granules were confined to proximal tubule (PT) segments, including S1, S2, S3 (Figure 2; Supplementary Figure S3). These argyrophyllic granules were not found in distal tubules, identified by more closely apposed and cuboidal epithelial cells and absence of apical brush border (Figure 2a and b). PT cells (PTCs) containing large argyrophilic granules had a variety of appearances ranging from preserved height to flattening and simplification with and without loss of brush border in a diffuse to focal distribution (Figure 3). Focally, PTCs had apical blebs, with and without silver positive granules, protruding into the tubular lumen (Figure 3c; Supplementary Figure S4). These membrane-bound fragments appeared to shed in the lumen, although tangential cutting must be considered (Figure 3c; Supplementary Figure S4). Atrophic tubules often but not always were more likely to contain enlarged argyrophilic granules, although they also could be seen in preserved tubulointerstitial areas. Affected PTCs had a nonproliferative, nonapoptotic phenotype, in contrast to distal tubular cells (Figure 410Nayak A. Raikar A. Kotrashetti V. et al.Histochemical detection and comparison of apoptotic cells in the gingival epithelium using hematoxylin and eosin and methyl green-pyronin: a pilot study.J Indian Soc Periodontol. 2016; 20: 294-298PubMed Google Scholar,11Singh N.P. A simple method for accurate estimation of apoptotic cells.Exp Cell Res. 2000; 256: 328-337Crossref PubMed Scopus (156) Google Scholar).Figure 4Proximal tubules (PTs) with lysosomal accumulation have minimal to no proliferative activity or apoptosis. Autofluorescence revealing Chronic Interstitial Nephritis in Agricultural Communities (CINAC)–affected proximal tubules (a). The same section as in (a) stained for the proliferation marker proliferating cell nuclear antigen (b). There were very few scattered proliferating epithelial cells in the affected PTs; in contrast, distal nephron cells demonstrated prominent proliferative activity. In 25% of distal tubules (DTs), the proliferative distal tubular epithelium has scattered apoptotic cells (arrows) as evidenced by the typical condensed nuclei surrounded by a bright halo on hematoxylin and eosin counterstain10Nayak A. Raikar A. Kotrashetti V. et al.Histochemical detection and comparison of apoptotic cells in the gingival epithelium using hematoxylin and eosin and methyl green-pyronin: a pilot study.J Indian Soc Periodontol. 2016; 20: 294-298PubMed Google Scholar,11Singh N.P. A simple method for accurate estimation of apoptotic cells.Exp Cell Res. 2000; 256: 328-337Crossref PubMed Scopus (156) Google Scholar (c,d). Apoptosis was not present in PT cells. Images (a) and (b) are from a patient with CINAC from El Salvador. Images (c) and (d) are from a patient with CINAC from Sri Lanka. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PTC granules in patients with CINAC demonstrated autofluorescence clearly contrasting with the cellular background (Figure 5). Comparing the same deparaffinized renal tissue section with autofluorescence and after Jones staining demonstrated that almost all autofluorescent granules were argyrophylic (Figure 5). Staining of serial sections demonstrated cathepsin B–positive granules in PTCs with argyrophylic granules (Figures 5b and 6a ). Immunofluorescent staining for cathepsin B followed by Jones staining on the same slide revealed the that cathepsin B–positive lysosomes are argyrophylic (Figure 6c and d). The same strategy with a nuclear marker (PCNA) did not result in argyrophylic nuclei, demonstrating specificity of the Jones reaction for lysosomes (data not shown). In patients with CINAC, autofluorescent granules were further characterized by staining for cytochrome c oxidase subunit 6C (COX6C), lysosomal associated membrane protein 1 (LAMP1), and cathepsin B. COX6C was absent in affected tubules, making it unlikely that the autofluorescent/argyrophylic granules are mitochondria, and may indicate injured/dysfunctional mitochondria (Supplementary Figure S5). Confocal microscopy with LAMP1 and cathepsin B staining identified an important subset of the granules as lysosomes (Figure 7; Supplementary Figure S6). Additional staining for enzymatic activity of lysosomal enzyme acid phosphatase revealed a granular pattern in tubules with autofluorescent granules (Supplementary Figure S7).Figure 7Autofluorescent granules are lysosomes. Confocal microscopy of red-stained cathepsin B demonstrating that a subset of the enlarged green autofluorescent granules are positive for this lysosomal enzyme (a). Detail of (a) is shown in (b). Confocal microscopy of red-stained lysosomal-associated membrane protein 1 demonstrating that a subset of the enlarged green autofluorescent granules are delineated by this lysosomal membrane marker (c). Detail of (c) is shown in (d). Images are from a Sri Lankan patient with Chronic Interstitial Nephritis in Agricultural Communities. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EM revealed that PTCs in patients with CINAC focally contain few to many large inclusions delineated by a single (often indistinctive) membrane (Figure 8a and b ; Supplementary Figure S8). The inclusions lacked cristae or other features of mitochondria, autophagic vacuoles, lipofuscin/ceroid droplets, or peroxisomes (marginal plate) but were consistent with lysosomes.12Eskelinen E.L. Kovács A.L. Double membranes vs. lipid bilayers, and their significance for correct identification of macroautophagic structures.Autophagy. 2011; 7: 931-932Crossref PubMed Scopus (21) Google Scholar, 13Eskelinen E.-L. Reggiori F. Baba M. et al.The impact of electron microscopy on autophagy research.Autophagy. 2011; 79: 935-956Crossref Scopus (182) Google Scholar, 14Eskelinen E.-L. To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells.Autophagy. 2008; 4: 257-260Crossref PubMed Scopus (130) Google Scholar, 15Frank A.L. Christensen A.K. Localization of acid phosphatase in lipofuscin granules and possible autophagic vacuoles in interstitial cells of the guinea pig testis.J Cell Biol. 1968; 36: 1-13Crossref Scopus (116) Google Scholar, 16Perše M. Injac R. Erman A. Oxidative status and lipofuscin accumulation in urothelial cells of bladder in aging mice.PLoS One. 2013; 8e59638Crossref PubMed Scopus (37) Google Scholar The EM appearance corresponded to argyrophylic lysosomes on Jones stained slides (Figure 8c–e). Transmission EM energy-dispersive x-ray spectroscopy on EM grids prepared from Jones stained 3-μm sections confirmed the presence of Jones-stain derived silver (Ag) and gold (Au) atoms specifically in the intracellular inclusions, thereby linking light microscopy (LM) to EM morphology and corroborating the lysosomal nature of the inclusions (Figure 9).Figure 9Transmission electron microscopy (EM)–energy dispersive x-ray analysis confirmed, on the ultrastructural level, that lysosomes (either enlarged/dysmorphic or not) are argyrophylic. Pictures are EM grids prepared from Jones-stained 3-μm sections. Numbered regions depict electron-beam exposure for elemental analysis. The table shows the atomic percentage of silver (Ag) and gold (Au), which are derived from the Jones staining. Lysosomes contained increased amounts of Ag and Au, as did the basement membrane (region 7) that is known for its argyrophylic properties. Images are from a patient with Chronic Interstitial Nephritis in Agricultural Communities from El Salvador. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These lysosomes were mildly to markedly larger than mitochondria, often with irregular/dysmorphic, sometimes very distorted, shapes (Figure 10a and b ). The longest diameter was 5.3 μm, and the average longest diameter of the 140 largest lysosomes from the Sri Lanka CINAC cohort was 1.75 ± 0.69 μm, larger than the normal lysosomal diameter of 0.1 to 1.0 μm (Supplementary Figure S9).17Appelqvist H. Wäster P. Kågedal K. et al.The lysosome: from waste bag to potential therapeutic target.J Mol Cell Biol. 2013; 5: 214-226Crossref PubMed Scopus (455) Google Scholar,18Bandyopadhyay D. Cyphersmith A. Zapata J.A. et al.Lysosome transport as a function of lysosome diameter.PLoS One. 2014; 9e86847Crossref PubMed Scopus (67) Google Scholar Nearly all enlarged/dysmorphic lysosomes contained homogenous, nonmembrane bound, dark electron dense rounded/irregular "aggregates" dispersed throughout the lysosomal matrix, which was of light to medium uniform electron density; a small percentage of lysosomes additionally contained dark electron dense punctate material (Figures 8 and 10). The CINAC lysosomes did not resemble lysosomal meyloid bodies because no lamellar pattern was observed in any enlarged lysosomes.19De Broe M.E. Giuliano R.A. Verpooten G.A. Choice of drug and dosage regimen. Two important risk factors for aminoglycoside nephrotoxicity.Am J Med. 1986; 80: 115-118Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 20Costa R.M. Martul E.V. Reboredo J.M. et al.Curvilinear bodies in hydroxychloroquine-induced renal phospholipidosis resembling Fabry disease.Clin Kidney J. 2013; 6: 533-536Crossref PubMed Scopus (18) Google Scholar, 21Tulkens P.M. Nephrotoxicity of aminoglycoside antibiotics.Toxicol Lett. 1989; 46: 107-123Crossref PubMed Scopus (124) Google Scholar, 22De Broe M.E. Paulus G.J. Verpooten G.A. et al.Early effects of gentamicin, tobramycin, and amikacin on the human kidney.Kidney Int. 1984; 25: 643-652Abstract Full Text PDF PubMed Scopus (143) Google Scholar Also, CINAC lysosomes are not to be mistaken for degenerating mitochondria, which can contain swollen dark electron dense cristae (Supplementary Figure S10). In addition, cases with large dysmorphic lysosomes typically contained clusters of at least 3 smaller (<1.2 μm in greatest dimension) lysosomes, with the same features as their larger counterparts (Figure 10c and d). The CINAC lysosomal diagnostic criteria are provided in Table 1.Table 1Diagnostic criteria of the aberrant lysosomal phenotypeObserved phenotypeDiagnostic interpretationLight microscopyPresence of enlarged proximal tubular cell argyrophylic granules (identified as lysosomes) in association with varying degrees of epithelial simplification, tubular atrophy, luminal cell fragment shedding with or without tubulointerstitial expansion (inflammation, edema, fibrosis)Suggestive (requiring electron microscopy confirmation!)Electron microscopyPresence of enlarged (>1.2 μm) dysmorphic lysosomes containing dispersed rounded/irregular electron dense aggregatesConfirmedPresence of 2 or more clusters of ≥3 lysosomes that meet the aforementioned criteria of intralysosomal aggregates and mild to moderate dysmorphia but are smaller than 1.2 μm in greatest diameterSuspicious Open table in a new tab To determine whether the PTC lesion is specific for CINAC, we analyzed renal biopsy specimens from healthy control subjects (n = 10), from patients with CINAC from Sri Lanka (n = 18), El Salvador (n = 11), India (n = 1), and France (n = 4), 5 proteinuric nephropathies (n = 16), non-CINAC Sri Lankan glomerulopathies (n = 6), interstitial nephritis (n = 19), and 6 drug/toxin-induced nephropathies, including calcineurin inhibitor treatment (n = 49; Tables 2 and 3). All were evaluated by Jones stained LM sections and EM.Table 2Relevant clinical data and assessment of the lysosomal phenotype by electron microscopy of patients with CINACPatient no.SexAge, yrS-creatinine, mg/dlProteinuriaaProteinuria: nil (negative on dipstick), trace < moderate (1–3.4 g/ml or <1500 mg/24 hr) < severe.Blood pressure, mm HgeGFR, MDRDHeat exposurebHeat stress was defined as working under direct sun exposure during the day while working in the field in Sri Lanka. In El Salvador, heat stress was determined as working under conditions over 30 °C for more than 4 hours.Lysosomal phenotype (EM confirmation)Sri Lanka1M471Moderate140/9085NoYes2M371.8Nil100/7046NoYes3M492Trace110/8038YesNo4F342.1Nil140/9029NoYes5M681.81Trace130/8038NoYes6M593.09Trace150/8021YesYes7M381.28Trace140/8070YesNo8F304.56Severe150/8012NoInadequate samplecInadequate sample: medullary tissue, no proximal tubules, autolysis, no reprocessing possible.9M611.65Trace130/8044NoYes10F340.81Severe110/6094NoYes11F282.34Severe130/8027NoYes12M422.6Trace140/8029YesYes13M452.28Trace130/8033NoInadequate samplecInadequate sample: medullary tissue, no proximal tubules, autolysis, no reprocessing possible.14M601.5Nil110/7051YesYes15M531.7Nil110/6045yesNo16M611.52Trace120/7050YesYes17dPatients with type 2 diabetes.M712.3Nil150/9030YesNo18dPatients with type 2 diabetes.M582.7Severe130/8026NoYesEl Salvador19F380.8Trace120/7080NoYes20M532Moderate120/8053YesYes21M541.3Nil130/7058YesSuspiciouseSuspicious is defined by the presence of 2 or more clusters of ≥3 lysosomes that meet the criteria of aggregates and dysmorphia but are smaller than 1.2 μm in greatest diameter.22F410.8Nil120/8079NoSuspiciouseSuspicious is defined by the presence of 2 or more clusters of ≥3 lysosomes that meet the criteria of aggregates and dysmorphia but are smaller than 1.2 μm in greatest diameter.23M501.4Nil130/8054NoYes24M361.1Nil100/6076YesYes25M591.4Trace120/8059YesYes26M491.9Nil150/8033NoYes27M341.4Nil100/6058NoYes28M361Nil100/7085NoYes29M311.3Nil110/7068NAYesIndia30M542.3Moderate130/8532NoYesFrance31F735.7ModerateNA30NoYes32M452.03NA90/706NoYes33M472.07Trace150/9658NoYes34F721.9TraceNA20NoYesCINAC, Chronic Interstitial Nephritis in Agricultural Communities; eGFR, estimated glomerular filtration rate; EM, electron microscopy; F, female; M, male; MDRD, Modification of Diet in Renal Disease; NA, not available.a Proteinuria: nil (negative on dipstick), trace < moderate (1–3.4 g/ml or <1500 mg/24 hr) < severe.b Heat stress was defined as working under direct sun exposure during the day while working in the field in Sri Lanka. In El Salvador, heat stress was determined as working under conditions over 30 °C for more than 4 hours.c Inadequate sample: medullary tissue, no proximal tubules, autolysis, no reprocessing possible.d Patients with type 2 diabetes.e Suspicious is defined by the presence of 2 or more clusters of ≥3 lysosomes that meet the criteria of aggregates and dysmorphia but are smaller than 1.2 μm in greatest diameter. Open table in a new tab Table 3Healthy control subjects and non-CINAC nephropathies investigated for the aberrant lysosomal phenotypeClinical conditionnLysosomal phenotype (EM-confirmed)Drug exposure and toxic nephropathies22YesSuspiciousaSuspicious is defined by the presence of 2 or more clusters of ≥3 lysosomes that meet the criteria of aggregates and dysmorphia but are smaller than 1.2 μm in greatest diameter. Tenofovir-treated HIVAN710 Cisplatinum400 Lithium970 Lomustine110 Clomiphene110Calcineurin inhibitor treatment27 Renal transplant indication biopsy21162 Nonrenal transplant kidney biopsy660Interstitial nephritis19 Acute, chronic, or mixed interstitial nephritis154bPositive cases were drug induced: allopurinol (n = 2), cocaine (n = 1), African herbs (n = 1).3 Pyelonephritis402Proteinuric nephropathies16 Light chain disease520 Diabetes500 Minimal change disease300 Lupus200 Tip variant focal segmental glomerulosclerosis100Non-CINAC Sri Lankan nephropathies6 Mixtures of focal segmental glomerulosclerosis, minimal change, glomerulonephritis, tubulointerstitial nephritis611Healthy control101cPositive control case was from Sri Lanka.0CINAC, Chronic Interstitial Nephritis in Agricultural Communities; EM, electron microscopy; HIVAN, HIV-associated nephropathy.a Suspicious is defined by the presence of 2 or more clusters of ≥3 lysosomes that meet the criteria of aggregates and dysmorphia but are smaller than 1.2 μm in greatest diameter.b Positive cases were drug induced: allopurinol (n = 2), cocaine (n = 1), African herbs (n = 1).c Positive control case was from Sri Lanka. Open table in a new tab CINAC, Chronic Interstitial Nephritis in Agricultural Communities; eGFR, estimated glomerular filtration rate; EM, electron microscopy; F, female; M, male; MDRD, Modification of Diet in Renal Disease; NA, not available. CINAC, Chronic Interstitial Nephritis in Agricultural Communities; EM, electron microscopy; HIVAN, HIV-associated nephropathy. Of the 32 CINAC biopsies with EM, 26 (81.3%) contained the dysmorphic lysosomal phenotype (Table 2 and Figure 11). EM analysis of healthy and pathologic control conditions revealed that the CINAC-lysosomal phenotype occurred in a substantial number of renal transplant indication biopsies on calcineurin inhibitor (CNI) therapy (79%), lithium exposure (78%), light chain disease (40%), drug-induced interstitial nephritis (27%), and in cases of lomustine, clomiphene, and rarely tenofovir nephrotoxicity (Table 3, Figure 12, and Supplementary Figure S11). Healthy and proteinuric control subjects rarely had the EM lysosomal lesion. The intralysosomal electron-dense structures appear slightly coarser in CINAC cases compared with CNI exposure, yet the overall morphology is the same (Supplementary Figure S12). In contrast, PTC lysosomes from patients with moderate or overt