Wastewater surveillance of SARS-CoV-2 in Bangladesh: Opportunities and challenges.

The ongoing pandemic of the coronavirus disease 2019 (COVID-19) is a public health crisis of global concern. The progression of the COVID-19 pandemic has been monitored in the first place by testing symptomatic individuals for SARS-CoV-2 virus in the respiratory samples. Concurrently, wastewater carries feces, urine, and sputum that potentially contains SARS-CoV-2 intact virus or partially damaged viral genetic materials excreted by infected individuals. This brings significant opportunities for understanding the infection dynamics by environmental surveillance. It has advantages for the country, especially in densely populated areas where individual clinical testing is difficult. However, there are several challenges including: 1) establishing a sampling plan and schedule that is representative of the various catchment populations 2) development and validation of standardized protocols for the laboratory analysis 3) understanding hydraulic flows and virus transport in complex wastewater drainage systems and 4) collaborative efforts from government agencies, NGOs, public health units and academia.

Quantitative assessment of exposure to fecal contamination in urban environment across nine cities in low-income and lower-middle-income countries and a city in the United States.

BACKGROUND: During 2014 to 2019, the SaniPath Exposure Assessment Tool, a standardized set of methods to evaluate risk of exposure to fecal contamination in the urban environment through multiple exposure pathways, was deployed in 45 neighborhoods in ten cities, including Accra and Kumasi, Ghana; Vellore, India; Maputo, Mozambique; Siem Reap, Cambodia; Atlanta, United States; Dhaka, Bangladesh; Lusaka, Zambia; Kampala, Uganda; Dakar, Senegal. OBJECTIVE: Assess and compare risk of exposure to fecal contamination via multiple pathways in ten cities. METHODS: In total, 4053 environmental samples, 4586 household surveys, 128 community surveys, and 124 school surveys were collected. E. coli concentrations were measured in environmental samples as an indicator of fecal contamination magnitude. Bayesian methods were used to estimate the distributions of fecal contamination concentration and contact frequency. Exposure to fecal contamination was estimated by the Monte Carlo method. The contamination levels of ten environmental compartments, frequency of contact with those compartments for adults and children, and estimated exposure to fecal contamination through any of the surveyed environmental pathways were compared across cities and neighborhoods. RESULTS: Distribution of fecal contamination in the environment and human contact behavior varied by city. Universally, food pathways were the most common dominant route of exposure to fecal contamination across cities in low-income and lower-middle-income countries. Risks of fecal exposure via water pathways, such as open drains, flood water, and municipal drinking water, were site-specific and often limited to smaller geographic areas (i.e., neighborhoods) instead of larger areas (i.e., cities). CONCLUSIONS: Knowledge of the relative contribution to fecal exposure from multiple pathways, and the environmental contamination level and frequency of contact for those "dominant pathways" could provide guidance for Water, Sanitation, and Hygiene (WASH) programming and investments and enable local governments and municipalities to improve intervention strategies to reduce the risk of exposure to fecal contamination.

A method for correcting underestimation of enteric pathogen genome quantities in environmental samples.

Exposure to enteric pathogens in the environment poses a serious risk for infection and disease. The accurate detection and quantification of enteric pathogens in environmental samples is critical for understanding pathogen transport and fate and developing risk assessment models. In this study, we successfully applied TaqMan real-time PCR assays to quantitatively detect five human-specific pathogens (Shigella/EIEC, Salmonella Typhi, Vibrio cholera, Norovirus, and Giardia) in samples from open drains, canals, floodwater, septic tanks, and anaerobic baffled reactors (ABR) collected in Mirpur, Dhaka, Bangladesh from April to October 2019. Overall, the grab and sediment samples showed low inhibition but the ultrafiltration samples collected from open drain had significantly higher (P = 0.0049) degree of PCR inhibition (median Ct = 31.06) compared to the extraction controls (Ct = 28.54). We developed a two-step method to adjust underestimation of pathogen quantities due to PCR inhibition and non-optimum PCR efficiency. Compared to other sample types, ultrafiltration samples demonstrated a wide range of concentration increase (1.0%-182.5%) by pathogens after adjusting for PCR inhibition and non-optimum efficiencies. These quantitative qPCR assays are successful in quantifying multiple enteric pathogens in environmental samples, and the adjustment method would be useful for correcting underestimates of pathogen quantities due to partial PCR inhibition and non-optimum efficiency.

Evaluation of Low-Cost Phage-Based Microbial Source Tracking Tools for Elucidating Human Fecal Contamination Pathways in Kolkata, India.

Phages, such as those infecting Bacteroides spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated B. fragilis strain GB-124, newly isolated Bacteroides strains (K10, K29, and K33) and recently isolated Kluyvera intermedia strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (Escherichia coli) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting Bacteroides strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log10PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log10PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples. E. coli, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log10 PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log10 PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India.

Modelling faecal pathogen flows and health risks in urban Bangladesh: Implications for sanitation decision making.

Faecal-oral infections are a major component of the disease burden in low-income contexts, with inadequate sanitation seen as a contributing factor. However, demonstrating health effects of sanitation interventions - particularly in urban areas - has proved challenging and there is limited empirical evidence to support sanitation decisions that maximise health gains. This study aimed to develop, apply and validate a systems modelling approach to inform sanitation infrastructure and service decision-making in urban environments by examining enteric pathogen inputs, transport and reduction by various sanitation systems, and estimating corresponding exposure and public health impacts. The health effects of eight sanitation options were assessed in a low-income area in Dhaka, Bangladesh, with a focus on five target pathogens (Shigella, Vibrio cholerae, Salmonella Typhi, norovirus GII and Giardia). Relative to the sanitation base case in the study site (24% septic tanks, 5% holding tanks and 71% toilets discharging directly to open drains), comprehensive coverage of septic tanks was estimated to reduce the disease burden in disability-adjusted life years (DALYs) by 48-72%, while complete coverage of communal scale anaerobic baffled reactors was estimated to reduce DALYs by 67-81%. Despite these improvements, a concerning health risk persists with these systems as a result of effluent discharge to open drains, particularly when the systems are poorly managed. Other sanitation options, including use of constructed wetlands and small bore sewerage, demonstrated further reductions in local health risk, though several still exported pathogens into neighbouring areas, simply transferring risk to downstream communities. The study revealed sensitivity to and a requirement for further evidence on log reduction values for different sanitation systems under varying performance conditions, pathogen flows under flooding conditions as well as pathogen shedding and human exposure in typical low-income urban settings. Notwithstanding variability and uncertainties in input parameters, systems modelling can be a feasible and customisable approach to consider the relative health impact of different sanitation options across various contexts, and stands as a valuable tool to guide urban sanitation decision-making.

Pathogen flows from on-site sanitation systems in low-income urban neighborhoods, Dhaka: A quantitative environmental assessment.

BACKGROUND: Despite wide usage of on-site sanitation, there is limited field-based evidence on the removal or release of pathogens from septic tanks and other primary treatment systems, such as anaerobic baffled reactors (ABR). In two low-income areas in Dhaka, we conducted a cross-sectional study to explore pathogen loads discharged from commonly used on-site sanitation-systems and their transport in nearby drains and waterways. METHODS: We collected samples of drain water, drain sediment, canal water, and floodwater from April-October 2019. Sludge, supernatant, and effluent samples were also collected from septic tanks and ABRs. We investigated the presence and concentration of selected enteric pathogens (Shigella, Vibrio cholerae (V. cholerae), Salmonella Typhi (S. Typhi), Norovirus Genogroup-II (NoV-GII), and Giardia) and presence of Cryptosporidium in these samples using quantitative polymerase chain reaction (qPCR).The equivalent genome copies (EGC) of individual pathogens were estimated in each sample by interpolation of the mean Ct value to the corresponding standard curve and the dilution factor for each sample type. Absolute quantification was expressed as log10 EGC per 100 mL for the water samples and log10 EGC per gram for the sediment samples. RESULTS: Among all samples tested (N = 151), 89% were contaminated with Shigella, 68% with V. cholerae and NoV-GII, 32% with Giardia, 17% with S. Typhi and 6% with Cryptosporidium. A wide range of concentration of pathogens [range: mean log10 concentration of Giardia = 0.74 EGC/100 mL in drain ultrafiltration samples to mean log10 concentration of NoV-GII and Giardia = 7.11 EGC/100 mL in ABR sludge] was found in all environmental samples. The highest pathogen concentrations were detected in open drains [range: mean log10 concentration = 2.50-4.94 EGC/100 mL], septic tank effluent [range: mean log10 concentration = 3.32-4.65 EGC/100 mL], and ABR effluent [range: mean log10 concentration = 2.72-5.13 EGC/100 mL]. CONCLUSIONS: High concentrations of pathogens (particularly NoV-GII, V.cholerae and Shigella) were frequently detected in environmental samples from two low-income urban neighbourhoods of Dhaka city. The numerous environmental exposure pathways for children and adults make these findings of public health concern. These results should prompt rethinking of how to achieve safe sanitation solutions that protect public health in dense low-income areas. In particular, improved management and maintenance regimes, further treatment of liquid effluent from primary treatment processes, and appropriate application of onsite, decentralised and offsite sanitation systems given the local context.

The SaniPath Exposure Assessment Tool: A quantitative approach for assessing exposure to fecal contamination through multiple pathways in low resource urban settlements.

Inadequate sanitation can lead to exposure to fecal contamination through multiple environmental pathways and can result in adverse health outcomes. By understanding the relative importance of multiple exposure pathways, sanitation interventions can be tailored to those pathways with greatest potential public health impact. The SaniPath Exposure Assessment Tool allows users to identify and quantify human exposure to fecal contamination in low-resource urban settings through a systematic yet customizable process. The Tool includes: a project management platform; mobile data collection and a data repository; protocols for primary data collection; and automated exposure assessment analysis. The data collection protocols detail the process of conducting behavioral surveys with households, school children, and community groups to quantify contact with fecal exposure pathways and of collecting and analyzing environmental samples for E. coli as an indicator of fecal contamination. Bayesian analyses are used to estimate the percentage of the population exposed and the mean dose of fecal exposure from microbiological and behavioral data. Fecal exposure from nine pathways (drinking water, bathing water, surface water, ocean water, open drains, floodwater, raw produce, street food, and public or shared toilets) can be compared through a common metric-estimated ingestion of E. coli units (MPN or CFU) per month. The Tool generates data visualizations and recommendations for interventions designed for both scientific and lay audiences. When piloted in Accra, Ghana, the results of the Tool were comparable with that of an in-depth study conducted in the same neighborhoods and highlighted consumption of raw produce as a dominant exposure pathway. The Tool has been deployed in nine cities to date, and the results are being used by local authorities to design and prioritize programming and policy. The SaniPath Tool is a novel approach to support public-health evidence-based decision-making for urban sanitation policies and investments.

Variation in E. coli concentrations in open drains across neighborhoods in Accra, Ghana: The influence of onsite sanitation coverage and interconnectedness of urban environments.

Alongside efforts to improve safe management of feces along the entire sanitation chain, including after the toilet, global sanitation efforts are focusing on universal access 'basic' services: onsite facilities that safely contain excreta away from human contact. Although fecal sludge management is improving in urban areas, open drains remain a common fate for feces in these often densely-populated neighborhoods in low-income countries. To-date, it is unclear to what extent complete coverage of onsite sanitation reduces fecal contamination in the urban environment and how fecal contamination varies within urban drains across neighborhoods by sanitation status within a city. We assessed how neighborhood levels of environmental fecal contamination (via spatially-representative sampling of open drains for E. coli) varied across four neighborhoods with varying income, type and coverage of household sanitation facilities, and population density in Accra, Ghana. Neighborhoods with very high sanitation coverage (≥89%) still had high (>4 log10 CFU/100 mL) E. coli concentrations in drains. Between-neighborhood variation in E. coli levels among the high coverage neighborhoods was significant: drain concentrations in neighborhoods with 93% and 89% coverage (4.7 (95% CI: 4.5, 4.9) & 4.9 (95% CI: 4.5, 5.3) log10 CFU/100 mL, respectively) were higher than in the neighborhood with 97% coverage (4.1 log10 CFU/100 mL, 95% CI: 3.8, 4.4 log10 CFU/100 mL). Compared with the highest coverage neighborhood, the neighborhood with lowest coverage (48%) also had higher E. coli concentrations (5.6 log10 CFU/100 mL, 95% CI: 5.3, 5.9 log10 CFU/100 mL). Although fecal contamination in open drains appeared lower in neighborhoods with higher onsite sanitation coverage (and vice versa), other factors (e.g. fecal sludge management, animals, population density) may affect drain concentrations. These results underscore that neighborhood-level onsite sanitation improvements alone may not sufficiently reduce fecal hazards to public health from open drains. These findings supporting the need for integrated, city-level fecal sludge management alongside multifaceted interventions to reduce fecal contamination levels and human exposure.

Quantitative assessment of fecal contamination in multiple environmental sample types in urban communities in Dhaka, Bangladesh using SaniPath microbial approach.

Rapid urbanization has led to a growing sanitation crisis in urban areas of Bangladesh and potential exposure to fecal contamination in the urban environment due to inadequate sanitation and poor fecal sludge management. Limited data are available on environmental fecal contamination associated with different exposure pathways in urban Dhaka. We conducted a cross-sectional study to explore the magnitude of fecal contamination in the environment in low-income, high-income, and transient/floating neighborhoods in urban Dhaka. Ten samples were collected from each of 10 environmental compartments in 10 different neighborhoods (4 low-income, 4 high-income and 2 transient/floating neighborhoods). These 1,000 samples were analyzed with the IDEXX-Quanti-Tray technique to determine most-probable-number (MPN) of E. coli. Samples of open drains (6.91 log10 MPN/100 mL), surface water (5.28 log10 MPN/100 mL), floodwater (4.60 log10 MPN/100 mL), produce (3.19 log10 MPN/serving), soil (2.29 log10 MPN/gram), and street food (1.79 log10 MPN/gram) had the highest mean log10 E. coli contamination compared to other samples. The contamination concentrations did not differ between low-income and high-income neighborhoods for shared latrine swabs, open drains, municipal water, produce, and street foodsamples. E. coli contamination levels were significantly higher (p <0.05) in low-income neighborhoods compared to high-income for soil (0.91 log10 MPN/gram, 95% CI, 0.39, 1.43), bathing water (0.98 log10 MPN/100 mL, 95% CI, 0.41, 1.54), non-municipal water (0.64 log10 MPN/100 mL, 95% CI, 0.24, 1.04), surface water (1.92 log10 MPN/100 mL, 95% CI, 1.44, 2.40), and floodwater (0.48 log10 MPN/100 mL, 95% CI, 0.03, 0.92) samples. E. coli contamination were significantly higher (p<0.05) in low-income neighborhoods compared to transient/floating neighborhoods for drain water, bathing water, non-municipal water and surface water. Future studies should examine behavior that brings people into contact with the environment and assess the extent of exposure to fecal contamination in the environment through multiple pathways and associated risks.

Associations between open drain flooding and pediatric enteric infections in the MAL-ED cohort in a low-income, urban neighborhood in Vellore, India.

BACKGROUND: Open drains are common methods of transporting solid waste and excreta in low-income urban neighborhoods. Open drains can overflow due to blockages with solid waste and during rainfall, posing exposure risks. The goal of this study was to evaluate whether pediatric enteric infection was associated with open drains and flooding in a dense, low-income, urban neighborhood. METHODS: As part of the MAL-ED study in Vellore, India, a cohort of 230 children provided stool specimens at 14-17 scheduled home visits and during diarrheal episodes in the first two years of life. All specimens were analyzed for enteric pathogens. Caregivers in 100 households reported on flooding of drains and households and monthly frequency of contact with open drains and flood water. Household GPS points were collected. Monthly rainfall totals for the Vellore district were collected from the Indian Meteorological Department. Clustering of reported drain and house flooding were identified by Kulldorff's Bernoulli Spatial Scan. Differences in enteric infection were assessed for household responses and spatial clusters, with interactions between reported flooding and rainfall to approximate monthly drain flooding retrospectively, using multivariable, mixed-effects logistic regression models. RESULTS: Coverage of household toilets was low (33%), and most toilets (82%) discharged directly into open drains, suggesting poor neighborhood fecal sludge management. Odds of enteric infection increased significantly with total monthly rainfall for children who lived in households that reported that the nearby drain flooded (4% increase per cm of rain: OR: 1.04, 95% CI: 1.00-1.08) and for children in households in a downstream spatial cluster of reported drain flooding (5% increase per cm of rain: OR: 1.05, 95% CI: 1.01-1.09). There was no association between odds of enteric infection and frequency of reported contact with drain or floodwater. CONCLUSIONS: Children in areas susceptible to open drain flooding had increased odds of enteric infection as rainfall increased. Results suggested that infection increased with rainfall due to neighborhood infrastructure (including poor fecal sludge management) and not frequency of contact. Thus, these exposures may not be mitigated by changes in personal behaviors alone. These results underscore the importance of improving the neighborhood environment to improve children's health in low-income, urban settings.

Multipathway Quantitative Assessment of Exposure to Fecal Contamination for Young Children in Low-Income Urban Environments in Accra, Ghana: The SaniPath Analytical Approach.

Lack of adequate sanitation results in fecal contamination of the environment and poses a risk of disease transmission via multiple exposure pathways. To better understand how eight different sources contribute to overall exposure to fecal contamination, we quantified exposure through multiple pathways for children under 5 years old in four high-density, low-income, urban neighborhoods in Accra, Ghana. We collected more than 500 hours of structured observation of behaviors of 156 children, 800 household surveys, and 1,855 environmental samples. Data were analyzed using Bayesian models, estimating the environmental and behavioral factors associated with exposure to fecal contamination. These estimates were applied in exposure models simulating sequences of behaviors and transfers of fecal indicators. This approach allows us to identify the contribution of any sources of fecal contamination in the environment to child exposure and use dynamic fecal microbe transfer networks to track fecal indicators from the environment to oral ingestion. The contributions of different sources to exposure were categorized into four types (high/low by dose and frequency), as a basis for ranking pathways by the potential to reduce exposure. Although we observed variation in estimated exposure (108-1016 CFU/day for Escherichia coli) between different age groups and neighborhoods, the greatest contribution was consistently from food (contributing > 99.9% to total exposure). Hands played a pivotal role in fecal microbe transfer, linking environmental sources to oral ingestion. The fecal microbe transfer network constructed here provides a systematic approach to study the complex interaction between contaminated environment and human behavior on exposure to fecal contamination.

The Influence of Household- and Community-Level Sanitation and Fecal Sludge Management on Urban Fecal Contamination in Households and Drains and Enteric Infection in Children.

AbstractUrban sanitation necessitates management of fecal sludge inside and outside the household. This study examined associations between household sanitation, fecal contamination, and enteric infection in two low-income neighborhoods in Vellore, India. Surveys and spatial analysis assessed the presence and clustering of toilets and fecal sludge management (FSM) practices in 200 households. Fecal contamination was measured in environmental samples from 50 households and household drains. Enteric infection was assessed from stool specimens from children under 5 years of age in these households. The two neighborhoods differed significantly in toilet coverage (78% versus 33%) and spatial clustering. Overall, 49% of toilets discharged directly into open drains ("poor FSM"). Children in households with poor FSM had 3.78 times higher prevalence of enteric infection when compared with children in other households, even those without toilets. In the neighborhood with high coverage of household toilets, children in households with poor FSM had 10 times higher prevalence of enteric infection than other children in the neighborhood and drains in poor FSM clusters who had significantly higher concentrations of genogroup II norovirus. Conversely, children in households with a toilet that contained excreta in a tank onsite had 55% lower prevalence of enteric infection compared with the rest of the study area. Notably, households with a toilet in the neighborhood with low toilet coverage had more fecal contamination on floors where children played compared with those without a toilet. Overall, both toilet coverage levels and FSM were associated with environmental fecal contamination and, subsequently, enteric infection prevalence in this urban setting.

Household sanitation is associated with lower risk of bacterial and protozoal enteric infections, but not viral infections and diarrhoea, in a cohort study in a low-income urban neighbourhood in Vellore, India.

OBJECTIVE: This study examined associations between household sanitation and enteric infection - including diarrhoeal-specific outcomes - in children 0-2 years of age in a low-income, dense urban neighbourhood. METHODS: As part of the MAL-ED study, 230 children in a low-income, urban, Indian neighbourhood provided stool specimens at 14-17 scheduled time points and during diarrhoeal episodes in the first 2 years of life that were analysed for bacterial, parasitic (protozoa and helminths) and viral pathogens. From interviews with caregivers in 100 households, the relationship between the presence (and discharge) of household sanitation facilities and any, pathogen-specific, and diarrhoea-specific enteric infection was tested through mixed-effects Poisson regression models. RESULTS: Few study households (33%) reported having toilets, most of which (82%) discharged into open drains. Controlling for season and household socio-economic status, the presence of a household toilet was associated with lower risks of enteric infection (RR: 0.91, 95% CI: 0.79-1.06), bacterial infection (RR: 0.87, 95% CI: 0.75-1.02) and protozoal infection (RR: 0.64, 95% CI: 0.39-1.04), although not statistically significant, but had no association with diarrhoea (RR: 1.00, 95% CI: 0.68-1.45) or viral infections (RR: 1.12, 95% CI: 0.79-1.60). Models also suggested that the relationship between household toilets discharging to drains and enteric infection risk may vary by season. CONCLUSIONS: The presence of a household toilet was associated with lower risk of bacterial and protozoal enteric infections, but not diarrhoea or viral infections, suggesting the health effects of sanitation may be more accurately estimated using outcome measures that account for aetiologic agents.

Genetic studies of type 2 diabetes in South Asians: a systematic overview.

Diabetes Mellitus, which affects 366 million people worldwide, is a leading cause of mortality, morbidity, and loss of quality of life. South Asians, comprising 24% of the world's population, suffer a large burden of type 2 diabetes. With intriguing risk phenotypes, unique environmental triggers, and potential genetic predisposition, South Asians offer a valuable resource for investigating the pathophysiology of type 2 diabetes. Genomics has proven its potential to underpin some of the etiology of type 2 diabetes by identifying a number of susceptibility genes, but such data are scarce and unclear in South Asians. We present a systematic review of studies on the genetic basis of type 2 diabetes or its complications in South Asians published between 1987-2012, and discuss the findings and limitations of the available data. Of the 91 eligible studies meeting our inclusion criteria, a vast majority included Indian populations, followed by a few in those of Pakistani origin, while other South Asian countries were generally under-represented. Though a large number of studies focused on the replication of findings from genome-wide association studies (GWAS) in European populations, a few studies explored new genes and pathways along with GWAS in South Asians and suggested the potential to unravel population- specific susceptibility genes in this population. We find encouraging improvements in study designs, sample sizes and the numbers of genetic variants investigated over the last five years, which reflect the existing capacity and scope for large-scale genetic studies in South Asians.

Acid stress increases gene expression of proinflammatory cytokines in Madin-Darby canine kidney cells.

Metabolic acidosis is thought to exacerbate chronic kidney disease in part by stimulating the release of potentially injurious substances. To define the genes whose expression is affected by exposure to an acidic milieus, we examined the effect of exposure of MDCK cells to pH 7.4 and pH 7.0 for 24 h on gene expression using a canine derived microarray. Exposure to this pH stress for 24 h led to increased expression of 278 genes (2.2% of the transcriptome) by at least 2-fold and 60 of these (21%) were upregulated by >3-fold. On the other hand, 186 genes (1.5% of the transcriptome) were downregulated by at least 2-fold and 16 of these (9%) were downregulated by 3-fold or more. Ten percent of the genes upregulated by at least threefold encode proinflammatory cytokine proteins, including colony stimulating factor 2, chemokine ligand 7, chemokine ligand 20, chemokine ligand 8, and interleukin-1α. Two others encode metallopeptidases. The most highly upregulated gene encodes a protein, lubricin, shown to be important in preventing cartilage damage and in tissue injury or repair. Upregulation of four genes was confirmed by quantitative PCR. Housekeeping genes were not increased. To examine the effect of decreasing medium pH, we measured intracellular pH (pH(i)) using 2,7-bis (2-carboxyethyl)5-carboxyfluorescein. With extracellular pH (pH(o)) of 7.0, pH(i) fell and remained depressed. These findings suggest that a pH stress alone can increase renal expression of proinflammatory and other genes that contribute to renal injury.