Chronic dehydration induces injury pathways in rats, but does not mimic histopathology of chronic interstitial nephritis in agricultural communities

CINAC-patients present renal proximal tubular cell lysosomal lesions which are also observed in patients experiencing calcineurin inhibitor (CNI) nephrotoxicity, suggesting that CINAC is a toxin-induced nephropathy. An alternative hypothesis advocates chronic dehydration as a major etiological factor for CINAC. Here, we evaluated histological and molecular changes in dehydrated versus toxin exposed rats. Wistar rats were divided in 3 groups. Group 1 (n = 6) had free access to drinking water (control group). Group 2 (n = 8) was water deprived for 10 h per 24 h, 5 days/week and placed in an incubator (37 °C) for 30 min/h during water deprivation. Group 3 (n = 8) underwent daily oral gavage with cyclosporine (40 mg/kg body weight). After 28 days, renal function, histopathology and proteomic signatures were analysed. Cyclosporine-treated rats developed focal regions of atrophic proximal tubules with associated tubulo-interstitial fibrosis. PASM staining revealed enlarged argyrophilic granules in affected proximal tubules, identified as lysosomes by immunofluorescent staining. Electron microscopy confirmed the enlarged and dysmorphic phenotype of the lysosomes. Overall, these kidney lesions resemble those that have been previously documented in farmers with CINAC. Dehydration resulted in none of the above histopathological features. Proteomic analysis revealed that dehydration and cyclosporine both induce injury pathways, yet of a clear distinct nature with a signature of toxicity only for the cyclosporine group. In conclusion, both cyclosporine and dehydration are injurious to the kidney. However, dehydration alone does not result in kidney histopathology as observed in CINAC patients, whereas cyclosporine administration does. The histopathological analogy between CINAC and calcineurin inhibitor nephrotoxicity in rats and humans supports the involvement of an as-yet-unidentified environmental toxin in CINAC etiology.


Table of Contents
Table S1: Differentially expressed renal proteins in response to dehydration protocols Table S2: Differentially expressed renal proteins in response to cyclosporine treatment Table S3: Venn diagram analysis of differentially expressed renal proteins in response to either dehydration or cyclosporine treatment Table S4: Gene Ontology-based interpretation of dehydration-induced proteomic perturbations Table S5: Gene Ontology-based interpretation of cyclosporine-induced proteomic perturbations Table S6: Ingenuity Canonical Signaling Pathway Analysis comparison of renal proteomic responses to dehydration or cyclosporine treatment Table S7: Overlapping of dehydration-and cyclosporine exposure DEPs with a literaturederived protein list Table S8: Ingenuity Disease/Bio-Function comparison analysis of renal proteomic responses to dehydration or cyclosporine treatment Table S9: Ingenuity Toxicity Function analysis of renal proteomic responses to dehydration Table S10: Ingenuity Toxicity Function analysis of renal proteomic responses to cyclosporine treatment Table S11: Heatmaps of Gene Symbol-Biomedical Text word correlations using input DEP lists from the dehydration cohort showing associated text matrices revealing a clustering of proteins with biomedical terms.
Table S12: Heatmaps of Gene Symbol-Biomedical Text word correlations using input DEP lists from the cyclosporine cohort showing associated text matrices revealing a clustering of proteins with biomedical terms.Table S1.Differentially expressed renal proteins in response to dehydration protocols.Proteins that demonstrated a significant (p<0.05)deviation from background expression levels, when comparing samples from dehydrated animals compared to controls are depicted in the table.For each differentially regulated protein the Uniprot accession #, description, official Gene Symbol and the log2 transformed expression ratio (Dehydration:Control) is given.Table S3.Venn diagram analysis of differentially expressed renal proteins in response to either dehydration or cyclosporine treatment.For each significantly-regulated protein the respective log2 transform of the expression change (positive for upregulation versus control rats, negative for downregulation versus control rats) and the official Gene Symbol are given.Table S8.Ingenuity Disease/Bio-Function comparison analysis of renal proteomic responses to dehydration or cyclosporine treatment.For each IPA-defined Disease/Bio-Function (n>2 proteins per pathway, p<0.05 for enrichment) the predicted Disease/Bio-Function z-score is given.Functions that are considered to be activated are given a positive z score while those considered to be inhibited are given a negative z score.S11.Heatmaps of Gene Symbol-Biomedical Text word correlations using input DEP lists from the dehydration cohort showing associated text matrices revealing a clustering of proteins with biomedical terms.Table S12.Heatmaps of Gene Symbol-Biomedical Text word correlations using input DEP lists from the cyclosporine cohort showing associated text matrices revealing a clustering of proteins with biomedical terms.

Table S2 .
Differentially expressed renal proteins in response to cyclosporine treatment.Proteins that demonstrated a significant (p<0.05)deviation from background expression levels, when comparing samples from cyclosporine-treated animals compared to controls are depicted in the table.For each differentially regulated protein the Uniprot accession #, description, official Gene Symbol and the log2 transformed expression ratio (Cyclosporine:Control) is given.

Table S4 .
Gene Ontology-based interpretation of dehydration-induced proteomic perturbations.Gene Ontology terms that demonstrated a significant (p<0.05)enrichment using data from dehydrated animals compared to controls are indicated.For each differentially regulated Gene Ontology (GO) term the process name, GO term ID, enrichment p value (Pvalue) as well as the proteins form the input data set that populate the specific GO term are represented.

Table S5 .
Gene Ontology-based interpretation of cyclosporine-induced proteomic perturbations.Gene Ontology terms that demonstrated a significant (p<0.05)enrichment using data from cyclosporine-treated animals compared to controls are indicated.For each differentially regulated Gene Ontology (GO) term the process name, GO term ID, enrichment p value (P-value) as well as the proteins form the input data set that populate the specific GO term are represented.

Table S6 .
Ingenuity Canonical Signaling Pathway Analysis comparison of renal proteomic responses to dehydration or cyclosporine treatment.For each defined pathway (n>2 proteins per pathway) the negative log10 of the enrichment p value is given.Statistically significant pathway enrichment is observed from a log10 transformed p value of >1.3.

Table S9 .
Ingenuity Toxicity Function analysis of renal proteomic responses to dehydration.For each IPA-defined Toxicity Function (n>2 proteins per pathway) the negative log10 of the enrichment p value is given.Statistically significant pathway enrichment is observed from a log10 transformed p value of >1.3.

Table S10 .
Ingenuity Toxicity Function analysis of renal proteomic responses to cyclosporine treatment.For each IPA-defined Toxicity Function (n>2 proteins per pathway) the negative log10 of the enrichment p value is given.Statistically significant pathway enrichment is observed from a log10 transformed p value of >1.3.