| Background:People started to realize cadmium-caused health damages since nineteen forties.Initial studies have mainly focused on the populations that are occupationally exposed to cadmium.Since the 21st century,chronicle injuries due to low level cadmium exposure from the daily environment have gained increasing attention.Once entering the body,cadmium mainly accumulate in the kidney,which is its major toxicity organ.Previously,epidemiology studies have indicated that the diabetic population may be more susceptible to cadmium accumulation-induced renal injury,which involves complicated physiological and pathological processes.It has been reported that oxidative stress,cell death,immunological responses,and mutations of metallothionein MT1 and MT2 are all mechanistically related to cadmium induced renal injury.Other than that,little is known about the underlying genetics.While these hypothesis-driven studies have greatly advanced our understanding of the mechanisms of cadmium induced renal injury,they may be biased toward prior knowledge.To have a more comprehensive view,open-ended unbiased studies may be more appropriate.In this light,to better understand the characteristics and differential susceptibility to cadmium induced renal injury in type-2 diabetic and non-diabetic populations,establishment of suitable animal models and exploration of molecular insights into the effects of diabetic conditions on cadmium-induced renal injury are much needed.Objective:1.To investigate the effects of cadmium accumulation on renal injury of residents who were non-occupationally exposed to cadmium in the community through epidemiological investigation and comparing cadmium accumulation and the degree of associated renal injury in diabetic and non-diabetic populations.2.To establish a suitable animal model to study chronic cadmium accumulation-induced renal injury and the susceptibility mechanism of type-2diabetes to cadmium accumulation induced renal injury by comparing the renal injury,laboratory examination,and histopathology in cadmium treated ob/ob mice and C57BL/6J mice of the same brood.3.To explore the molecular mechanisms of the susceptibility of type-2 diabetes to cadmium accumulation-induced renal injury by functional genomics approaches.Methods:1.Epidemiological investigation:1,000 permanent residents in the Yangpu community of Shanghai were randomly selected from August 2015 to August 2017,and a face-to-face questionnaire survey was conducted.Urinary cadmium（UCD）、urinary cadmium metallothionein（UCD-MT）、urinary albumin（UAlb）、urinary N-acetyl-?-D-glucosidase（UNAG）、urinary retinol-binding protein（URBP）、urinary albumin（UALB）、Urinary creatinine（UCR）、serum creatinine（CR）、urea（BUN）、and uric acid（UA）were examined accompanied with ultrasound examination of the urinary system.2.Establishment of the animal model:18 male,Ob/Ob mice,aged 6 months,and18 male C57BL/6J,6 months littermate control mice were selected and randomized into 2 groups treated with different doses of cadmium chloride and 1 control group.Animals in the cadmium chloride-treated group were injected intraperitoneally with cadmium chloride solution every other day for 4 weeks.After the last cadmium or vehicle treatment,the animals were kept for another four weeks.Blood and urine samples were collected prior to these mice were euthanized.Renal tissue was also collected for histopathology analysis and snap frozen for whole transcriptome sequencing analysis.Urinary cadmium（UCd）,urinary albumin（UALB）,urinary N-acetyl-B-D-glucosidase（UNAG）,urinary retinal-binding protein（URBP）,urinary albumin（UALB）,urinary creatinine（UCr）,serum creatinine（Cr）),urea（BUN）,uric acid（UA）were examined.The renal-injury characteristics of 2 kinds of mice injected with the same doses of cadmium chloride were compared based on the laboratory examination and pathological results.3.Genomics analysis:Total RNA was extracted from the mouse renal tissue and submitted to RNA-Seq by second-generation sequencing service provider.The sequencing library（Tru Seq RNA Sample Preparation Kit）was generated from the total RNA through the kit and then sequenced on the Hi Seq2000 platform.The raw data were rigorously quality controlled and subject to further bioinformatics analysis,including Differentially Expressed Genes（DEGs）identification,Principal Component Analysis（PCA）,as well as Gene Ontology（GO）analysis,gene network analysis,and signaling pathway enrichment analysis.Results:1.A total of 683 subjects in the epidemiological of the community were included in the final analysis.The mean urinary cadmium concentration was0.77 g/g（0.41,1.32）after correctingby urinary creatinine.The UCD concentration was significantly higher in the groups of female subjects（1.06±0892ug/g,P=0.0220）,more than 60 years old（1.05±0.932ug/g,P=0.0045）,and with a BMI≤24（1.10±0.979ug/g,P=0.0088）.148 subjects with dominant albuminuria（urinary albumin/urine creatinine ratio≥30 mg/g）had higher urinary cadmium than those without dominant albuminuria（urinary albumin/urine creatinine ratio<30mg/g）（0.98 g/g vs.0.72 g/g;P<0.001）.In the group with urinary albumin lower than 30mg/g,the urinary cadmium of the subjects with lower level albuminuria（13.84mg/g≤urinary albumin/creatinine ratio<30mg/g）was higher than that with normal urinary albumin（urinary albumin/creatinine ratio<13.84mg/g）（0.83/g vs.0.69/g;P<0.001）.The urinary cadmium level was found to be associated with low-level albuminuria(OR:2.98;95%CI（1.74-5.10）,even after the correction of a variety of confounding factors,.There was no significant difference in urinary cadmium（0.90±0.40/g vs.1.26±0.58/g;P=0.177）or urinary cadmium metallothionein level（1.50±0.31 vs.1.35±0.37;P=0.70）between the diabetic and non-diabetic groups.The diabetic population also had a significantly higher percentage of subjects with abnormal urinary albumin level（P=0.0072）.The urinary cadmium level was found to be associated with GFR in diabetic people（P=0.045）,While in the low cadmium exposed group（population with uninary cadmium levels in the lower two quarters）;the diabetic people had a higher GFR than the non-diabetic ones（P=0.0176）。2.High-dose cadmium-treated ob/ob mice had obviously decreased viability after4-week feeding:1died in the 7thweek 1 and one was found to have renal failure during the serum examination in the 8th week.Low-dose cadmium-treated ob/ob mice looked better than the high-dose cadmium-treated ob/ob mice,without death or renal failure.C57BL/6J mice were in better general conditions,without death or renal failure.High-dose cadmium-treated ob/ob mice had significantly higher ratio of urinary albumin/urinary creatinine than the baseline,（P=0.001）,however,there is no significant change in urinary albumin level in C57BL/6J mice upon cadmium treatment.Pathological examination of the renal tissue showed different degrees of corpuscle and tubule injury in ob/ob and C57BL/6J mice exposed to higher doses of cadmium-treated mice.The ob/ob mice had uneven glomerular outer margin and severer tubule interstitial injury compared with C57BL/6J mice,more damaged lesion of tubules and inflammatory cells were found in ob/ob mice.The 2 types of mice exposed to low-dose cadmium had similar tubule injury,relatively milder than the high cadmium dose group,and the control groups not exposed to cadmium had hypertrophy of glomeruli only in the ob/ob mice..3.Transcriptome profiling through RNA-Seq showed that cadmium treatment induced dose-dependent gene expression change in ob/ob mice and the wild type C57BL/6J littermate controls.The number of Differentially Expressed Genes（DEGs,1496）in ob/ob mice far exceeded that in C57BL/6J mice（800）at the same dose.This data are suggestive of the possibility that diabetic mice were more sensitive to cadmium-induced renal injury.Further principal component analysis（PCA）of the differentially expressed genes suggested that cadmium could cause more harm in ob/ob mice than in C57BL/6J mice at the same dose.Network analysis of gene expression identified three major relatively independent sub-network nodules in ob/ob mice,including oxidative stress,cell cycle,and extracellular matrix remodeling.However,the sub-network of cell cycle did not exist in the network established by using the DEGs identified from the C57BL/6J mice,suggesting that cell cycle regulation change may play a more significant role in cadmium renal injury in diabetic mice.Gene function enrichment analysis revealed that oxidative stress was the most significant mechanism in both mice types at high or low dose.However,in ob/ob mice,in addition to the known mechanisms associated with cadmium-induced renal injuries,such as oxidative stress,cytokine signaling,and MAPK signaling pathway,etc.,glucuronidation and Flt3 signaling pathways were also significantly altered,suggesting that these two pathways may contribute to the molecular mechanisms of diabetes caused susceptibility to cadmium nephrotoxicity,thus warrant for future studies.Conclusions:1.Community residents who are non-occupationally exposed to cadmium have already had cadmium accumulation,which is likely to have adverse health consequences.Urinary cadmium level is associated with low level urinary albumin,and the diabetic population is likely more susceptible to cadmium accumulation-induced renal injury;2.For type-2 diabetic and non-diabetic mice injected with the same dose of cadmium,diabetic mice have a more serious renal injury,suggesting that diabetic mice are more susceptible to cadmium accumulation-induced renal injury;3.Whole transcriptome profiling by using RNA-Seq further support that diabetes could increase the susceptibility to cadmium-induced renal injury in mice.Gene function enrichment and network analyses revealed,in addition to oxidative stress,cytokine signaling,MAPK signaling pathway and other known mechanisms associated with cadmium-induced renal injury,some novel insights into the mechanisms underlying the diabetes associated susceptibility to cadmium nephrotoxicity,includingcell cycle regulation,glucuronidation pathway,and Flt3signaling pathway,etc. |
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