Journal of The Egyptian Society of Nephrology and Transplantation

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 19  |  Issue : 1  |  Page : 19--23

Regulatory T-cells dysregulation in type 2 diabetic nephropathy


Walaa H.M Ibrahim1, Mostafa G Aly1, Ahmad B Ahmad2, Noha G Sayed3, Heba S Galal4, Muhammad H Al-Maghraby2,  
1 Nephrology Unit, Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt
2 Cardiology Unit, Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt
3 Department of Clinical Pathology, South Egypt Oncology Institute, Assiut University, Assiut, Egypt
4 Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt

Correspondence Address:
Dr. Walaa H.M Ibrahim
Nephrology Unit, Faculty of Medicine, Assiut University Hospital, Assiut, 71515
Egypt

Abstract

Background An immune-mediated process has been postulated to be involved in the pathogenesis of diabetic nephropathy (DN). Regulatory T-cells (Tregs) mediate self-tolerance and allogeneic tolerance and alteration in its expression was supposed to mediate the development of type 2 DN. We are interested in studying the expression of Treg cells in type 2 DN patients. Patients and methods We conducted a cross-sectional study carried out on 25 type 2 DN patients and 23 nondiabetic control patients. Demographic data were recorded in predefined data sheets. Blood samples for laboratory variables were collected and measured using the standard methods. Flow cytometry analysis was used to assess the phenotype and function of Treg cells from blood samples. Results In the current study, DN patients had a statistically significant higher Treg expression (P=0.038). There were insignificant associations between Treg cells and both proteinuria and duration of diabetes mellitus (P>0.05). Although univariate analysis showed that absolute Treg cell count and duration of diabetes were independent predictors of estimated glomerular filtration rate (P=0.028, 0.039, respectively), multivariate analysis showed that only the absolute Treg cell count was an independent predictor of estimated glomerular filtration rate with a just borderline statistical significance (P=0.048). Conclusion The current study did not support the immunological base of DN and we recommend testing the expression of other T-cell subtypes and cytokines in type 2 DN patients to be more confident about this thinking.



How to cite this article:
Ibrahim WH, Aly MG, Ahmad AB, Sayed NG, Galal HS, Al-Maghraby MH. Regulatory T-cells dysregulation in type 2 diabetic nephropathy.J Egypt Soc Nephrol Transplant 2019;19:19-23


How to cite this URL:
Ibrahim WH, Aly MG, Ahmad AB, Sayed NG, Galal HS, Al-Maghraby MH. Regulatory T-cells dysregulation in type 2 diabetic nephropathy. J Egypt Soc Nephrol Transplant [serial online] 2019 [cited 2019 Oct 17 ];19:19-23
Available from: http://www.jesnt.eg.net/text.asp?2019/19/1/19/255242


Full Text



 Introduction



Diabetic nephropathy (DN) is a leading cause of chronic kidney disease, resulting in end-stage renal disease [1]. Traditionally, it was known that metabolic and hemodynamic factors play a main role in the development of DN, but recently conflicting data about the involvement of inflammation and immunological processes in the development and progression of type 2 DN has emerged [2],[3]. Furthermore, increased infiltration of monocytes/macrophages and activated T lymphocytes, as well as augmented expression of inflammatory cytokines in the kidneys have also been found in patients with DN [4],[5]. The CD4+CD25+Foxp3+ is the best characterized and studied regulatory T-cells (Tregs) in the development of autoimmune diseases and organ transplantation rejection [6],[7]. We are interested in studying the expression of Treg cells in type 2 DN patients.

 Patients and methods



Patient groups

We conducted a cross-sectional study on 25 type 2 DN patients who had proteinuria or renal impairment after exclusion of other causes for renal impairment and 23 nondiabetic patients as a control group. Patients with acute or chronic infection, autoimmune diseases, and those on dialysis were excluded. All patients were recruited from the Outpatient Nephrology and Endocrinology Clinic of Assiut University Hospital from the period between May 2017 and May 2018. Written informed consents were obtained from the patients. Illiterate participants provided their consent by fingerprints. The study was approved by the ethics committee of the Faculty of Medicine of Assiut University.

Demographic and clinical data

Patients’ demographic data and medical history including age, sex, duration of diabetes, history of hypertension, and its duration were recorded. BMI was calculated, and systolic and diastolic blood pressures of the patients were recorded with a mercury sphygmomanometer after 15 min of recumbency. Fundus examination was done for all patients.

Laboratory data

Venous blood samples were collected from our patients and analyzed in the Clinical Pathology Department Laboratory of Assiut University Hospital. Laboratory measurements included serum level of creatinine, blood urea nitrogen, and serum albumin. Urinalysis was done for all patients for the detection of proteinuria and exclusion of active sediments. Estimated glomerular filtration rate (eGFR) was calculated using online CKD-EPI calculator.

Flow cytometry analysis

Currently, isolating CD4+CD25 high/bright T-cells is the most common strategy to assess the phenotype and function of nTreg cells from the human blood or tissue [8]. When looking solely at antigen expression, Tregs are often defined as CD3+, CD4+, CD25hi, and FOXP3+. Using these markers, a clear population is visible from samples such as human peripheral blood mononuclear cells (PBMC). Fluorochrome-conjugated antibodies against human CD4 monoclonal antibody, clone [SK-3 (SK-3) PerCP eBioscience, FITC mouse antihuman CD25 (Clone M-A251 (RUO)], and cytoplasmic FOXP3 Monoclonal Antibody, clone (150D/E4) PE, eBioscience (eBioscience (invitrogen) by Thermo Fisher Scientific; Waltham, Massachusetts, US) were used for the detection of Treg cell population.

Samples were acquainted and blotted to analysis by multicolor flow cytometry (FACS Calibur; Becton Dickinson, USA). Appropriate immunoglobulin FITC, PE, and PerCP-conjugated antibodies were used as isotype controls and blocking IgG (Becton Dickinson, Franklin Lakes, New Jersey, USA).

About 10 000 events were acquired. Gate of mononuclear cells (lymphocytes) was first drawn (R1) excluding the debris and dead cells. Gated cells were analyzed for the expression of CD4 and CD25 and the cells were classified according to the CD25 fluorescence intensity into three regions: low, intermediate, and high fluorescence intensity (R1, R2, and R3) where we assess the positivity percent of gated cells. Accordingly, the CD4+ and CD25 high fluorescent intensity region (CD25 high) are sequentially assayed for the cytoplasmic FOXP3 percent and mean fluorescence intensity.

Statistical analysis

The statistical analysis was carried out using the statistical package for the social sciences (version 19.0; SPSS Inc., Chicago, Illinois, USA). The continuous variables were presented as the means±SD and categorical variables were presented as number and percentage. χ2 test was used to compare between categorical variables and continuous variables were compared using a Mann–Whitney test. Pearson’s correlation was used to assess the association between Treg variables and both duration of diabetes mellitus (DM) and proteinuria. Regression analysis was used to assess the independent predictors eGFR in the DN patient group. A P value of less than 0.05 was considered statistically significant.

 Results



Clinical characteristics

There were statistically insignificant differences between the patients’ groups regarding demographic data which denote a good control matching ([Table 1]).{Table 1}

Laboratory investigations

The patient group had statistically significant higher absolute Treg cell count, Treg/lymphocyte percentage, and serum creatinine and statistically significant lower hemoglobin and eGFR than the control as shown in [Table 2].{Table 2}

Correlations

There were statistically insignificant associations between Treg variables and both duration of DM and proteinuria ([Table 3] and [Table 4]).{Table 3}{Table 4}

Regression analysis

Univariate analysis showed that both duration of diabetes and absolute Treg cell count were the independent predictors of eGFR; however, multivariate analysis showed that only Treg count was an independent predictor of eGFR with a just borderline statistical significance ([Table 5]).{Table 5}

 Discussion



Multiple mechanisms were well known as contributing mechanisms for the development of DN such as metabolic disorders, hemodynamic changes, genetic redisposition, inflammatory milieu, and oxidative stress [9]. Adiposity-associated inflammatory process has been proposed as a pathogenic mechanism of DM [10],[11].

Tregs mediate self-tolerance and allogeneic tolerance by suppressing the effector T-cells and exerting anti-inflammatory effects [6] and alteration in its expression was postulated to mediate DM development and progression.

The immune-mediated hypothesis of DN was supported by animal studies which showed that db/db mouse with type 2 diabetes, CD4+CD25+Foxp3 Treg depletion with anti-CD25 monoclonal antibody, enhanced insulin resistance, albuminuria, and glomerular hyperfiltration [12], while adoptive transfer of CD4+CD25+Foxp3 Tregs increased FoxP3 mRNA synthesis in the recipients and improved insulin sensitivity and type 2 DN [12]. However, clinical studies have shown conflicting data regarding CD4+CD25+Foxp3 cells in type 2 diabetic patients [13].

Zhang et al. [13] have found that the level of CD4+CD25+Foxp3+ Treg cells was markedly decreased in patients with type 2 DN, and that urinary albumin creatinine ratio was negatively related to the proportion of Treg cells. This study demonstrated for the first time that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in type 2 DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of type 2 DN [13]. However, Xu et al. [14] have found that there is no significant difference in the expression of CD4+CD25+Foxp3+ Treg cells between the control group and type 2 DN patients with microalbuminuria or macroalbuminuria.

In the current study type, two DN patients had statistically significant higher absolute Treg cell count, Treg/lymphocyte percentage than the control; however, there was no statistically significant association between Treg variables and both duration of DM and proteinuria and multivariate analysis showed that Treg count was an independent predictor of eGFR with a borderline statistical significance. These results may solidify the traditional thinking about the metabolic mechanism of DN. However, the current study asked an unanswered question, why type 2 DN patients has this high Treg expression than the control group? Does Treg involve − by unknown pathway − in the development of DN despite no association with proteinuria or is type 2 DN immunomodulatory?

In contrast to these conflicting results about the role of Treg cells in the development of DN, the conventional metabolic pathway for diabetic kidney disease (DKD) has the superiority in explaining the development of DKD. Activation of advanced glycation end products (AGEs)-AGE-binding receptors axis in persistent hyperglycemia leads to the activation of transcription factor nuclear factor-kappa B and generation of reactive oxygen species. Nuclear factor-kappa B in turn leads to the release of cytokines and growth factors, including the transforming growth factor-β1, interleukin-1β (IL) and IL-6, insulin-like growth factor-1, tumor necrosis factor, and platelet-derived growth factor [15] and vascular endothelial growth factor and its dimeric transmembrane tyrosine kinase receptor [16]. Collectively, these changes result in glomerular hyperfiltration, glomerular hypertension, renal hypertrophy, and altered glomerular composition, which is manifested clinically as albuminuria and by a decline in GFR. Kidney pathology shows mesangial deposition of extracellular matrix, glomerular basement membrane thickening, proliferative changes, and tubular atrophy, ultimately resulting in interstitial fibrosis and glomerulosclerosis [17].Added to this argument, innate immunity also had been emerged to be involved in the development of DN. Activation of AGEs-AGE-binding receptors axis causes macrophage activation and recruitment [18] which then facilitates the recruitment of leukocytes and mast cells in the kidney [19]. These immune responses accelerate renal damage and a decline in renal function in DKD. Hence, the inflammation in DKD is involved in the activation of innate immune responses [20] rather than adaptive immune responses that are involved in the DN development.

Limitations and recommendations

However, our study had some limitations including the small sample size, by financial issues, which may decrease the study power; and the need for testing expression of other T cell types such as Th1, Th2, and Th17 and cytokines levels such as interferon γ, tumor necrosis factor-α, IL-2, IL-6, IL-4, IL-10, and IL-17. We recommend the conduction of other large prospective studies testing the expression of these cell types and cytokines in type 2 DN patients.

Acknowledgements

The authors acknowledge all the nursing team in the Nephrology and Endocrinology Outpatient Clinic, nursing team in the Internal Medicine Department, and the technicians in the Clinical Pathology Labs in South Egypt Institute.

Support: Kits for Treg cells were granted by the Research Funding Unit, Faculty of Medicine, Assiut University.

Walaa H.M. Ibrahim: concept and design of the study, drafting of the article, and gave final approval of the version to be published. Mostafa G.O. Aly: concept and design of the study, analysis and interpretation of data, drafting the article, and gave final approval of the version to be published. Ahmad B. Ahmad: analysis of data, revising the article critically for important intellectual content, final approval of the version to be published. Noha G. Sayed: conducted the laboratory tests for the study group, drafted the clinical pathology part in the article. Heba S. Galal: acquisition of data, drafting of the article. Muhammad H. Al-Maghraby: concept and design of the study and gave final approval of the version to be published.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.

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