Expression of Selectins in the Development of Diabetic Nephropathy in Patients with type 2 Diabetes

When platelets are stimulated, P-selectin is rapidly redistributed from -granules to the platelet surface [22]. E-selectin (CD62e), a membrane glycoprotein, is produced in large quantities and is expressed only on activated endothelium, promoting the adhesion of monocytes and neutrophils to the endothelium [23].

The purpose of the research was to study the role of the expression of P- and E-selectins (CD62p and CD62e) in the glomerular capillary endothelium and peritubular capillaries in patients with different morphological classes of DN and to evaluate their influence on the development and progression of histological changes in renal tissue.

I. PARENTS AND METHOD

The study included 50 patients with type 2 diabetes mellitus (DM), complicated by the development of diabetic nephropathy. The average age of the patients was years. There were 35 women, 14 men. The duration of disease in patients with diabetes was years. The duration of DN from the moment of detection of microalbuminuria to the morphological examination of the renal tissue and diagnosis was years.

The material for histological examination was obtained through intravita percutaneous kidney biopsies and was subsequently examined by light and immunofluorescence microscopy. Morphological changes in tissue were assessed in accordance with the latest international classification of diabetic nephropathy developed by the Scientific Committee of the Pathology Society, USA [24].

Light microscopy of kidney biopsy tissue was assessed using the following indicators.

1. The presence of global and segmental glomerular sclerosis;

2. Cellularity of the glomerulus;

3. Severity of expansion of the mesangial matrix (less than and more than 25%);

4. GBM thickening;

5. Kimmelstiel–Wilson nodules;

6. Presence of hyaline caps;

7. Periglomerular sclerosis;

8. Sclerotic changes in the interstitium;

9. The presence and severity of mononuclear inflammatory infiltrates in the interstitium;

10. The presence of protein masses in the lumens of the tubules;

11. Atrophy and dystrophy of the epithelium of the urinary tubules (thickness of the apical edge and height of the epithelium of the tubules);

12. Hyalinosis of afferent and efferent arterioles.

The severity of morphological changes was assessed using a semi-quantitative method in points (0–3). Global and segmental glomerular sclerosis was assessed as the percentage of globally and segmentally sclerotic glomeruli from the total number of glomeruli in the nephrobiopsy section. Interstitial fibrosis and tubular atrophy (IFTA) was scored (0–3) as a percentage of the total interstitial and tubular area in the biopsy specimen. Mononuclear infiltration (IM), afferent and efferent hyalinosis (AH) were also scored (0–2 and 0–2, respectively) according to the criteria of the international classification of DN [24].

According to light microscopy, class IIa (mild mesangial expansion) was detected in 12 patients, class IIb (severe mesangial expansion) in 14 patients, class III (nodular Kimmelstiel-Wilson lesions) in 19 patients, class IV in 5 patients (advanced diabetic glomerulosclerosis).

In addition to light microscopy, the expression of CD62P (P-selectin) and CD62E (E-selectin) in the glomerular endothelium and peritubular capillaries was determined in all patients using monoclonal antibodies labeled Fitc (FITC anti-human CD62P (P-Selectin) Antibody, clone AK4 Cat#304904 and FITC anti-human CD62E (E-Selectin) Antibody, clone HCD62E Cat#322606, Biolegend (USA)). The intensity of expression was assessed in points (0–4) [25], the nature and location of the expression of CD62P and CD62E in the glomerular endothelium and in peritubular capillaries.

II. STATISTICAL ANALYSIS

Statistical processing of the obtained data was carried out using the IBM SPSS Statistics software package, version 26 (Armonk, NY: IBM Corp.). Group results are presented as the arithmetic mean ± standard error (M ± Standard Error).

Statistical comparison of data between groups of patients was carried out using the nonparametric Mann–Whitney U test. Differences in continuous variables were assessed using the independent sample Student's t test and were considered significant if .

For statistical processing, parametric (Pearson's method) and non-parametric (Spearman's method, Kendall's tau ( ) method) were used. To verify compliance with the condition of independence of observations, linear regression analysis was carried out (with the calculation of the coefficient of determination (R Square) and the Durban–Watson test) and analysis of variance (ANOVA Analisis of Variance) with the calculation of the Fisher test (F) to test the significance of the model. The standardized coefficient with 95% confidence intervals was calculated. The critical level of significance for the difference in indicators was taken equal to 0.05.

III. RESULT

Analysis of the expression of P- and E-selectins in renal tissue showed that expression is present in the area of glomerular endothelium and periglomerular capillaries. The results are presented in Table 1.

Table 1: Intensity of expression of CD62p and CD62e in the glomerular endothelium and peritubular capillaries in patients with type 2 diabetes with DN

Table 2: Intensity of CD62P expression in the glomerular endothelium and peritubular capillaries in groups of patients with different classes of DN

Table 3: Intensity of CD62E expression in the glomerular endothelium and peritubular capillaries in groups of patients with different classes of DN

From the presented data it is clear that the expression of selectins in renal tissue depends on the morphological class of DN.

Next, a correlation analysis of the relationship between the expression of selectins in renal tissue and morphological changes in the general group of patients was carried out. The obtained data are presented in Table 4.

Table 4: Correlations between the expression of CD62P and CD62E in the glomerular endothelium with morphological changes in the general group of patients with DN

To identify the prognostic significance of the expression of CD62p and CD62e and their role in the progression of morphological changes in renal tissue during the development of DN, linear regression analysis was carried out with the calculation of determination coefficients R2 (R

Square) and analysis of variance (ANOVA Analysis of Variance) using the F test with 95% confidence interval. The obtained values, indicating the significance of the regression models, are presented below (Table 5, 6).

Table 5: Regression models of the significance of CD62P and CD62E expression in the glomerular endothelium in the general group of patients with DN

Table 6: Regression models of the significance of CD62P and CD62E expression in the peritubular capillaries in the general group of patients with DN

tissue in patients with DN. Next, we analyzed the influence of the expression of CD62P and CD62E on the progression of the stage of DN in the general group of patients (Table 7).

Table 7: Regression models of the significance of CD62P and CD62E expression in the glomerular endothelium on the progression of the stage of DN in the general group of patients

IV. DISCUSSION

In the pathogenesis of glomerular diseases, platelets play an active role through mechanisms involved in the development of the inflammatory process. Numerous studies have shown increased platelet activation in patients with diabetes (2With increased platelet hyperreactivity in patients with diabetes, proteins are released from -granules, dense granules and lysosomal granules, which act as pro-inflammatory mediators, pro-fibrotic mediators, growth factors and vasoactive mediators that contribute to the pathophysiological mechanisms of DN development [26, 18]. Proinflammatory mediators increase the expression of adhesion molecules on the endothelium and initiate the migration of leukocytes to the site of inflammation. The migration of leukocytes from blood to tissues involves several stages: coagulation, adhesion, diapedesis, and chemotaxis (27). The earliest and necessary event is coagulation, which initiates leukocyte extravasation and inflammation. Blood coagulation is mediated by a family of adhesion molecules, including various ligand/receptor molecules that promote intercellular and intercellular-ECM adhesion [28]. Selectins include endothelial E-selectin, platelet P-selectin and leukocyte L-selectin [29].

Selectins are involved in the constitutive return of leukocytes and in chronic and acute inflammatory processes . E- and P-selectins are expressed by endothelial cells. P-selectin is the main mediator of platelet and leukocyte aggregation. When P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-I), which is expressed on the membrane of monocytes and neutrophils, adhesion of platelets and endothelial cells to leukocytes occurs [2]. PSGL-1 has high affinity for P-selectin. E-selectin is produced upon pro-inflammatory stimulation and is considered important for leukocyte transport [30, 31]. Gotoh R. et al. showed in their work that E-selectin regulates adventitial inflammation through leukocyte adhesion and promotes the process of intimal hyperplasia [32].

The most comprehensive and one of the first studies of the expression of P- and E-selectins in renal tissue from T2DM patients with DN was published by Roy-Chaudhury Prabir et al. in 1996. The study was conducted on 119 biopsy blocks of kidneys taken from patients with different morphological diagnoses. The expression of P- and E-selectins on extraglomerular vascular endothelium was assessed. The authors showed that the expression of adhesion molecules in the tubulointerstitium is associated with interstitial fibrosis and tubular atrophy and may contribute to the progression of kidney disease (E-selectin (0.71, P <0.0001) and P-selectin (0.72, P <0,0001)). Spearman's correlation was found regardless of the morphological form of the primary diagnosis, but was clearly associated with histological damage. Expressions of E-selectin (P < 0.0001) and P-selectin (P < 0.0001) were dramatically increased in extraglomerular capillaries. The authors of the work showed that there is a common pathway of tubulointerstitial damage, regardless of the primary diagnosis, and the expression of adhesion molecules within the tubulointerstitium may be an important mechanism in the pathogenesis of DN [33].

In our study, regression analysis showed that the expression of CD62P and CD62E contribute to the development of interstitial sclerosis and atrophy of the tubular epithelium. This confirms previously published data on morphological changes in diabetic nephropathy . Also, the mechanism of tubulointerstitial damage and progression of DN is confirmed by our regression model of the relationship between the expression of CD62P and CD62E with the progression of the stage of DN in the general group of patients.

Taken together, these data support the hypothesis of Roy-Chaudhury Prabir et al. about the unified mechanism of tubulointerstitial damage and the significant role of the expression of CD62P and CD62E molecules in its development.

The literature contains data on a comparative analysis of the intensity of selectin expression in DN, lupus nephritis, membranous proliferative glomerulonephritis and IgA nephropathy .

There is also evidence of increased serum levels of E-selectin and P-selectin in patients with diabetes . Several cross-sectional studies have assessed the expression of cell adhesion molecules in blood and renal tissue and identified them as markers of endothelial dysfunction associated with the incidence of various microvascular complications of diabetes. In our study, the expression of P- and E-selectins was detected in the glomerular endothelium and in the peritubular capillaries of the renal tissue of patients. Regression analysis showed the influence of CD62P expression on the development of expansion of the mesangial matrix, arteriolar hyalinosis, and basement membrane thickening. CD62E expression promotes the development of mesangial matrix expansion, Kimmelstiel–Wilson nodules, arteriolar hyalinosis, and basement membrane thickening. Our data confirm and agree with data published by other authors.

It is known from the literature that in the early stages of DN, matrix expansion is formed due to the expansion of glycoproteins, collagen I, collagen III, collagen IV ( and chains), collagen V, collagen VI, laminin, fibronectin and small leucine-rich (SLR) proteoglycans and other structural components. That is, expansion of the mesangial matrix is the result of metabolic disturbances caused by chronic hyperglycemia, which leads to an imbalance between the synthesis of extracellular matrix (ECM) glycoproteins and their degradation [39, 40, 41].

An inverse relationship was shown between the relative volume of the mesangium and the surface filtration density of peripheral capillaries, as well as an inverse relationship between the surface filtration density and the density of the endothelial-mesangial border (r=-0.86 p=0.0005 – correlation of the percentage of total mesangium and peripheral S/V surface of capillaries) [42]. This mechanism can probably explain the regression models we obtained.

V. CONCLUSION

A study of biopsy tissue from patients with DN demonstrated the role of the expression of P- and E-selectins in the development of histological changes in renal tissue. P-selectin plays a greater role in the development of expansion of the mesangial matrix and the formation of Kimmelstiel–Wilson nodules with arteriolar hyalinosis. Both selectins play a role in the development of tubulointerstitial lesions.

In the future, it is planned to study the expression of other adhesion molecules and their role in the morphological changes of renal tissue in DN.

Funding: The study had no sponsorship

Conflict of interest: The authors declare no conflict of interest

Authors' contribution

All the authors have made substantial contribution to this study and approved final script version.