54
Artigo 3
Endothelin-1 and Endothelin A Receptor Expression are Increased in
55 Abstract
Background. Endothelin-1 (ET-1) is associated with progression of renal disease, acting as a potent vasoconstrictor and as a growth factor of mesangial cells. In normal human kidney, ET-1 and endothelin A receptor (ETRA) are more markedly expressed in vessels, and at a lower intensity in glomerular structures. Animal models of diabetes mellitus (DM) showed that glomerular expression of ET-1 was five-fold greater, suggesting a potential association between endothelin and diabetic nephropathy (DN). The aim of this study was to determine the level of ET-1 and ETRA expression in patients with DN as compared to IgA Nephropathy (positive comparison group) and to normal kidney tissue.
Methods. Cross-sectional study comprising thirteen patients with type 2 DM and DN, ten patients with proteinuric IgA nephropathy and thirteen samples of normal kidney from tumor nephrectomies. The distribution and intensity of ET-1 and ETRA expression in renal biopsies and normal kidney tissue were determined by immunohistochemistry.
Results. Patients with DN and IgA nephropathy on biopsy had markedly increased staining for ET-1 in endothelial cells of glomerular capillaries and peritubular capillaries as compared to controls (p <0.001). ETRA staining was also more intense and more diffuse in DN and IgA nephropathy than in control (p= 0.019). Patients with higher proteinuria levels had a greater expression of ET-1 but not of ETRA.
Conclusion. A higher expression of ET-1 and ETRA was found both in DN and IgA nephropathy, suggesting a potential role for the endothelin system in diabetic nephropathy and probably in other non diabetic glomerular diseases.
56 Introduction
Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is the most potent vasoconstrictor known (1). There is much evidence showing that ET-1 is related to the progression of renal disease, acting as a potent growth factor of mesangial cells (2-4). The kidney is an important ET-1 production site and it is also a target for ET-1 effects. Mesangial cells are the main cells that produce and suffer the actions of ET-1 in the kidney (5), however, endothelial cells of glomerular capillaries and tubular epithelial cells also produce ET-1 (6).
Endothelin A receptor (ETRA) is present in arcuate arteries and vasa recta, and endothelin B receptor (ETRB) is predominantly found in the collecting ducts, suggesting different roles in the reabsorption of salt and water. Both receptors are found in the glomerulus (2).
Animal studies showed that ET-1 is not only more markedly expressed in kidney of diabetic rats (7), but can also mediate the effect of hyperglycemia on cellular mesangial hypertrophy and synthesis of extracellular matrix proteins (8). ET-1 growth-promoting and arteriolar vasoconstricting effects in glomeruli and renal cortex, reducing renal blood flow and glomerular filtration rate (GFR), are mediated via ETRA (9; 10).
In normal human kidney, ET-1 and ETRA are expressed in greater quantity in the vascular tissue and at a lower intensity in glomerular structures (10-13). Studies in animal models of diabetes mellitus (DM) showed a five-fold increase in the expression of ET-1 in the glomerulus, suggesting a role in diabetic nephropathy (DN) (8). Additionally, experimental blockade of ET-1 receptor with antagonists corrects the initial hemodynamic changes and decreases proteinuria in rats with DN (13; 14). Angiotensin II is a potent stimulus for ET-1 secretion and its effects on mesangial matrix synthesis are attenuated by antagonists of ET-1 (15).
Studies in patients with IgA nephropathy showed significantly increased expression of ET-1 and ETRA in the kidney. In those patients, proteinuria correlated positively with ET-1
57 expression, and when receiving angiotensin-converting enzyme inhibitors (ACEi) a significant reduction in ET-1 expression is observed (15). In kidney transplant recipients, intragraft ET-1 expression is positively correlated with post-transplant hypertension and proteinuria, and higher levels of proteinuria are also associated with more intense expression of ET-1 and ETRA (16). Similar changes are seen in patients with lupus nephritis (17). So far, no study has evaluated ET-1 and its receptor expression in patients with DM and DN.
Patients with type 2 DM have elevated plasma levels of ET-1, independent of the presence of microvascular disease. Moreover, patients with DM and microalbuminuria have higher plasma ET-1 levels than those with DM and normoalbuminuria or hypertension and microalbuminuria without DM (18). In a previous study in type 2 DM, we reported a progressive increase in plasma ET-1 levels from normo-, micro and macroalbuminuria (19).
The aims of this study were to determine the renal expression of ET-1 and ETRA in patients with DN and to correlate this expression with renal function and proteinuria.
Patients and Methods Subjects
Thirteen type 2 DM patients with diabetic nodular glomerulosclerosis on kidney biopsy were selected from the Nephrology Division database at Hospital de Clínicas de Porto Alegre (HCPA). Ten patients with mesangial proliferative IgA glomerulonephritis were included as a positive control group, because a high expression of endothelin was previously demonstrated in this nephropathy (15). Renal biopsies in patients with DN and IgA nephropathy were performed according to clinical indication. IgA nephropathy was defined by the presence of mesangial cell proliferation and/or matrix expansion (or focal segmental glomerular sclerosis in advanced stages) with predominant mesangial granular deposits of IgA (2+ or more) on immunofluorescence. Diabetic nephropathy was defined by diffuse capillary
58 basement membrane thickening with peripheral hyaline PAS positive nodules (Kimmelstiel-Wilson), with segmental or global glomerulosclerosis at advanced stages, and thickened arterioles with hyaline deposits. Histologically normal renal tissue from 13 patients who had unilateral nephrectomy for renal tumor (3 were diabetic and 10 were not diabetic) was included as control. Paraffin blocks with enough tissue for immunohistological analysis were located in the Pathology Division archive at HCPA.
The study protocol was approved by the HCPA Research Ethics Committee and registered in the Institutional Review Board (IRB) under number 00000921.
Methods
Demographic and clinical variables were collected retrospectively in patient medical records. Age, gender, ethnicity, serum creatinine, proteinuria (total protein /creatinine ratio in a morning urine sample). Proteinuria was available only for subjects with DN and IgA nephropathy. No proteinuria data were available in controls. GFR was estimated by the Modification of Diet in Renal Disease (MDRD) equation (20).
The distribution and intensity of ET-1 and ETRA expression were determined by immunohistochemistry in formalin-fixed, paraffin-embedded sections. To detect protein expression of ET-1 in kidney biopsies, a mouse monoclonal antibody (ab2786, Abcam Limited, Cambridgeshire, UK) with aorta sections as the positive control for the technique was used. This antibody shows little cross-reactivity to ET-2 and ET-3. For detection of ETRA protein expression, a rabbit polyclonal antibody (ab12977, Abcam Limited, Cambridgeshire, UK) was used. The positive control for ETRA was human large bowel.
Immunohistochemistry techniques are described in detail below.
Immunohistochemistry for ET-1
59 Paraffin sections were cut to a 4 mm thickness and placed on pre-treated slides with Histogrip (Zymed, US), heat-fixed at 60°C for 24 hours. Deparaffinization and dehydration in alcohols were followed by washing in distilled water. Antigen retrieval was done with citrate pH 9.0 solution (Target Retrieval Solution, Dako, US) in a pressure cooker designed for use in a microwave oven (NordicWare Microwave Tender Cooker, Biogenex, US). Endogenous peroxidase was blocked with hydrogen peroxide 3% in methanol twice, for 15 min each, and washed in PBS pH 7.2. Nonspecific blocking was done with Protein Block Serum-Free solution (Dako, US) for 30 min at room temperature. Slides were then incubated overnight with primary monoclonal anti-mouse antibody to ET-1 (ab2786, Abcam Limited, Cambridgeshire, UK) at 1:1000 dilution. The reaction was amplified with the peroxidase polymer Picture Max (HRP Polymer Conjugate Broad Spectrum, Invitrogen, US) according to the manufacturer’s instructions. To develop the reaction, diaminobenzidine was used for 5 min (Dako Liquid DAB Substrate Chromogen System, US). The slides were counterstaining with Harris hematoxylin for 1 min, followed by dipping in ammonia water 37 mM for 15 seconds and dehydration in alcohols. Slides were mounted and coverslipped with non permanent Entellan mounting medium (Merck, Germany).
Immunohistochemistry for ETRA
Paraffin sections were cut to a 4 mm thickness and placed on pre-treated slides with Histogrip (Zymed, US), heat-fixed at 60°C for 24 hours. Kidney tissue was deparaffinized and dehydrated in alcohols followed by washing in distilled water. Endogenous peroxidase was blocked with hydrogen peroxide 4.5% in methanol. Washing was done with TBS/Tween20 buffer between steps. Antigen retrieval was done with citrate buffer (pH 6.0) in an automated pressure cooker (Decloaking Chamber, Biocare Medical, US). Nonspecific blocking was done with normal goat serum at 1:50 and avidin D at 1:10 dilutions. Slides were then incubated
60 overnight with primary polyclonal anti-rabbit antibody to ETRA (ab12977, Abcam Limited, Cambridgeshire, UK) at 1:200 dilution in TBS / 1% BSA for 45 minutes, followed by incubation with secondary biotinylated anti-rabbit antibody (Biocare Medical, US) for 30 minutes. The next step was to apply the enzyme alkaline phosphatase (AK 5000, Vector Laboratories, US) for 30 minutes (1 ml of TBS / BSA 1% + l0 ml solution A + 10 ml solution B). The final step was to wash with TBS alone, and to develop the reaction with blue chromogen substrate of alkaline phosphatase (Vector Blue, Vector Laboratories, US) The slides were counterstained with Harris hematoxylin for 1 min, followed by dipping in ammonia water 37 mM for 15 seconds and dehydration in alcohols. Slides were mounted and coverslipped with non permanent Entellan mounting medium (Merck, Germany).
ET-1 and ETRA expression
Quantification of the expression of ET-1 and ETRA was performed by digital image analysis using Image Pro Plus software, version 4.5 (Media Cybernetics). Images were visualized through a Zeiss microscope (model AXIOSKOP-40, Carl Zeiss, Oberkochen, Germany), and captured using the Cool Snap-Pro CS (Media Cybernetics) camera. After selecting the hot spot areas with higher expression of ET-1 and ETRA using low power objectives, non-consecutive random fields were chosen using the greek line method to avoid field overlap (21). Thirty-five to forty fields were captured in nephrectomy tissue (mean, 37 fields) and for the entire fragment of percutaneous biopsies, 21 fields in average.
ET-1 expression
ET-1 expression was detected exclusively in endothelial cells of peritubular capillaries and glomerular capillaries, with the strongest intensity of staining in dark brown by DAB chromogen as compared to background color. Positive control was considered as the brown
61 staining in red blood cells, and negative control as the background. Positive events were considered when spherical or elliptical shaped stained structures, with or without the presence of red blood cells, stained by DAB at the same intensity of red blood cells. Contiguous objects were counted as a single event.
Positive event counting was performed with 200X magnification and a resolution of 1392 x 1040 pixels per 0.76 mm². Image analysis was performed by unbiased counting, inserting a correction grid with 1312x960 pixel amplitude, corresponding to the area of 0.66 mm². Positive staining was counted within the area of the grid in the captured field, and all events that touched the continuous line of the grid were also counted, those that touched the dotted line were excluded. The area (in pixels) from the field without tissue or with non-renal tissue was subtracted from the total area of the field, thereby correcting for positive events per area of renal tissue in that field. Counting was performed on an ordinal scale, and the arithmetic mean of positive events was calculated for the 35-40 selected fields in tissue nephrectomies, or for all fields in fragments of percutaneous biopsies, at each hot spot (21).
ETRA expression
Endothelin A receptor was expressed only in the cytoplasm of tubular epithelial cells, with the strongest intensity of staining in dark blue compared to background color. If one or more epithelial cells in a tubular section stained positive, that tubule was considered one event. Positive event counting and calculation of the mean to obtain a final score for ETRA (on an ordinal scale) were performed the same way as for ET-1.
Statistical analysis
Data were presented as mean and standard deviation (SD) (or median and interquartile ranges) or proportions. Comparisons between different groups were done by ANOVA.
62 Nominal data were analyzed using the Chi-square and Fischer exact test. For statistical analysis, ET-1 and ETRA expressions were logarithmically transformed to reduce skewness.
Correlations between immunohistochemical scores of ET-1 and ETRA with renal function and proteinuria were performed using Spearman’s correlation coefficient. A p value <0.05 was considered significant. Data were processed and analyzed using Statistical Package for Social Sciences for Windows, version 16.0 (SPSS Inc. Chicago, IL).
Results
Clinical and laboratory data
Clinical and laboratory data of the patients are presented in Table 1. The prevalence of arterial hypertension and the use of ACEi were similar in all three groups. Patients with IgA nephropathy were younger compared to controls and DN.
Control patients had lower serum creatinine levels and higher GFR as compared to DN and IgA nephropathy groups. Proteinuria was of a greater magnitude in patients with DN but it was also high in IgA nephropathy, as these patients had an indication for biopsy.
Immunohistochemical staining for immunoreactive ET-1
Kidney tissue from control subjects had no or only a weak expression of ET-1 in endothelial cells of glomerular and peritubular capillaries. Among patients with IgA nephropathy and DN, staining was markedly increased in endothelial cells of glomerular capillaries and was slightly increased in peritubular capillaries (Figure 1). There was a stronger staining and higher scores in DN and IgA nephropathy biopsies when compared to controls (p <0.001) as illustrated in Figure 2. ET-1 expression was also detected in renal vessels, mainly in endothelial cells and vascular smooth muscle cells of arteries and arterioles, and it was more prominent in biopsies of DN patients.
63 Immunohistochemical staining for immunoreactive ETRA
ETRA was only expressed in the cytoplasm of tubular epithelial cells, mainly in distal tubules. No staining was detected in glomeruli, peritubular capillaries and vessels. Control sections demonstrated only weak and focal expression, whereas in most of the DN and IgA nephropathy cases the staining was much more intense and diffuse (Figure 1). There were stronger staining and higher scores in DN and IgA nephropathy biopsies when compared to controls (p = 0.019) as illustrated in Figure 3.
ET-1 and ETRA correlation with renal function and proteinuria
Proteinuria was available for 18 subjects (11 ND and 7 IgA nephropathy). There was a positive correlation between proteinuria and ET-1 expression (r = 0.634, p = 0.027). No correlation was observed between ETRA expression and proteinuria (r = 0.095, p = 0.77).
Additionally, no correlation was found between the magnitude of ET-1 or ETRA expression and GFR or serum creatinine.
Discussion
This comprehensive immunohistological analysis of the two main components of the endothelin system showed an up-regulation of ET-1 as well as ETRA expression in DN compared to normal renal tissue. The magnitude of this expression was similar to that observed in IgA nephropathy, and in both conditions ET-1 expression correlated positively with proteinuria.
Elevated plasma ET-1 levels have been reported in patients with DM (18; 22; 23). In a previous study we showed that there is a progressive increase in plasma ET-1 levels which correlated positively with increased urinary albumin excretion (19). The gene expression of ET-1 is increased in kidneys of diabetic rats, as well as in mesangial cell culture of rats
64 exposed to high glucose concentrations (7). Endothelial dysfunction in diabetic patients, increases ET-1 production leading to vascular hypertrophy, atherogenesis and, in the kidney, glomerulosclerosis. Endothelin receptor blockade has a nephroprotective effect, correcting both the initial hyperfiltration and its progression to clinical DN (13; 14).
In normal human kidneys, immunoreactive ET-1 was localized segmentally in endothelium of glomerular capillary loops, vessels, and peritubular capillaries, suggesting that its secretion is compartimentalized and locally regulated (24). In disease, several studies reported that renal expression of ET-1 is increased in both animal models and human chronic nephropathies (24; 25). In human subjects, increased ET-1 expression was observed in several conditions, e.g., lupus nephritis, IgA nephropathy and membranous glomerulonephritis (15), acute graft rejection, chronic allograft nephropathy, and post-operative acute tubular necrosis (16; 26). This study is the first to demonstrate consistently the increased ET-1 and ETRA expression in human DN.
Endothelin-1 can be over expressed in kidney tissue in several situations. However, the pattern of ET-1 expressions seems to vary. Lehrke et al (15) showed a markedly increased ET-1 expression in proximal tubular epithelial cells and less marked but still increased expression in glomerular endothelium of subjects with IgA nephropathy. Expression of ETRA was not significantly increased in biopsy samples, but immunoreactive ETRB was greatly increased in proximal tubules but less intense in glomeruli. In lupus nephritis, mesangial staining for ET-1 was elevated suggesting that under certain conditions (i.e., inflammation) ET-1 is also present in glomerular mesangium (24). In dysfunctional kidney allografts, expression of ET-1 and ETRA was markedly increased in glomeruli and tubuli irrespective of the underlying histological diagnosis, and there was a strong correlation between proteinuria and ET-1 expressed in glomeruli and tubuli (16). In the present study, ET-1 expression was segmental and restricted to endothelial cells of glomerular and peritubular capillaries,
65 suggesting a local production and regulation. Unexpectedly, ETRA staining was restricted to tubular epithelial cells, mainly in distal segments, a finding to be further explored.
Previous studies in patients with IgA nephropathy demonstrated an association between proteinuria levels and intensity of ET-1 expression in glomeruli and in proximal tubular cells. Patients with high-grade proteinuria (≥2 g/24h) have a significantly higher ET-1 expression than patients with low-grade proteinuria (<2 g/24h) (15). Furthermore, a strong correlation was described between proteinuria and ET-1 expression in glomeruli and proximal tubules of recipients of kidney transplants (16). In accordance with these previous reports, in the present study there was a significant correlation between proteinuria and ET-1 expression in glomeruli and peritubular capillaries in patients with DN and IgA nephropathy (r = 0.634, p
= 0.027).
Douglas et al (27) analyzed ET-1 expression in two different renal cell carcinomas (clear cells vs. papillary) showing that the endothelin axis was expressed differently in these two main subtypes. We considered normal renal tissue derived from nephrectomies due to tumors as controls and we included cases of both clear cells and papillary carcinoma subtypes.
ET-1 and ETRA expression did not differ between the two subtypes (data not shown).
Therefore, due to over expression of ET-1 in many kidney diseases, increased ET-1 expression could be a non specific phenomenon secondary to kidney injury. However, studies with experimental models of DM where ETRA and ETRB were blocked by specific antagonists demonstrated a normalization of the glomerular pressure, a decrease of protein deposition in mesangial extracellular matrix and a decrease in urinary albumin excretion suggesting that the endothelin system does in fact play a role in the pathogenesis of DN (28;
29).
In conclusion, our results suggest a potential role for the endothelin system in DN and probably in other non diabetic glomerular diseases such as IgA nephropathy. These findings
66 are of interest and must be further explored, in view of the experimental observation that ET system blockade with specific antagonists could prevent the progression of DN.
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70 Table 1. Clinical and laboratory characteristics of patients with normal kidney tissue,
diabetic nephropathy or IgA nephropathy
Normal Kidney n = 13
Diabetic Nephropathy
n = 13
IgA Nephropathy
n = 10
P
Age (years) 62 ± 20 52 ± 15 39 ± 14 0.013*
Sex male, n (%) 8 (61.5) 8 (61.5) 7 (70.0) 1.0
White patients, n (%) 11 (84.6) 10 (76.9) 10 (100) 0.184 Serum creatinine (mg/dL)
Proteinuria (g/24h)
1.0 ± 0.2 NA
2.8 ± 2.5 294 (107-570)
2.8 ± 2.7 193 (33-219)
0.041£ 0.056
GFR (mL/min/1.73 m2) 78.5± 25 52.1 ± 42 50.1 ± 36 0.115 Hypertension, n (%) 7 (53.8) 11 (84.6) 3 (37.5) 0.112 ACE inhibitor use, n (%) 7 (53.8) 9 (75) 6 (75) 0.265
ET-1 score‡ 1.52
(1.02 - 2.12)
8.86 (5.41 - 15.12)
3.16 (2.41 – 3.88)
<0.001£
ETRA score‡ 2.50
(1.36 - 2.37)
4.90 (2.80 - 7.70)
4.60 (3.10 - 5.40)
0.019£
Data are expressed as mean ± SD or median (interquartile ranges 25 -75%); *IgA vs. normal kidney; £DN and IgA nephropathy vs. controls vs.; NA: not available; ‡Data were logarithmically transformed before analysis.
71 Figure 1. Representative microphotographs of the immunostaining of ET-1 in normal kidney tissue (A1), IgA nephropathy (A2) and diabetic nephropathy (A3); magnification: X200. Cells stained dark brown indicate the expression of ET-1. Representative microphotographs of the immunostaining of ETRA expression in normal tissue (B1), IgA nephropathy (B2) and diabetic nephropathy (B3); magnification: X100. Cells stained dark blue indicate the expression of ETRA. Arrowhead: increased ET-1 expression in endothelial cells of glomerular and peritubular capillaries (A2, A3) which was more intense in diabetic nephropathy (A3); no or only weak expression of ET-1 in normal renal tissue (A1). Arrow:
diffuse and strong expression of ETRA in the cytoplasm of tubular epithelial cells in IgA nephropathy (B2) and diabetic nephropathy (B3); a focal and weak expression of ETRA in tubuli of normal tissue (B1).
72 Figure 2. Score analysis of ET-1 positive events per area of renal tissue (PEART) according to renal disease.
0.00 8
<0.00 1
0.51 9
73 Figure 3. Score analysis of ETRA positive events per area of renal tissue (PEART)
according to renal disease.
0.03 6 0.02
7
0.97 2