ASSESSMENT OF INTRARENAL VASCULARIZATION IN DIFFUSE SYSTEMIC SCLEROSIS

Systemic sclerosis is an autoimmune disease, characterized by fi brosis and vascular abnormalities, which determine the clinical manifestations and prognosis. The aim of this study was the assessment of intrarenal vascularization in diffuse systemic sclerosis patients and its correlations with nailfold capillaroscopy abnormalities. Material and methods. The study was performed on 11 patients with diffuse systemic sclerosis without any renal abnormalities and 11 sexand age-matched controls. In all patients were determined: glomerular fi ltration rate (GFR), proteinuria, urinary sediment, resistive (RI) and pulsatility (PI) index of interlobar renal arteries and the number of nailfold capillaries/mm. All the data were presented as mean ± standard deviation. The statistically analysis was done using Pearson’s test and Student’s t-test, p < 0.05 was considered statistically signifi cant. Results. The mean age of diffuse systemic sclerosis patients was 46.90 ± 4.98 years, and the mean length of disease was 4.63 ± 2.96 years. GFR, RI, and PI were signifi cantly lower in diffuse systemic sclerosis patients than in controls (p < 0.001). It was identifi ed a strong correlation between nailfold capillaries density/mm and GFR (r = 0.719, p < 0.05), RI (r = 0.784, p < 0.01), PI (r = 0.748, p < 0.01), and the mean length of the disease (r = 0.85, p < 0.001). Conclusion. In patients with systemic sclerosis, intrarenal vascularization and glomerular fi ltration rate correlate with the capillaroscopic fi ndings.

Systemic sclerosis (SSc) is an autoimmune multisystem disease, characterized by immune activation, autoantibody production, fi broblast dysfunction, extracellular matrix deposition, and vasculopathy (1). Its incidence is about 20 cases/1 million/year, rising in the last years. The SSc prevalence varies from 250 to 350 cases/million people (2,3).
Major organ involvement (lung, kidney, heart, gastrointestinal tract) is associated with decreased survival in these patients (1). In their study, Steen and Medsger revealed that the 9-year survival in sclerodermic patients with major organs involvement is reduced at 39%, compared with the patients without these comorbidities, in whom the survival is about 72% (4).
Renal involvement represents an important cause of morbidity and mortality in SSc patients. The spectrum of renal complications in SSc includes: sclero-derma renal crisis, normotensive scleroderma renal crisis, myeloperoxidase-antineutrophil cytoplasmic antibodies associated glomerulonephritis and vasculitis, penicillamine-associated renal disease, nephropathy induced by antiphospholipid antibodies, isolated urinary abnormalities (proteinuria, microscopic haematuria), isolated reduction in glomerular fi ltration rate. Another kidney involvement in scleroderma, less studied, is represented by abnormal increase of intrarenal resistance indices, without any clinical or biological signs (5). Evidence of renal disease may identify a subset of patients with scleroderma, who will experience signifi cant morbidity and mortality (6). Subclinical renal impairment affects approximately 50% of scleroderma patients and may be associated with other vascular manifestations. Subclinical renal involvement rarely progresses to end-stage renal failure; however, Shanmu-gam and Steen suggest that it may predict mortality in patients with other vasculopathic manifestations (5).
Microcirculation of patients with SSc is assessed at the nailfold skin of the fi ngers of the hands by means of nailfold capillaroscopy. Based on the capillaroscopic abnormalities, Cutolo classifi ed the sclerodermic capillaroscopic pattern into three stages: early (few giant capillaries, few capillary microhaemorrhages, no evident loss of capillaries, and a relatively well-preserved capillary distribution), active (frequent giant capillaries, frequent capillary microhaemorrhages, moderate loss of capillaries, absence of or mildly ramifi ed capillaries with slight disorganisation of the capillary architecture), and late (irregular enlargement of the capillaries, almost absent giant capillaries and microhaemorrhages, severe loss of capillaries with extensive avascular areas, ramifi ed/bushy capillaries, and intense disorganisation of the normal capillary array) (7).
Intrarenal vascularisation may be assessed by means of Doppler ultrasonography of interlobar renal arteries, determining the resistive index (RI) and pulsatile index (PI) (8).
The aim of this study is represented by the assessment of intrarenal vascularization and its correlation with the nailfold capillaroscopy fi ndings in diffuse SSc patients.

MATERIAL AND METHODS
The study was performed on a group of 11 patients with systemic sclerosis without clinical and biological renal abnormalities and 11 sex-and agematched controls. The diagnosis of SSc was established based on 2013 Classifi cation Criteria for Systemic Sclerosis (9). Exclusion criteria were: isolated urinary abnormalities (microhaematuria and/or proteinuria), primary or secondary kidney diseases (glomerulonephritis, tubular-interstitial nephropathies, nephrolithiasis, renal cysts, vascular nephropathies), arterial hypertension, diabetes mellitus, heart failure, generalized atherosclerosis.
Serum and urinary creatinine were determined using Jaffe method, proteinuria by biuret method, and urinary sediment by Addis-Hamburger method.
Intrarenal vascularization was characterized by resistive (RI) and pulsatility (PI) indices of interlobar renal arteries. They were determined by means of Doppler ultrasonography, with Siemens Acuson X300 Ultrasound System, with convex 3,5 MHz probe.
Capillaroscopy is a non-invasive imaging technique that is used for in vivo assessment of the microcirculation. In our study, nailfold capillaroscopy was performed with USB Digital Microscope 2.0 mega Pixel Digital Camera.
Before starting this procedure, the patients took place in a room with a stable temperature of 20-22°C for at least 15 minutes, in order to avoid capillaries vasoconstriction, which can induce false positivity for avascular areas. The 2 nd , 3 rd , 4 th and 5 th fi ngers of both hands were examined. Giant capillaries, capillaries haemorrhages, avascular areas, and capillary architecture were the recorded nailfold capillaroscopic parameters (10,11). Nailfold capillaries density/mm was the parameter used in the statistical analysis.
All the data were presented as mean ± standard deviation. The statistically analysis was done using Pearson's test and Student's t-test, p < 0.05 was considered statistically signifi cant.

RESULTS
The group of patients with SSc was formed by 8 females and 3 males, with the mean age of 46.90 ± 4.98 years. All the patients had diffuse form of SSc, with the mean length of evolution of 4.63 ± 2.96 years. Antinuclear antibodies were present in all the patients. The assessed parameters are presented in the Table 1. In the SSc group, the capillaries density/mm was signifi cantly reduced than in controls (Fig. 1, 2). Our study reveals a strong inverse correlation between the capillaries density and the mean length of disease evolution (r = -0.85, p < 0.001) (Fig. 3).
Proteinuria and urinary sediment didn't show any changes in sclerodermic group. But glomerular fi ltration rate was signifi cantly reduced in these patients (p = 0.0000072). Resistive and pulsatility indices were higher in studied patients than in controls (p = 0.0000079 for RI, and p = 0.000053 for PI), demonstrating the presence of intrarenal vasoconstriction (Fig. 4). These parameters were correlated with the capillaries density. We found a signifi cant correlation between the capillaries density and GFR (r = 0.719, p = 0.012). Other correlations were established between the capillaries density and RI (r = -0.784, p = 0.004), respective PI (r = -0.748, p = 0.008). (Fig. 5, 6).

DISCUSSION
Vascular pathology represents one of the main factors involved in pathogenesis and organ dysfunction in SSc. Vascular involvement in SSc affects predominantly the microcirculation (arterioles and capillaries). Vascular dysfunction and disorganised microvasculature occur early in the disease evolution, and may predict the prognosis (3).
Sclerodermic vessels pathology shows concentric intimal proliferation, marked luminal obstruction, and lymphocyte infi ltration (12). Microvasculature changes, such as those assessed by nailfold capillaroscopy, are seen in all involved organs (lungs, heart, kidneys, and gastrointestinal tract), demonstrating the widespread nature of capillary changes in this disease (13).
Renal involvement in SSc could exhibit several forms of different severity, from asymptomatic renal involvement to sclerodermic renal crisis. Trostle et al. reported that the asymptomatic renal pathology was identifi ed from autopsy studies in 60-80% of  sclerodermic patients. They demonstrated that the intimal thickening was present in small and medium-sized arteries of patients with diffuse SSc, but only in small arteries of limited SSc patients, com-pared with those in controls. The authors concluded that renal vascular structural changes represent an important feature in SSc, with major impact on prognosis (14). Histopathologic renal injury is common  in scleroderma, even without clinically evident sclerodermic renal crisis (5).
In our study, GFR was reduced in scleroderma patients, compared to healthy controls (p < 0.001). However, the lowest value of GFR wasn't below of 69 ml/min. These values of GFR weren't accompanied by pathological proteinuria, urinary sediment, or elevated serum creatinine. Kingdon et al. showed that the impairment of renal function can be present in patients with diffuse SSc without clinical and biological sign of renal involvement (15). In another study, Scheja et al. identifi ed that the decreased glomerular fi ltration rate was found in 11% of limited SSc and 8.6% of diffuse SSc patients (16). In their study, performed on 675 patients with diffuse SSc, Steen el al. reported reduced renal function in 32% of patients. During a period of 10 years, none of the patients developed severe renal failure, suggesting that the asymptomatic reduced of renal function is common in scleroderma, having a benign clinical course (17). It was revealed a correlation between GFR and capillaries density in SSc patients (r = 0.719, p = 0.012). This relation between GFR and capillaroscopic damage was described by Rosato et al. in their study (18).
Resistive and pulsatility indices of interlobar renal arteries represent markers of severity renal damage. They evaluate the intrarenal elasticity and compliance. Their elevated value identifi es vascular and interstitial nephropathies, because glomeruli are only responsible for 10% of the intraparenchymal fl ow resistances. Intrarenal vessels remodeling leads to elevation of RI as renal vascular disease progresses (8,18). In scleroderma patients, renal involvement appearance, even subclinical, is accompanied by the increase of these parameters (18). Our study reveals that these indices were increased in patients with SSc sclerosis, compared to controls (p < 0,001). On the other hand, between these parameters and capillaries density, signifi cant negative correlations were demonstrated (r = -0.784, p = 0.004 for the correlation between capillaries density and RI, respectively r = -0.748, p = 0.008 for the correlation between capillaries density and PI). Rosato et al. showed that the intrarenal indices were signifi cantly increased in sclerodermic patients than in controls. The authors revealed that these indices increased with the progression of capillaroscopic damage progression (18). Rivolta et al. showed that in sclerodermic patients, RI was signifi cantly increased than in controls (19). Aikimbaev et al. demonstrated the increase of Doppler indices of intrarenal vascular resistance in sclerodermic patients, compared with healthy controls, the values of these indices correlating with the disease duration, the age of patients and the plasma renin activity (20). Increased intrarenal resistive index signifi es the presence of renal vascular involvement and correlates with GFR and digital microvascular damage in SSc patients (5).
In SSc, some of types of renal involvement do not have an immediate clinical signifi cance. However, in the presence of severe capillaroscopic damage, intrarenal vascularization assessment can show signifi cant vasoconstriction, even in absence of clinical or biological signs of renal involvement (21).

CONCLUSION
In patients with diffuse SSc, assessment of intrarenal vascularization shows a vasoconstrictive pattern. Signifi cant correlations were demonstrated between capillaroscopic fi ndings and the indices which characterizes the intrarenal vascularization and glomerular fi ltration rate.
In this context, fi nding severe capillaroscopic damage in a patient with SSc should be followed by the assessment of renal vascular involvement, even in the absence of any clinical or biological signs.