Correlation Analysis between Intrarenal Small Artery Intimal Thickening and Clinicopathological Features and Prognosis in Primary Membranous Nephropathy Patients Free

Primary membranous nephropathy (PMN) is the most common pathological type of nephrotic syndrome in adults [1]. Its incidence is increasing year by year [2, 3]. The main mechanism of PMN is the shedding of podocyte antigens on the basement membrane followed by the binding of antibodies. Its pathology is characterized by extensive deposition of immune complexes on the subepithelial area [4]. Phospholipase A2 receptor and thrombospondin domain-containing 7A are the target antigens most commonly found in PMN patients [5, 6]. In addition, many new target antigens have been discovered in recent years. For example, exotoxin 1 and exotoxin 2 are primarily prevalent in young females; neural EGF-like-1 protein is associated with malignancy; semaphorin 3B has a familial tendency; and protocadherin 7 is mainly detected in elderly patients [7]. Patients with different target antigens have different renal prognoses [5]. In addition, studies have confirmed that impaired renal function at baseline, severe proteinuria, hypertension, glomerulosclerosis, and interstitial fibrosis all contribute to the poor prognosis of membranous nephropathy [8, 9].

Intrarenal arteriolar lesions are common pathological changes in renal biopsies of patients with kidney disease [10]. It can affect the interlobular artery, the afferent arteriole, and the efferent arteriole [11]. Arterioles are structurally divided into three layers: intima, media, and adventitia [12]. The function and structure of renal arterioles are altered in many renal diseases [13]. A Chinese study showed that more than 60% of PMN had intimal thickening, which is usually associated with luminal stenosis [14]. And the incidence and severity were higher in older patients [15]. Compared to patients with IgA nephropathy (IgAN), PMN patients had a higher percentage of arteriolar wall thickening alone and a lower percentage of concomitant hyaline changes [16]. In IgAN patients, intrarenal arterial lesions (including arteriolar thickening) were associated with hypertension, decreased renal function, and poor renal prognosis [17]. However, studies on intrarenal small artery intimal thickening and renal prognosis in PMN patients are still insufficient. The objectives of this study were to (1) evaluate the incidence of intrarenal small artery intimal thickening; (2) investigate clinicopathological features in PMN patients; (3) explore the correlation between intimal thickening and renal composite outcome. Therefore, in order to prevent the development and delay the progression of this lesion in PMN patients, early detection and treatment can be administered to those who are susceptible to intrarenal small artery intimal thickening.

This study was a single-center retrospective study. Patients hospitalized at Shenzhen Second People’s Hospital between January 2008 and December 2020 diagnosed with membranous nephropathy through renal biopsy were included. Exclusion criteria included (1) age <14 years; (2) the number of biopsied glomeruli <8; (3) patients with renal pathology without a description of vascular lesions; (4) secondary membranous nephropathy, including lupus nephritis, viral hepatitis, and malignancy. For the cross-sectional investigation, baseline data were obtained from renal biopsies taken at the time of hospitalization in patients who satisfied the inclusion and exclusion criteria. Patients were divided into two groups according to the presence or absence of intrarenal small artery intimal thickening. The renal function of the patients in the subsequent follow-up was collected into the cohort study, with exclusion criteria of (1) baseline estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m² and (2) follow-up time <6 months. The study followed the Declaration of Helsinki and was reviewed and approved by the Medical Ethics Committee of Shenzhen Second People’s Hospital (ethical approval document number: 201408186).

All data were collected by accessing the electronic case system and pathology report.

Demographic data including sex, age, medical history, and body mass index (BMI) were collected at the time of the initial renal biopsy. Laboratory data included serum creatinine, eGFR, hemoglobin, albumin, total cholesterol, triglycerides, 24-h urine protein quantitation, and renal biopsy results. Initial drug use after renal biopsy was collected, including the use of angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin receptor blockers (ARBs) and steroids/immunosuppressants (e.g., methylprednisolone and cyclosporin). Mean arterial pressure equals 1/3 systolic pressure + 2/3 diastolic pressure. Glomerular filtration rate (GFR) was estimated using the CKD-EPI equation [18].

Renal biopsy specimens were routinely processed and examined by light microscopy, immunofluorescence, and electron microscopy. Pathological staging was divided into five stages according to Ehrenreich-Churg staging [19]. Hematoxylin-eosin, periodic acid-Schiff, periodic acid-silver methenamine, and Masson staining were used for light microscopy. The number of glomeruli, global sclerosis, segmental sclerosis, crescent, mesangial proliferation, interstitial infiltration, tubular atrophy, and intimal thickening were observed. Intimal thickening was determined by comparison with the thickness of adjacent media [20]. Immunofluorescence was used to observe the deposition site and extent of IgG, IgA, IgM, C3, C1q, and M-type phospholipase A2 receptor (PLA2R) by direct method. Electron microscopy was used to observe the ultrastructure including the glomerular basement membrane, pedicles of the podocytes, and deposition of electron-dense material. All renal pathology results were read independently by two pathologists from Guangzhou Jinyu Medical Laboratory Center. A consensus was reached through mutual consultation if there were divergent pathological conclusions.

During follow-up, patients were defined to have reached the study endpoint and the renal composite outcome if their eGFR <15 mL/min/1.73 m² or had decreased by 30%, underwent a kidney transplant, or converted to hemodialysis. The remaining patients were not to be tracked until June 30, 2021.

R (http://www.R-project.org, the R Foundation), Empower Software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA, USA), and GraphPad Prism 8 (GraphPad Software Inc, La Jolla, CA, USA) were used for statistical processing. Data conforming to a normal distribution were conveyed in the form of mean ± standard deviation (mean ± SD), and data with a non-normal distribution were conveyed in the form of mean (1/4 quartile, 3/4 quartile). Categorical variables were expressed as numeral (percentage). The χ² test was used for the comparison of categorical variables. The independent sample t test was used for the comparison of normally distributed variables between groups. Univariate and multivariate analyses of risk factors affecting intrarenal small artery intimal thickening were performed using logistic regression models. The predictors for kidney composite outcome were assessed by Cox regression models. The results were expressed as odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI). Kaplan-Meier curves and log-rank tests were used to analyze patients’ renal survival. The difference was considered statistically significant at p < 0.05.

A total of 354 patients were diagnosed with membranous nephropathy by renal biopsy. According to exclusion criteria, 54 patients were excluded including 15 with membranous lupus nephritis, 13 with hepatitis B-associated nephritis, 17 with atypical membranous nephropathy with full-house immunofluorescence [21], 1 with membranoproliferative nephritis, 3 with combined tumor, 2 with combined diabetic nephropathy, 1 with lack of arterioles on renal biopsy, and 2 with end-stage renal disease at the time of renal biopsy (Fig. 1). Therefore, 300 patients with PMN were enrolled in the cross-sectional study. Patients with baseline eGFR <30 mL/min/1.73 m² (n = 3) or follow-up <6 months (n = 90) were excluded. Finally, 214 patients entered the retrospective cohort study for survival analysis. There were 165 patients (55.00%) with intrarenal small artery intimal thickening (Fig. 2). Patients with intimal thickening were older, with higher BMI, serum uric acid, systolic and diastolic blood pressure, a higher proportion of combined hypertension and acute kidney injury, more urine protein, and lower eGFR. There were no significant differences in gender, hemoglobin, serum albumin, serum triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, or blood PLA2R antibodies between the two groups (Table 1).

A total of 283 patients underwent staging for membranous nephropathy through renal biopsy. There were 49 (17.31%) patients in stage I, 203 (71.73%) in stage II, 31 (10.95%) in stage III, and no patients in stage IV (not listed in the table). 9 (3.00%) patients had crescentic lesions. 28 (9.33%) patients had segmental sclerotic changes. 138 (46.00%) patients had global sclerosis. 152 (50.67%) patients had mild to moderate tubular atrophy. 219 (73.00%) patients had interstitial infiltration. 281 (93.67%) patients had mild to moderate mesangial proliferation. Seventy-six patients were tested for serum PLA2R antibodies, among which 47 (15.67%) were positive (Table 1).

Univariate regression analysis showed that age, BMI, hypertension, eGFR, renal function stage, global and segmental sclerosis, mesangial proliferation, tubular atrophy, and interstitial infiltration were associated with intimal thickening. Multivariate regression analysis showed that age (OR = 1.06, 95% CI: 1.03–1.09), global sclerosis (OR = 2.13, 95% CI: 1.05–4.33), and interstitial infiltration (OR = 3.21, 95% CI: 1.27–8.12) were independent risk factors for the formation of intimal thickening in PMN patients (Table 2).

From the time of renal biopsy, the average follow-up time was 34.20 ± 28.43 months. The time reaching the composite renal endpoint for patients with intimal thickening was 26.38 ± 21.38 months and for patients without intimal thickening was 35.82 ± 28.23 months, respectively. A total of 36 patients reached the endpoint, with 7 (7.45%) and 29 (24.17%) in the no-thickening and thickening groups, respectively. Kaplan-Meier survival curve analysis revealed a higher incidence of reaching the renal composite endpoint in PMN patients with intimal thickening (p < 0.05) (Fig. 3). In Cox regression analysis, intrarenal small artery intimal thickening was a risk factor for poor renal prognosis in PMN patients when not adjusting for other factors (HR = 4.68, 95% CI: 2.00–10.96, p < 0.001). After adjusting for age, gender, hypertension, BMI, urine protein, eGFR, use of ACEI/ARB, and steroids/immunosuppressants, intimal thickening remained a risk factor for poor renal prognosis in PMN patients (HR = 3.68, 95% CI 1.36–9.96, p < 0.05) (Table 3).

As common pathological findings in renal diseases, intrarenal arterial lesions can be caused by many factors [22‒24]. Previous research mainly focused on general vascular abnormalities, glomerulosclerosis, and arteriolar hyalinosis in PMN [25‒28]. In this study, we focused on the intimal thickening of the intrarenal small arteries. We investigated and compared clinical manifestations and pathological features of PMN patients with and without intimal thickening. The risk factors for intimal thickening were also investigated, and the relationship between this lesion and the renal composite outcome was examined. Results confirmed that intrarenal small artery intimal thickening was a risk factor for renal composite outcome and associated with poorer renal prognosis.

Atypical membranous nephropathy is pathologically manifesting as thickening of the basement membrane and deposition of electron-dense material at multiple sites which is characterized by “full house” after immunofluorescent staining [29]. It is considered to be a special kind of secondary membranous nephropathy [21]. Therefore, we did not include these patients in our research. Besides, we excluded eGFR <30 mL/min/1.73 m² from the follow-up study referred to the Oxford study since this group of patients was highly suspected to have vascular lesions [30].

Intimal thickening can be observed in various types of chronic kidney disease, while it is most commonly seen in IgAN patients [17, 31, 32]. A Chinese study including 1,683 IgAN patients reported that 60% of patients developed intimal thickening [17]. Another study reported a high prevalence of arterial-arteriolar wall thickening in IgAN patients, while such a lesion was found in 47.1% of MN patients [16]. In our study, 165 (55%) of the enrolled 300 PMN patients had intrarenal small artery intimal thickening. The discrepancy in incidence may be due to the inconsistency of the arteriolar thickening criteria. Concurrent diseases such as hypertension and diabetes generally accelerate disease progression. Diabetes can cause arteriolar injuries in individuals without chronic kidney disease or with preserved renal function [33]. In IgAN patients, hyperuricemia and hypertension were associated with arteriolar injuries and poor renal outcomes [17, 34]. In our study, although intimal thickening was observed more frequently in patients with higher serum uric acid levels, hypertension, and diabetes, these factors were unrelated to intrarenal intimal thickening. In general, intimal thickening is not a specific pathological manifestation in either PMN or chronic kidney disease. The effects of concurrent diseases on intimal thickening vary in different populations.

Previous studies have found that in PMN patients, arterial lesions were associated with renal function and prognosis. A retrospective analysis including 168 PMN patients suggested a higher incidence of arteriolar lesions in older PMN patients, which may contribute to a poorer prognosis [15]. Results of a Chinese study showed no significant differences in the incidence of intrarenal small artery intimal thickening among the young, middle, and older groups. Univariate logistic regression analysis revealed that intimal thickening was a risk factor for the prognosis of PMN patients [35]. In Japanese research of PMN patients with nephrotic syndrome, univariate analysis showed that intrarenal vascular lesions were risk factors for end-stage renal disease, but this effect was not significant in multivariate analysis [36]. In contrast, in our study, after adjusting for age, gender, hypertension, BMI, urine protein, eGFR, use of ACEI/ARB, and steroids/immunosuppressants, intimal thickening remained a risk factor for renal prognosis in PMN patients.

The mechanism of the formation of intrarenal small artery intimal thickening in PMN patients has not been well studied yet. Lhotta et al. [37] analyzed 135 renal biopsies and found a significant correlation between cigarette use and myointimal hyperplasia of small arteries. In IgAN patients, C3d deposition in the renal arterioles was correlated with different degrees of thickening and sclerosis. Arteriolar lesions spread from the intima to media with increasing amount of C3d deposits [38]. In another study, the concentration of serum von Willebrand factor and anti-endothelial cell antibodies was found to be associated with the severity of intrarenal arterial lesions [39]. Endothelial injury present in patients with chronic kidney disease was associated with micro-inflammation, vascular endothelial growth factor, tumor necrosis factors, and many other molecules [40, 41]. Similar to atherosclerosis, it may further develop into intimal thickening through the accumulation of smooth muscle cells [42, 43]. In our study, multivariate analysis showed that age, global sclerosis, and interstitial infiltration are independent risk factors of intrarenal small artery intimal thickening. Aging is a risk factor for many chronic diseases due to structural and functional changes in organs. Even in healthy aging, the incidence of vascular lesions increased with age [44]. In chronic glomerular lesions, injured capillaries were accompanied by loss of CD34+ glomerular endothelial cells [45]. This lesion was more prominent in PMN patients with focal segmental glomerulosclerosis [46]. Therefore, in some cases, global sclerosis may be one of the stages in the progression from endothelial injury to intimal thickening. Pei et al. [47] concluded that arterial lesions in IgAN patients were correlated with the severity of interstitial infiltration, which was mostly composed of CD68+ and DC-SIGN+ cells (a subset of dendritic cells). In PMN patients, the upregulated myofibroblasts accompanied by other molecules were observed in interstitium, especially in progressive patients [48]. The α-smooth muscle actin expressed by myofibroblasts may trigger the formation of intimal thickening [49, 50].

Intimal thickening and stenosis of the intrarenal small arteries can cause ischemia and hypoxia of the kidney tissue, resulting in glomerulosclerosis [51]. Glomerular damage may lead to alterations of the small arteries in turn [52]. This vicious cycle can be broken by ACEI to some extent. As early as 1989, an animal experiment done by Powell et al. [53] showed that rats receiving ACEI had less neointima formation and preserved luminal integrity after vascular injury. A recent study conducted by Sugiura et al. [54] among IgAN patients came to similar conclusions. IgAN patients with vascular lesions treated with ACEI were found to have a better renal prognosis, while such a beneficial effect was not found in patients without vascular lesions. Although there was little evidence that PMN patients with intimal thickening can benefit from ACEI, we hypothesize that this drug may play a similar role in slowing down the deterioration of kidney function because ACEI can relieve high perfusion, high pressure, and high filtration present in chronic kidney disease [55]. Further research is necessary to provide recommendations for clinical medicine regarding the effectiveness of ACEI used in such PMN patients.

Our study has several advantages. First of all, this is the first study to analyze the correlation between intrarenal small artery intimal thickening and clinicopathological characteristics as well as renal prognosis in PMN patients. Through regression and survival analysis, we identified intrarenal small artery intimal thickening as a risk factor for poor renal outcome. The prevalence of membranous nephropathy is high, which deserves further study to help prevent and avoid the rapid progression of the disease. Second, this study was a retrospective study with relatively little observational bias, which ensured the reliability of the data. Meanwhile, our study has some disadvantages. First, because of the prolonged follow-up duration, survival time may be affected by other unknown variables. Second, we did not conduct further studies to explain the interplay between risk factors. Molecular biological studies can be used to analyze the development of intimal thickening in PMN.

In conclusion, the results of this study suggest that intrarenal small artery intimal thickening is a risk factor for renal composite outcome and poor renal prognosis in PMN patients. Early intervention of this group of patients is very important.

We would like to thank Jinyu Institution for the assistance in analyzing the histological slides.

This study followed the Declaration of Helsinki and was reviewed and approved by the Medical Ethics Committee of Shenzhen Second People’s Hospital (ethics approval document number: 201408186). Written informed consent was obtained from participants in the study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This work was supported by a grant from the Basic Research Program of the Shenzhen Science and Technology R&D Fund (Y.C., Grant No. JCYJ20190806163801637), Shenzhen Second People’s Hospital Clinical Research Fund of Guangdong Province High-level Hospital Construction Project (Y.C., Grant No. 20203357012), and Shenzhen Key Medical Discipline Construction Fund (Grant No. SZXK009).

Yuan Cheng, Chen Jia, Qi Dongli, Guan Mijie, and Qingqing Rao investigated the patients; Yuan Cheng and Wangyang Li drafted the paper; Haofei Hu, Tao Cao, and Liling Wu performed the analysis; Qijun Wan planned and led the study.

Yuan Cheng and Wangyang Li have contributed equally to this work.

The datasets analyzed for this study can be found in the article.