Urinary podocyte microparticles are associated with disease activity and renal injury in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with multiple organ injuries. Lupus nephritis (LN) is recognized as one of the most severe organ manifestations of SLE and is characterized by proteinuria, haematuria and progressive renal dysfunction. The pathogenesis of LN involves the glomerular deposition of autoantibodies related to self-antigens, the activation of complement system, the infiltration of inflammatory cells, and the over-production of proinflammatory cytokines and chemokines. At present clarifying the pathogenesis of LN, monitoring the degree of disease activity and taking effective measures to slow the progression of LN are substantial challenges.

Microparticles (MPs), a subtype of extracellular vesicles (EVs), are released by outward blebbing of the plasma membrane after the externalization of phosphatidylserine (PS) during cell activation and apoptosis [1]. The MPs carry various bioactive molecules and thereby serve as vectors for intercellular interaction. Interestingly, MPs can display biological activities associated with thrombosis, inflammation, and immune response [2]. Thus, MPs may be involved in the pathological and physiological processes of some diseases. Recent studies found that podocyte-derived MPs played crucial roles in the pathogenesis of some glomerular and non-glomerular diseases, being a novel early biomarker reflecting the damage of glomerular cells [3]. Gilani et al. [4] reported that renal injury in preeclampsia was associated with an elevated urinary podocin+ EV-to-nephrin+ EV ratio. Compared with patients with essential hypertension and healthy volunteers, renovascular hypertensive patients had elevated urinary podocyte EV levels. Podocyte EVs may reflect primary kidney damage-related podocyte injury [5].

The detection and analysis of urinary podocyte-derived MPs could be a potential non-invasive method for the monitoring of the progression of glomerular diseases. Therefore, this study aimed to identify podocyte phenotype, detect podocyte-derived MP level in the urine of SLE patients with and without LN and examine this association with clinical parameters.

Podocytes are critical cell components of the glomerular filtration barrier, which belong to highly differentiated cells and are almost non-renewable. Podocytes play key roles in maintaining the selective filtration of the glomerulus. Once podocyte injury occurs, it results in worsening proteinuria and renal failure. In LN, a major complication of SLE is often manifested with mesangial cell proliferation, decreased podocyte density, foot process fusion, and the change of podocyte phenotype. More and more studies have focused on podocyte damage in LN [9, 10]. Specifically, podocyte lesions in LN are explained by the following patterns: one is focal and diffuse proliferative LN injuries, concerning the immune complex deposition with the structural podocyte damage; the other is non-proliferative LN, presenting with diffuse podocyte structural foot process effacement and dysfunction [11]. In both forms of LN, the diffused podocyte foot process effacement and reduced expression of podocyte-specific proteins [12], along with an increased excretion of podocyte-related products in the urine [10], are powerful evidence of podocyte, as a victim not to be ignored.

A main finding in this study was that urinary podocyte-derived MPs were increased in SLE patients compared with those in healthy controls, especially in those with active renal injuries characterized by proteinuria and higher SLEDAI scores. MPs are released from apoptotic or activated cells in response to various stimuli [13]. Urinary MPs, as a subtype of EVs, are enriched in functional cargo proteins and released from the different epithelial cells of the whole urinary tract. The cellular sources of urinary MPs can provide important information about disease pathogenesis. It is increasingly clear that EVs are present in urine and may be of significant clinical use. Subsequent studies have reported that the level of EVs in the urine is elevated in patients with glomerular diseases [14] such as focal segmental glomerulosclerosis [15], minimal change disease and diabetic nephropathy [16]. Moreover, the pathogenic roles of circulating MPs derived from various cellular sources, such as platelet-derived MPs in rheumatoid arthritis [14] and SLE patients [17], have been demonstrated. However, to date, there are only a few studies to reveal urinary MPs as new urine biomarkers [18, 19]. A increase of numbers of MPs observed in SLE patients most likely result from abnormal cell activation and apoptosis together with defective clearance of MPs, which are all mechanisms related to underlying SLE pathogenesis, as discussed elsewhere [20]. Healthy controls had certain levels of urinary MPs positive for podocalyxin. Podocalyxin is a transmembrane protein expressed on the apical cell membrane of glomerular podocytes [21]. Podocalyxin physically functions to maintain podocyte structure and the integrity of slit diaphragm. Upon damage, podocyte would experience various morphological changes, such as foot processes fusion, decrease of the cell density and the formation of pseudocysts, ultimately stripping from the glomerular basement membrane and falling off into the urine. It was reported that podocalyxin is shed from injured podocytes into the urine as small vesicles [22]. These results are in line with the idea that the shedding process of podocalyxin-related vesicles can be an indicator of the state of the podocyte in situ. Disease severity was identified by the SLEDAI score, and a SLEDAI score > 10 was a marker of highly active disease [23]. SLEDAI has been reported to be associated with the presence of active LN [24].

Our results, for the first time, demonstrated that the level of urinary podocyte-derived MPs was closely correlated to SLE disease activity, which was mainly shown by the strong positive association between the number of podocyte-derived MPs in the urine and the titre of anti-dsDNA antibodies. Serum levels of anti-dsDNA antibodies and Complement 3 are two important diagnostic indexes of SLE and play key roles in the pathogenesis of LN. Anti-dsDNA antibodies bind with circulating DNA and then form circulating immune complexes, thus resulting in renal damage. According to the report, anti-dsDNA antibodies can directly have cross reactions with the podocyte proteins such as α-actinin 4, inducing podocyte cytoskeleton reorganization and fused podocyte morphology, eventually resulting in LN [25]. Quantitative detection of anti-dsDNA antibodies can evaluate the disease activity of SLE. The complement system is intimately related to SLE. Low serum level of Complement 3, as a biomarker of active SLE, may reflect complement activation [26]. Although the sensitivity and specificity of Complement 3 (75%/71%) for identifying the degree of renal injuries is relatively low, there are certain implied values of this positive correlation for clinical decisions. The measurement of 24-h proteinuria in urine samples is the classical biomarker for assessing LN, and it somehow reflects eventual renal outcome. Proteinuria is partly consistent with histological index activity changes in LN, and a significant increase in urinary podocyte-derived MP levels with the gravity of histological features was observed. Serum creatinine (Cr) and eGFR are standardized tools for estimating renal function [27]. No possible association between podocyte-derived MPs and Cr and eGFR was acceptable. Vesicle shedding has been regarded as an early sign of podocyte injury. However, the patients we enrolled were not conditioned in the early stage of CKD.

Regardless of the different causes for podocyte injury, the characteristic morphological change of podocyte is the foot processes effacement. The cytoskeleton change of podocyte is often accompanied by the release of podocyte-derived MPs. Our results showed that the manifestations of foot process fusion varied among the pathological phenotypes of LN, especially for the combined membranous patterns (Type III + V or IV + V). There was more podocyte-derived MPs release in the pathological active LN groups. While the levels of podocyte-derived MPs were no difference among LN patients with increased chronic indices. It meaned that as the podocyte foot process fusion is aggravating from the acute to chronic damage, the number of podocytes in situ was reduced and the corresponding shedding of podocyte-derived MPs was also decreased.

ROC analysis was performed to calculate the area under the curve (AUC) for evaluating the diagnostic performance of the models. Our data demonstrated that the AUC of podocyte-derived MPs was 0.789, when SLEDAI score ≥ 5 was designated as light or moderate clinical disease activity of LN. The AUC of podocyte-derived MPs was 0.962, when proteinuria (> 0.5 g/24 h) was designated a diagnostic LN pathological activity indicator. In summary, podocyte-derived MPs can be a reliable indicator that reflect the disease activity of SLE.

There are an increasing number of urine proteins, including chemokines, cytokines, growth factors and adhesion molecules [26]. These proteins have been evaluated as candidates for SLE biomarkers, but few have been validated and implied for clinical practice. EVs are not only the witnesses, but the contributors to the diseases. The biological information of the particles and the effect of the downstream target cells, especially tubular epithelial cells, need to be further discussed.

The limitations of this study include the following aspects. First, the small sample size may affect the reliability of the results and further analysis. Thus, a large sample and prospective follow-up studies are also needed. Second, this study could not link higher podocyte-derived MP counts to the progression of kidney disease, which would require longitudinal studies.

To summarize, our studies firstly demonstrated that the levels of urinary podocyte-derived MPs were dramatically increased in patients with SLE. Moreover, the levels of urinary podocyte-derived MPs were positively associated with SLE disease activities. Further studies will be required to determine the release mechanisms of podocyte-derived MPs and to explore whether the MPs released from glomerular cells may be used as novel biomarkers for the early prediction and monitoring of disease activity in SLE.