ELISA Estimation of Urinary Podocalyxin and Nephrin as Non-invasive Modality for Disease-severity grading of Patients with Lupus Nephritis
Ahmed T Abu ghanima MD1 -Mohammed F Almaghraby MD2 - Hossam M Elsaadany MD3- Mohammed Ahmed Hosny MRCP UK4 - Roobina Khan Kumar,DCP,MD5
Abstract
Objectives: To evaluate the impact of systemic lupus erythmatosus (SLE) on urinary levels of podocalyxin and nephrin in urine and to determine its relationship to pathological diagnosis of renal biopsy in lupus nephritis (LN) patients.
Patients & Methods: The study included 50 LN patients diagnosed by renal biopsy according to the International Society of Nephrology (INS) classification. Disease activity was determined using the British Isles LupusAssessment Group (BILAG). All patients underwent clinical and laboratory evaluation. Urine samples were collected for ELISA estimation of urinary podocalyxin, nephrin, protein and creatinine concentrations and urinary podocalyxin to creatinine (UPxC), urinary nephrin to creatinine (UNC) and urine total protein-to-creatinine (UPC) ratios were calculated.
Results: Urinary levels of nephrin, podocalyxin and protein were significantly higher, while urinary creatinine levels were significantly lower compared to control levels. Consequently, UNC, UPxC and UPC ratios were significantly higher in patients compared to control ratios. Studied patients showed stepwise increase of estimated ratios with increased pathological disease severity grade and ISN grading showed positive significant correlation with the estimated ratios and with BILAG scores, but correlation was most significant with UPxC ratio. ROC curve and regression analyses defined UPxC ratio as the specific significant predictor of pathological grade.
Conclusion: SLE deleteriously affects fine glomerular structure as reflected by increased urinary levels of podocyte-related proteins; podocalyxin and nephrin. Urinary podocalyxin/creatinine ratio significantly predicts the pathological impact of SLE on kidney and could be used as non-invasive marker for such effect and its progression.
Keywords: Lupus nephritis, Urinary markers, Podocalyxin, Nephrin
Introduction
Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by production of a number of antinuclear antibodies. Proteinuria in patients with SLE has generally been associated with immune complex deposition in the sub-epithelial and sub-endothelial glomerular capillary wall as well as endo-capillary proliferation and inflammation. It is a characteristic finding in all stages of lupus nephritis (LN) but is particularly characteristic of proliferative class IV and membranous class V(1).
The kidney glomerulus is a highly specialized structure ensuring the selective ultrafiltration of plasma so that essential proteins are retained in the blood. Within the kidney, the function and survival of major cell constituents of the glomerular filtration barrier, i.e., podocytes, heavily depends on the integrity of lipid rafts.Podocytes are highly differentiated glomerular epithelial cells that line the outside of the glomerular capillary and are formed of a body with extending major processes that further branch into foot processes (FP). FP from neighboring cells are bridged by a 40-mm wide extracellular structure known as the slit diaphragm(SD) (2).
The FPof podocytesare linked to the glomerular basement membrane with their actin cytoskeleton. Processes from neighboring podocytes form a characteristic inter-digitating pattern that leaves filtration slits in between them. The latter are bridged by the slit diaphragmthat togetherwith the glomerular basement membrane and the fenestrated endothelium plays an important role in the selective permeability of the filtration barrier of the glomerulus. Integrity of this filtration barrier is important in order to prevent the loss of protein into the urine(3).
Podocyte injury is an important feature of several renal diseases that involvesorganization of the FP structure with fusion of filtration slits and apical displacement of the SD which is required to control actin dynamics, response to injury, endocytosis, and cell viability. Regulation of the podocyteactin cytoskeleton is therefore of critical importance for sustained function of the glomerularfilter. The connection of the actin cytoskeleton to the SD is mediated by several podocyte proteins such as Nephrin and Podocin (4, 5).
Several markers of podocyte injury are available and include nephrin, sinaptopodin, podocalyxin, podocin. Nephrinispartiallyassociatedwithpodocytelipidraftsandco-immunoprecipitateswitha podocytespecific9-O-acetylatedganglioside. Injection of an antibody against the 9-O-acetylated ganglioside causes morphological changes of the filtration slits, resembling FP effacement(6, 7).
The relationship between urinary podocytes and renal diseases is supported by the detection of podocytes in patients with immunoglobulin A nephropathy, Henoch-Schönleinpurpura nephritis, lupus nephritis, diabetic nephropathy, and focal segmental glomerulosclerosis, and the use of technology for detecting podocytes in the urine would have broad implications for the evaluation of disease activity, the degree of dedifferentiation, and the possibility of regeneration (8).
Thus, the current study tried to evaluate the impact of SLE on fine glomerular architecture using non-invasive ELISA estimation of podocyte-related markers; podocalyxin and nephrin in urine and to determine its relationship to pathological diagnosis of renal biopsy.
Patients & Methods
This study was conducted at Internal Medicine, Rheumatology and Clinical Pathology departments, Hospital, KSA. The study included 50 lupus nephritis (LN) patients diagnosed by previous renal biopsy. Systemic lupus erythmatosus(SLE) was diagnosed depending on the American College of Rheumatology Criteria for Classification of SLE; 1997 Update of the 1982 ACR revised criteria for classification of SLE. The classification is based on 11 criteria; a person is defined as having SLE if any 4 or more of the 11 criteria are present, serially or simultaneously, during any interval of observation(9). All patients underwent clinical and laboratory evaluation. Laboratory tests included immunologic assessment of blood levels of C3, C4, CRP and anti-dsDNA, and total leucocytic count (TLC). Hemoglobin concentration (Hb conc.) was determined and anemia was defined by Hb conc. of ≤12 g/dl for women and of ≤13.5 g/dl for men(10).Proteinuria was measured by a dipstick method. The study also included 20 normal subjects free of renal disease as control group for estimated urinary biomarkers.
Disease activity was determined using the British Isles LupusAssessment Group (BILAG) consisted of evaluation of 8 points of interest: general, muco-cutaneous, neurological, musculoskeletal, cardio-respiratory and renal manifestations, manifestations of vasculitis and hematological findings. To obtain a global score, BILAG component scores can be assigned numerical values: A=9 (most active disease), B=3 (intermediate activity), C=1 (mild, stable disease activity), D=0 (inactive disease) and E=0 (no activity), resulting in a potential summed range of 0-72 points with 72=most active disease affecting the 8 organs (11).
Lupus nephritis was diagnosed depending on the presence of proteinuria and hematuria which are considered as characteristic features in patients with LN (12) and were classified depending on the findings at histology according to the International Society of Nephrology (INS) and Renal Pathology Section (RPS) classification (13) that involves deposition of immunoglobulin in glomerular and tubular basement membrane-enhanced inflammatory response and renal fibrosis as minimal mesangial (ISN class I), mesangial proliferative LN (ISN class II), focal LN (ISN class III), diffuse LN (ISN class IV), membranous LN (ISN class V) and advanced sclerosis (ISN class VI)
Investigations
Urine sample collection: Random urine (10 ml) was collected in plastic tubes, without preservative. Samples were clarified by centrifugation at 3,000 rpm for 5 min and supernatant was collected in Eppendorff tubes and kept frozen at -80°C till be assayed.
Estimated parameters
-Urinary podocalyxin was estimated using commercially available Podocalyxin ELISA test (Exocell Inc., Philadelphia, PA). Urine samples were diluted with dilution buffer provided by the ELISA kits in a ratio of 1:2. Each sample was measured in duplicate. The values are expressed as ng/ml (2).
-Urinary nephrin using was estimated using commercially available nephrin ELISA test (Exocell Inc., Philadelphia, PA). Urine samples were diluted with dilution buffer provided by the ELISA kits in a ratio of 1:10. Each sample was measured in duplicate. The values are expressed as ng/ml(14).
-Urine protein concentrations were measured by the Bradfordmethod.
-Estimation of urinary creatinine: Urine creatinine was measured by the Jaffe reaction on the same aliquot of urine to adjust the ratio of urinary podocalyxin to creatinine (UPXC, expressed as ng/mg), urinary nephrin to creatinine (UNC, expressed as ng/mg) and urine total protein-to-creatinine ratio (UPC, expressed as g/mg).
Statistical analysis
Obtained data were presented as mean±SD, ranges, numbers and ratios. Results were analyzed using Wilcoxon; ranked test for unrelated data (Z-test) and Chi-square test (X2 test). Sensitivity & specificity of estimated parameters as predictors were evaluated using the receiver operating characteristic (ROC) curve analysis judged by the area under the curve (AUC) compared versus the null hypothesis that AUC=0.05. Regression analysis (Stepwise method) was used for stratification of studied parameters as specific predictors. Statistical analysis was conducted using the SPSS (Version 15, 2006) for Windows statistical package. P value <0.05 was considered statistically significant.
Results
The current study included 50 SLE patients; 39 females and 11 males with mean age of 45.7±11.8; range: 22-69 years. All patients had LN diagnosed clinically and was documented by renal biopsy that showed varied diagnoses with mesangial proliferative and focal LN were the most frequent diagnosis. Details of patients' distribution according to result of renal biopsy are shown in table 1.
Table (1): Patients distribution according to pathological diagnosis of renal biopsy of studied patients
ISN class / Diagnosis / Number (%)I / Minimal mesangial LN / 9 (18%)
II / Mesangial proliferative LN / 16 (32%)
III / Focal LN / 18 (36%)
IV / Diffuse LN / 3 (6%)
V / Membranous LN / 3 (6%)
VI / Advanced sclerosis / 1 (2%)
Mean duration of disease was 5.1±1.1; range: 3-8 years. Mean BILAG score was 17±9.6; range: 8-38; 24 patients had mean BILAG score of 9.1±0.7; range 8-10, 6 patients had mean BILAG score of 13.5±2.4; range: 11-17, 13 patients had mean BILAG score of 23.8±2.8; range: 20-29 and 7 patients had mean BILAG score of 34.6±2.4; range: 32-38; details of patients' distribution according to systematic BILAG grading was shown in table 2.
Table (2): Patients distribution according to systematic BILAG grading of SLE patients enrolled in the study
ManifestationBILAG grade / General / MC / N / MS / CR / Renal / Vasculitis / Hematological
A (Severe=9) / 5 / 6 / 3 / 3 / 3 / 7 / 10 / 11
B(Intermediate=3) / 8 / 12 / 10 / 8 / 12 / 18 / 13 / 12
C (Mild=1) / 13 / 20 / 11 / 29 / 23 / 19 / 10 / 16
D (inactive=0) / 15 / 7 / 17 / 6 / 7 / 4 / 11 / 7
E (No activity=0) / 9 / 5 / 9 / 4 / 5 / 2 / 6 / 4
MC: Mucocutaneous lesions; N: Neurological manifestations; MS: Musclo-skeletal manifestations; CR: Cardio-respiratory manifestations
Hemogram detected 11anemic patients (22%) with mean Hb conc. of 10±1.2 gm/dl, while the other 39 patients (78%) had mean Hb conc. of 13.2±0.5gm/dl.
Urinary levels of nephrin, podocalyxin and protein were significantly higher in LN patients compared to controls, while urinary creatinine levels were significantly lower compared to control levels. Consequently, urinary nephrin/creatine (UNC), urinary podocalyxin/protein (UPxC) and urinary protein/creatinine (UPC) ratios are significantly higher in studied patients compared to control levels (Table 3).
Table (3): Mean (±SD) levels of estimated urinary parameters and their ratios relative to urinary creatinine level in comparison to levels estimated in control subjects
Control (n=20) / Patients (n=50) / P valueUrinary nephrin (ng/ml) / 19.2±4.1 / 304.1±236.8 / =0.0003
Urinary podocalyxin (ng/ml) / 38.1±9 / 593.8±282.2 / =0.00008
Urinary protein (gm/l) / 0.341±0.125 / 2.36±0.56 / =0.00002
Urinary creatinine (mg/l) / 155.4±26.7 / 101.4±28.7 / =0.0009
Urinary nephrin/creatinine ratio / 12.7±3.2 / 287.4±163.3 / =0.0001
Urinary podocalyxin/creatinine ratio / 25±6.5 / 602±284.5 / =0.00001
Urinary protein/creatinine ratio / 0.226±0.09 / 2.46±0.72 / =0.00001
Data are presented as mean±SD; p<0.05: significant difference
Categorization of studied LN patients according to ISN pathological grades showed stepwise increase of estimated ratios with increased pathological disease severity grade (Fig. 1-3). Ratios of estimated parameters showed positive significant correlation with each other and with BILAG scores. Moreover, ISN grading showed positive significant correlation with the estimated ratios and with BILAG scores, but being most significant with UPxC ratio as shown in table 4.
Table (4): Correlation coefficient between the ratios of estimated urinary parameters relative to urinary creatinine level and BILAG clinical scoring with ISN pathological grading
UPxC ratio / UNC ratio / UPC ratio / BILAG score"r" / P / "r" / P / "r" / P / "r" / P
ISN score / 0.557 / 0.0008 / 0.379 / 0.007 / 0.364 / 0.009 / 0.286 / 0.044
UPxC ratio (ng/mg) / 0.429 / 0.002 / 0.371 / 0.008 / 0.311 / 0.028
UNC ratio (ng/mg) / 0.309 / 0.037 / 0.291 / 0.043
UPC ratio (g/mg) / 0.277 / 0.046
r: Pearson's correlation coefficient; UPxC ratio: Urinary podocalyxin/creatinine ratio; UNC ratio: Urinary nephrin/creatinine ratio; UPC ratio: Urinary protein/creatinine ratio
In trial to determine a predictor for pathological disease severity, UPxC ratio showed the highest AUC using ROC curve analysis that was the only significant area versus the null hypothesis (Table 5, Fig. 4). Regression analysis defined UPxC ratio as the specific significant predictor of pathological grade in two models of analysis and UNC ratio in one model, while UPC ratio and BILAG scoring were non-significant predictors as shown in table 6.
Table (5): ROC curve analysis of the ratios of estimated urinary parameters relative to urinary creatinine level and BILAG clinical scoring as predictors for ISN pathological grading
AUC / SE / P / CIBILAG score / 0.284 / 0.076 / 0.213 / 0.135-432
Urinary nephrin/creatinine ratio / 0.677 / 0.195 / 0.307 / 0.295-1.059
Urinary podocalyxin/creatinine ratio / 0.879 / 0.047 / 0.029 / 0.786-0.972
Urinary protein/creatinine ratio / 0.631 / 0.075 / 0.450 / 0.485-0.777
AUC: Area under curve; SE: Standard error; CI: Confidence interval; p<0.05: significant; p>0.05: non-significant
Table (6): Regression analysis of the ratios of estimated urinary parameters relative to urinary creatinine level and BILAG clinical scoring as predictors for ISN pathological grading
Parameter / β / t / PModel 1 / Urinary podocalyxin/creatinine ratio / 0.577 / 4.644 / =0.0009
Model 2 / Urinary nephrin/creatinine ratio / 0.347 / 3.147 / =0.003
Urinary podocalyxin/creatinine ratio / 0.536 / 4.863 / =0.0007
β: Standardized coefficient; t: paired t-test; p<0.05: significant result
Fig. (4): ROC curve analysis of the ratios of estimated urinary parameters relative to urinary creatinine level and BILAG clinical scoring as predictors for ISN pathological grading
Discussion
The current study detected elevated urinary levels of podocalyxin and nephrin and their relative concentration to urinary creatinine level in LN patients compared to normal control subjects. These findings indicated the impact of SLE on fine glomerulararchitecture. The obtained results showed positive significant correlation between urinary podocalyxin/creatinine(UPxC) and urinary nephrin/creatinine(UNC) ratios and BILAG severity scores of SLE and urinary protein/creatinine(UPC) ratio.
More interestingly, urinary podocalyxin/creatinine ratio and urinary nephrin/creatinine ratio showed positive significant correlation with ISN grade of pathological changes detected on renal biopsy and urinary podocalyxin/creatinine ratio was found to be the significant predictor for pathological change among other evaluated parameters, thus it could be considered as non-invasive predictor for pathological changes associated with LN.
These data supported that reported in literature wherein Perysinaki et al. (15) experimentally found glomerular expression of nephrin and podocin were significantly reduced in animal model of LN, starting from the earlier stages (mild mesangial LN) and becoming pronounced at advanced histological forms (focal and diffuse proliferative LN) and decreased expression correlated with electron microscopy findings of distorted slit diaphragms and clinically found that nephrin was decreased particularly in diffuse proliferative LN and expression of nephrin and podocin correlates with disease histology. Bollain-Y-Goytia et al. (16)reported that in LN patients, confirmed by renal biopsy, there is reduction of glomerularpodocytes that significantly correlated with the cumulative excretion of urinary podocytes and proteinuria.
In line with the obtained results,Sabino et al. (17) reported that podocyturia correlated statistically with the protein/creatinine ratio and both showed significant correlation with the degree of lupus disease activity and concluded that podocyturia with anti-podocin could be useful in monitoring disease activity in LN patients. Wang et al. (18)reported that podocyte damage was common in LN,urinary Podocytes met the histological criteria of lupuspodocytopathy, pure lupuspodocytopathy might act as an extreme form of lupuspodocyte lesion, and more patients might present with severe podocyte effacement concealed in different types of LN.Sir Elkhatim et al. (19)documented that urine podocyte-related protein markers have been used with varying degrees of success to study glomerulardiseases and the determination of urinary podocyte loss may become an important noninvasive tool in the evaluation of glomerulardiseases.Rezende et al. (20)reported that in proliferative forms of LN there seems to occur structural podocyte damage, whereas in the pure membranous the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome.
The present study relied on detection of podocyte-related markers using ELISA estimation of their urinary levels. Review of literature detected previous studies used ELISA estimation of urinary podocalyxin and nephrin in other kidney diseases, wherein Hara et al. (21)quantified urinary podocalyxin by ELISA in patients with glomerular diseases and patients with type 2 diabetes and found levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes; in patients with diabetes, urinary podocalyxinshowed positive correlations withlevels of HbA1c, urinary β2-microglobulin, α1-microglobulin and urinary N-acetyl-β-D-glucosaminidase, and concluded that urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes. Also, Palacios de Franco et al. (22) reported significantly higher levels of urinary podocalyxin as measured using by ELISA kits are seen in preeclampsia/eclampsia.
Recently, in 2015, Wang et al. (23) using indirect immunofluorescence, ELISA and Western blotting of urinary sediment found urinary podocyte number and nephrinlevel were significantly higher in patients with LN compared to patients with other glomerular diseases and were significantly higher in patients with severe proteinuria as compared to those with mild proteinuria, and urinary nephrin expression was positively related to podocyte and urinary albumin/creatinine ratio. Moreover, Wang et al. (23) found urinary podocyte number and nephrin level dramatically increased in the focal segmental glomerulosclerosis group as compared to those of the mesangial proliferative glomerulonephritis and minimal change disease groups and concluded that the detection of the urinary podocytes and nephrin could be taken as markers for glomerular disease, reflecting the type of the disease so can be used as a noninvasive method to evaluate the severity of the kidney disease.