Combined Urinary Excretion of IgG and α2-Macroglobulina Very Simple Marker to Assess Disease Severity, Outcome Prediction and Responsiveness to Steroids and Cyclophosphamide in Patients with Chronic Glomerulonephritis and Nephrotic Syndrome

Prediction of functional outcome and responsiveness to treatments in Glomerulonephritis (GN) with Nephrotic Syndrome (NS) is of paramount importance in clinical practice and has been the object of several studies in last decades. Some proteinuric biomarkers, mainly low and high Molecular Weight (MW) urinary proteins (α1-microglobulin, β2-microglobulin, IgG, IgM), have been useful to predict remission and progression to ESRD [1-12]. Fractional Excretion of IgG (FE IgG), according to cut offs assessed by ROC analysis, predicted Remission and ESRD in several types of GN (IMN, FSGS, IgAN, Crescentic IgAN, MPGN) [13-21], but no one of these proteinuric markers reachs 100% prediction. Thus it would be very useful for clinical practice to identify a new marker with 100% predictive power. A recent review by Remuzzi et al. [22], posed a question: “Novel Biomarkers for Renal Diseases?”; their answer was “None for the Moment (but One)” stating that “to date it is still uncertain whether and to what extent novel biomarkers will provide diagnostic and prognostic information over and above what is already granted by established, cheap and easily available biomarkers such as proteinuria”. Thus the assessment of urinary proteins excretion remains a useful target, taking into account that the excretion of high MW proteins (IgG/C and α2m/C) may be marker of damage of the final Glomerular Filtration Barrier (GFB) (podocytes and slit diaphragms) to filtration of macromolecules [23-26]. A reliable conclusion about predictors of responsiveness to various treatments in GN with NS has not been reached. A large systematic review and meta-analysis [27], of 36 clinical trials including 1762 patients with IMN compared several immuno-suppressive treatments (steroids, steroids in combination with alkylating agents, cyclosporine, tacrolimus, mycophenolate mofetil, adreno-corticotropic hormone, azathioprine and mizoribine) and concluded that corticosteroids in combination with alkylating agents significantly reduced all-cause mortality and ESRD. In NS the elevated urinary excretion of the high Molecular Weight (MW) proteins IgG (150 kDa) and α2-macroglobulin, (720 kDa) is associated with severe renal disease. The comparison of 4° to 1° quartile of IgG/C shows: highly significant eGFR increase (from 48.3 to 89.7 ml/min/1.73m2, p < 0.0001) and significant reduction of GGS% (from 17.5 to 8.3%, p = 0.005), TID score (from 2.57 to 1.17, p = 0.0001), AH score (from 0.83 to 0.26, p < 0.0001) and marked reduction of IgG/C (from 664 to 43, p < 0.0001) and α2m/C (from 24.0 to 9.0, p < 0.0001). Similar results comparing 4° to 1° quartile of α2m/C. Aim of this study is verify whether the combined excretion of these two proteins, also in association with normal or high Blood Pressure (BP), may be suitable for evaluation of disease severity, prediction of outcome and responsiveness to treatment with steroids and cyclophosphamide.

The patients cohort included in the study was not selected. The patients attending the Nephrology and Dialysis Unit of San Carlo Borromeo Hospital, Milan, Italy, between January 1992 and April 2006 with renal biopsy diagnosis of GN with NS were 204; 26 patients with Acute Reversible Renal Failure (ARF) at biopsy were excluded from analysis as do not meet the inclusion criterion (chronic glomerulonephritis); thus 178 patients were the object of this study with the following types of chronic primary Glomerulonephritis (GN) and Lupus Nephritis (LN) with NS (Table 1): Focal Segmental Glomerulosclerosis (FSGS, n. 34), Idiopathic Membranous Nephropathy (IMN, n.74), Minimal change disease (MCD, n. 14), Membrano-proliferative glomerulonephritis (MPGN, n. 18: type I n. 11; type II n. 1; type III n. 4; fibrillary type n. 2); IgA nephropathy (IgAN, n. 2), Crescentic IgAN (CIgAN, n. 13)] and Lupus Nephritis [LN, n. 23: (LN classes: 3+5 n. 3, 4 n. 13; 4+5 n. 2; 5 n. 5)]. Inclusion criteria: nephrotic syndrome (proteinuria ≥ 3.5 g/24h and/or serum albumin < 3.0 g/dL); at least six glomeruli in renal biopsy; typical features at light and immunofluorescence microscopy; no clinical signs of secondary GN except for LN. The functional outcome was available for 151 patients with rather long follow up: mean 85 ± 70 months (2-311). Five types of outcome were considered: 1) Remission of NS: complete: proteinuria ≤ 0.30 g/24h; partial: proteinuria ≤ 2.0 g/24h; 2) persistent NS with long lasting normal Renal Function (NRF) after a follow up of 80 ± 52 months; 3) progression to End-Stage Renal Disease (ESRD); 4) eGFR reduction ≤ 50% of baseline; 5) persistent NS with Chronic Renal Failure (CRF) and progressive eGFR reduction (from 49.3 to 39.1 ml/min/1,72 m2). Usually in prediction studies the outcomes considered are Remission and ESRD (often in combination with eGFR ≤ 50% of baseline). We decided to evaluate not only each type of outcome considered independently but also the combination of outcomes with similar prognostic significance: thus Remission was evaluated in combination with persistent NS with long lasting NRF, afterwards indicated as “Remission & persistent NRF”; ESRD and eGFR ≤ 50% were evaluated in combination with persistent NS with CRF characterized by eGFR reduction from 49.3 to 39.1 ml/min/1,72 m2 and thus candidate for progression to ESRD, afterwards indicated as “Progression & progression risk”. The diagnosis and clinical presentation of patients are reported in table 1. 

Table 1: Diagnosis and clinical, functional and proteinuricparameters in 178 patients with glomerulonephritis and nephrotic syndrome.        

Laboratory analysis 

Proteinuria was measured in 24 hour urine collection and second morning urine sample by the Coomassie blue method (modified with sodium-dodecyl-sulphate) and expressed as 24/hour proteinuria and protein creatinine/ratio (mg urinary protein/g urinary creatinine). Serum and urinary creatinine were measured enzymatically and expressed in mg/dL. Serum albumin and IgG and urinary IgG, α2-macroglobulin (α2m), Albumin and α1-microglobulin (α1m) were measured by immunonephelometry; urinary proteins were expressed as urinary protein/creatinine ratio (IgG/C, α2m/C, Alb/C, α1m/C). Estimated Glomerular Filtration Rate (eGFR) was measured by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula [28]. Three types of renal lesion that are markers of disease severity in any type of GN were evaluated: percentage of glomeruli with Global Glomerulosclerosis (GGS%); extent of Tubulo-Interstitial Damage (TID) evaluated semi-quantitatively by a score: tubular atrophy, interstitial fibrosis and inflammatory cell infiltration graded 0, 1 or 2 if absent, focal or diffuse (TID global score: 0-6) and extent of Arteriolar Hyalinosis (AH) evaluated semiquantitatively by a score: 0, 1, 2, 3 if absent, focal, diffuse, diffuse with lumen reduction, respectively (AH global score 0-4). For all the 178 patients and the 151 with functional outcome was calculated the median of IgG/C (IgG/C0 < median and IgG/C1 > median); the median of α2m/C was calculated independently in IgG/C1 and IgG/C0 patients, respectively and defined α2m/C0 and α2m/C1 if < or > the median; thus 4 groups were defined: IgG/C1 & α2m/C1, IgG/C1 & α2m/C0, IgG/C0 & α2m/C1, IgG/C0 & α2m/C0). 

Statistical analysis 

Continuous variables are expressed as means±SD. Categorical variables are expressed as the number of patients (%). The differences of mean were determined by t-test; categorical variables by the chi-square test. All statistical analyses were performed using Stata 15.1 (StataCorp LP, TX, USA). Two-sided p < 0.05 was considered statistically significant.

In the 178 patients those with high blood pressure (BP 1, n. 106) show significantly lower values of eGFR, and significantly higher values of TUP/C, IgG/C, α2m/C and α1m/C, GGS%, TID score and AH score in comparison with patients with normal blood pressure (BP 0, n. 72) (Table 2). The patients with IgG/C lower than median (IgG/C0, n. 89) show significantly higher values of eGFR and significantly lower values of TUP/C, IgG/C, α2m/C, Alb/C, α1m/C, GGS%, TID and AH scores in comparison with patients with IgG/C higher than median (IgG/C1, n. 89) (Table 3). The patients with α2m/C lower than median (α2m/C 0, n. 89) showed significant higher values of eGFR and significant lower values of all the proteinuric parameters and TID and AH score in comparison with patients with α2m/C higher than median (α2m/C1, n. 89). The 4 groups defined by combination of IgG/C and α2m/C (IgG/C1 & α2m/C1, IgG/C1 & α2m/C0, IgG/C0 & α2m/C1, IgG/C0 & α2m/C0) realize a reliable picture of disease severity in all 178 patients showing a progressive increase of eGFR and reduction of GGS%, TID score and AH score, TUP/C, IgG/C, α2m/C, Alb/C and α1m/C (Table 4). In 151 patients with functional outcome were reported the clinical, functional, proteinuric and histological data of the 5 type of functional outcome alone and in combination (Table 5); in the same patients were evaluated also the rates of functional outcomes (Table 6): the patients with IgG/C1 & α2m/C1 in combination with BP 1 show 15% of “Remission & persistent NRF” and 85% of “Progression & progression risk”; the patients with IgG/C0 & α2m/C0 in combination with BP 0 show 96% of “Remission & persistent NRF” and 4% of “Progression & progression risk”. In 84 patients treated with Steroids and Cyclophosphamide the treatment improves the functional outcome (Table 7): in patients IgG/C1 & α2m/C1 & BP 1 “Remission & persistent NRF” is 0% and “Progression & progression risk” is 100%; in patients with IgG/C0, α2m/C0 and BP 0 “Remission & persistent NRF” is 100% and “Progression & progression risk” is 0%. Very similar results in 30 patients with IMN and NS treated with Steroids and Cyclophosphamide (Table 8). 

Table 2: Baseline clinical, functional, histologic and proteinuric data in 178 patients with chronic glomerulonephritis (GN) and nephrotic syndrome (NS) with normal or high blood pressure. 

Table 3: Baseline clinical, functional, histologic and proteinuric data in 178 patients with chronic Glomerulonephritis (GN) and Nephrotic Syndrome (NS) with IgG/C < and > its median and α2m/C < or > its respective median.

Table 4: Disease severity in 178 patients with GN and NS classified in 4 groups according to the following combination:

1) (IgG/C1 & α2m/C1): IgG/C > median and α2m/C > median n. 45 patients.

2) (IgG/C1 & α2m/C0): IgG/C > median and α2m/C < median n. 44 patients.

3) (IgG/C0 & α2m/C1): IgG/C < median and α2m/C > median n. 44 patients. 4) (IgG/C0 & α2m/C0: IgG/C < median and α2m/C < median n. 45 patients. 

Table 5: Clinical, functional, proteinuric and histologic parameters in 151 patients with different functional outcome. 

Table 6: Disease severity in 151 patients with GN and NS and functional outcome evaluated by eGFR, proteinuric and histologic data according to the 4 groups of IgG/C and α2m/C in combination between them and with BP 1 or 0. 

Table 7: Baseline functional, proteinuric and histologic parameters and functional outcome according to the 4 groups of IgG/C and α2m/C also in combination with BP 1 or BP 0 in 84 patients treated with Steroids and cyclophosphamide. 

Table 8: Baseline functional, proteinuric and histologic parameters and functional outcome according to IgG/C and α2m/C groups in combination with BP 1 and BP 0 in 30 patients with IMN and NS treated with Steroids and cyclophosphamide.

The prediction of functional outcome and responsiveness to treatments in GN and NS is of paramount importance in clinical practice. Several studies in last decades evaluated the predictive power of functional outcome of several proteinuric and novel molecular biomarkers but none of them reached 100% prediction. The identification of a new marker with high outcome prediction would be useful also to assess responsiveness to new therapies introduced recently such as Rituximab. A recent article [28], “New insight into podocyte slit diaphragm, a therapeutic target of proteinuria” reviews the extracellular molecular components forming a molecular sieve of the slit diaphragm and the cytoplasmic molecules that link the slit diaphragm to the cytoskeleton; on the basis of increased knowledge of molecular components of podocytes and slit diaphragm, the Authors hope the definition of novel therapeutic strategy for proteinuria reduction but at present no one proteinuric or molecular marker may evaluate the extent of damage of podocytes and slit diaphragm and predicts outcome and treatment responsiveness. The proteinuric marker proposed for the first time in this study evaluating the combined excretion of two high MW proteins (IgG, 150 kDa and α2-macroglobulin, 720 kDa), may be considered on the basis of functional, histologic and proteinuric data a reliable marker of disease severity and damage of the epithelial cell layer of GFB (podocytes and slit-diaphragms) and may be useful to predict outcome and responsiveness to treatments. The IgG/C1 & α2m/C1 group, associated with the highest excretion of IgG/C (577) and α2m/C (29.52), suggest severe alteration of the epithelial cell layer of GFB (podocytes and slit-diaphragms) and the association with the lowest value of baseline eGFR (47.7 ml/min/1.72 m2) and the highest values of the histologic and proteinuric parameters (GGS% 17.7%, TID score 2.73 and AH score 0.78, TUP/C 6562, Alb/C 5023 and α1m/C 96.8). These data suggest the ability of this marker to identify the patients with the highest level of disease severity. By contrast the marker IgG/C0 & α2m/C0 associated with the lowest excretion of IgG/C (53) and α2m/C (0) and with the highest value of baseline eGFR (92.3 ml/min/1,72 m2) and the lowest values of the histologic and protenuric parameters (GGS%, 5.1%, TID score 0.90, AH score 0.24; TUP/C 2620, IgG/C 53, α2m/C0, Alb/C 2330 and α1m/C 17.5) suggest the ability of this marker to identify the patients with the lowest level of disease severity. The 4 groups of IgG/C and α2m/C in combination, show a high predictive value of functional outcome both for “Remission &persistent NRF” and “Progression & progression risk”. In 84 patients treated with Steroids and Cyclophosphamide the combination of IgG/C0 & α2m/0 with normal blood pressure (BP 0) is associated with the highest values of eGFR and the lowest values of histologic and proteinuric parameters; the functional outcome reachs 100% of “Remission & persistent NRF” and 0% of “Progression & progression risk”. The combination of IgG/C1 & α2m/C1 with high blood pressure (BP 1) is associated with worsening of all the parameters and 100% of “Progression & progression risk” and 0% of “Remission & persistent NRF”. To reach the highest rate of functional outcome it is necessary a combination of normal or high blood pressure also in patients treated with steroids and cyclophosphamide. Very similar results in 30 patients with IMN and NS treated with Steroids and Cyclophosphamide.

The combined urinary excretion of IgG/C and α2m/C realizes a reliable evaluation of disease severity in 178 patients with chronic glomerulonephritis and NS, in 151 patients with a long term functional outcome and in 84 patients treated with Steroids and Cyclophosphamide; in these last patients not only assess disease severity but also achieves 100%of outcome prediction: the patients with IgG/C1 & α2m/C1 & and BP 1 show 100% of “Progression & progression risk” and 0% of “Remission & persistent NRF”; the patients with IgG/C0 & α2m/C0 & and BP 0 show 100% of “Remission & persistent NRF” and 0% of “Progression & progression risk”. This new proteinuric marker could be very useful to assess functional outcome with new treatments such as Rituximab.

We thank Dr. Pietro Napodano for the analysis of renal biopsies.