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Research Article | DOI: https://doi.org/10.31579/2690-4861/206
Retired from Nephrology and Dialysis Unit, San Carlo Borromeo Hospital, Via Pio II, 3, Milan, Italy.
*Corresponding Author: Claudio Bazzi, Retired from Nephrology and Dialysis Unit, San Carlo Borromeo Hospital, Via Pio II, 3, Milan, Italy.
Citation: C Bazzi. (2022). Which is the most Powerful Proteinuric Marker in GN and NS for Outcome Prediction, Correlation with Functional and Histologic Parameters and Assessment of Responsiveness to Steroids and Cyclophosphamide?. International Journal of Clinical Case Reports and Reviews. 11(1); DOI:10.31579/2690-4861/206
Copyright: © 2022 Claudio Bazzi, This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 21 February 2022 | Accepted: 10 March 2022 | Published: 30 March 2022
Keywords: steroids; cyclophosphamide; urinary proteinuric biomarkers
Background: In glomerulonephritis (GN) with nephrotic syndrome (NS), proteinuric and novel molecular biomarkers do not achieve 100% accuracy in predicting outcome and treatment responsiveness.
Aim of study: To verify whether the combined excretion of IgG/C and α2m/C, markers of damage to the epithelial cell layer (podocytes and slit diaphragm) of the glomerular filtration barrier (GFB) can assess disease severity, and predict outcome and responsiveness to steroids and cyclophosphamide.
Methods: In 151 patients with glomerulonephritis and nephrotic syndrome a new proteinuric marker was developed based on a combination of different median values of urinary excretion of IgG/C and α2m/C: IgG/C < or > than the median was classified as IgG/C0 or IgG/C1, respectively. The median of α2m/C was calculated independently in IgG/C0 and IgG1, and classified as α2m/C0 and α2m/C1 if < or > than the median. Thus 4 groups were defined: IgG/C0 & α2m/C0, IgG/C0 & a2m/C1, IgG/C1 & α2m/C0, IgG/C1 & α2m/C1.
Results: The groups from 1+1 to 0+0 are characterized by increasingly severe disease, with progressively higher eGFR and progressively lower histologic (GGS%, TID and AH scores) and proteinuric parameters (TUP/C, IgG/C, α2m/C and α1m/C). In 151 patients the functional outcomes rates were: IgG/C1&α2m/C1&BP 1: “Remission and persistent NRF”, 15%; “Progression and eGFR reduction”, 85%. In IgG/C0&α2m/C0&BP 0: “Remission and persistent NRF”, 96%; “Progression and eGFR reduction”, 4%. In 84 patients treated with steroids + cyclophosphamide: IgG/C1&α2m/C 1&BP 1: “Progression and eGFR reduction”, 100%: “Remission and persistent NRF”, 0%; IgG/C0&α2m/C0& BP 0: “Remission and persistent NRF”, 100%; “Progression and eGFR reduction”, 0%.
Conclusions: Combined urinary excretion of IgG/C and α2m/C in association with blood pressure offers 100% prediction of outcome and responsiveness to steroid and cyclophosphamide treatment.
The prediction of functional outcome and responsiveness to treatment in glomerulonephritis (GN) with nephrotic syndrome (NS) is of paramount importance in clinical practice and has been the subject of several studies over recent decades. Some proteinuric biomarkers, mainly low and high molecular weight (MW) urinary proteins (α1-microglobulin, β2-microglobulin, IgG, IgM), have been useful for predicting remission and progression to ESRD (1-12). Fractional excretion of IgG (FE IgG), according to cut offs assessed by ROC analysis, can predict remission and ESRD in several types of GN (IMN, FSGS, IgAN, Crescentic IgAN, MPGN) (13-21), but none of these proteinuric markers predicts outcome with 100 percentage accuracy. A new marker with 100 percentage predictive power would therefore be useful in clinical practice. In a recent review, Remuzzi et al. (22) asked the 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”. Moreover, not all clinical laboratories have access to some novel biomarkers. Thus, the assessment of urinary protein excretion remains a useful target. Since excretion of high MW proteins [IgG/C (MW 150 kDa) and α2m/C (MW 720 kDa)] is a marker for damage to the final glomerular filtration barrier (GFB) (podocytes and slit diaphragms) that also regulates macromolecule movements (23-26) we recently developed a new proteinuric marker that combines median excretion of IgG/C with median excretion of α2m/C, with definition of 4 groups: IgG/C 1 & α2m/C 1, IgG/C 1 & α2m/C 0, IgG/C 0 & α2m/C 1, IgG/C 0 & α2m/C 0. These 4 groups assess renal disease severity, and accurately predict functional outcome and responsiveness to treatment with steroids and cyclophosphamide (27). A large systematic review and meta-analysis (28) of 36 clinical trials that included 1,762 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. The difference between the 4th and 1st quartile of IgG/C shows a highly significant eGFR increase (from 48.3 to 89.7 ml/min/1.73m2, p < 0 xss=removed p =0.0001),>
The aim of this study is to verify whether the combined excretion of these two proteins, also in association with normal or high blood pressure, might be useful for evaluating disease severity, and predicting outcome and responsiveness to treatment with steroids and cyclophosphamide.
The study included 151 patients with GN and NS attending the Nephrology and Dialysis Unit of San Carlo Borromeo Hospital, Milan, Italy, diagnosed by renal biopsy between January 1992 and April 2006. The patients with acute reversible renal failure (ARF) at biopsy were excluded from analysis as they did not meet the inclusion criterion (chronic glomerulonephritis). All patients had known functional outcomes with a rather long follow up (mean 85±70 months, range 2-311). The diagnosis of GN were breakdown: crescentic IgAN (12 pts), focal segmental glomerulosclerosis (FSGS, 32 pts), idiopathic membranous nephropathy (IMN, 66 pts), minimal change disease (MCD, 11 pts), membrano-proliferative glomerulonephritis (MPGN, 15 pts); IgA nephropathy (IgAN, 2 pts), and lupus nephritis [LN, 13 pts: (LN classes: 3+5, 2 pts; 4, 8 pts; 4+5 1 pt; 5, 2 pts)]. Inclusion criteria: nephrotic syndrome (proteinuria ≥3.5 g/24h and/or serum albumin <3>
Proteinuria was measured by 24-hour urine collection and second morning urine sample using the Coomassie blue method (modified with sodium-dodecyl-sulphate), expressed as 24-hour proteinuria and protein creatinine/ratio (mg urinary protein/g urinary creatinine). Serum and urinary creatinine concentrations 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 (29). Three types of renal lesions that are markers for 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 semi-quantitatively by a score: 0, 1, 2, 3 if absent, focal, diffuse, diffuse with lumen reduction, respectively (AH global score 0-4). In all 151 patients with functional outcomes were calculated the median of IgG/C (IgG/C0 < median> median); the median α2m/C was calculated independently in IgG/C1 and IgG/C0 patients, respectively, and defined as α2m/C0 and α2m/C1 if < or> than the median; thus 4 groups could be defined: IgG/C1 & α2m/C1, IgG/C1 & α2m/C0, IgG/C0 & α2m/C1, and IgG/C0 & α2m/C0) (abbreviated as 1+1, 1+0, 0+1, and 0+0) (27).
Data for continuous variables are expressed as the mean ± SD. Categorical variables are expressed as the number of patients (%). The difference in means was determined by t-test and in categorical variables by chi-square test. All statistical analyses were performed using Stata version 15.1 (StataCorp LP, TX, USA). Two-sided p<0>
In all the 151 patients the 4 groups defined by combination of IgG/C and α2m/C show from IgG/C1 & α2m/C1 to IgG/C0 & α2m/C0 a reliable picture of disease severity 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 and a high prediction of functional outcomes (Table 3). In patients with Groups 1+1 and 0+0 the functional outcome was also evaluated in combination with BP1 and BP0, respectively. The patients with IgG/C1 & α2m/C1 in combination with BP1 show 15% of “Remission & persistent NRF” and 85% of “Progression & progression risk”; the patients with IgG/C0 & α2m/C0 in combination with BP0 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 5): in patient’s IgG/C1 & α2m/C1 & BP1 “Remission & persistent NRF” is 0% and “Progression & progression risk” is 100%; in patients with IgG/C0, α2m/C0 and BP0 “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.
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% of outcome prediction. Thus the identification of a new marker with high outcome prediction would be markedly useful in clinical practice also to assess responsiveness to new therapies. A recent article (23) “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 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.7%, TID score 2.73 and AH score 0.78, TUP/C 6562, Alb/C 5023 and α1m/C 96.8). The data of this group suggest the ability of this marker to identify the patients with the highest level of disease severity. By contrast the marker IgG/C 0 & α2m/C 0 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/C 0, 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” (No progr.) and “Progression & progression risk” (Progr.). In 84 patients treated with Steroids and Cyclophosphamide the combination of IgG/C 0 & α2m/C 0 with normal blood pressure (BP 0) is associated with the highest values of eGFR and the lowest values of histologic and proteinuric parameters and the functional outcome reachs 100% of “Remission & persistent NRF” (No ptrogr.) 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” (Progr.) and 0% of “Remission & persistent NRF” (No progr.). To reach the highest rate of functional outcome it is necessary a combination with 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 151 patients with chronic glomerulonephritis and NS and 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/C 1 & α2m/C 1 & and BP 1 show 100% of “Progression & progression risk” and 0% of “Remission & persistent NRF”; the patients with IgG/C 0 & α2m/C 0 & 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.
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