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Introduction: C4d, an early product in the classical/lectin complement pathway has shown potential in the evaluation of C3 glomerulopathy where its absence would support an alternative pathway abnormality. As autoimmune/genetic complement testing is not readily available to most parts of the world, glomerular C4d staining may serve as a useful additional step toward the diagnosis. Methods: To test this hypothesis, C4d staining was performed on a large cohort of C3 glomerulopathy. Archival cases from 2011 to 2017 were reviewed and immunohistochemistry for C4d was performed, scored (scale of 0 to 3+), and correlated with the immunofluorescence and ultrastructural findings. Paraffin immunofluorescence was performed in cases of “discordant C4d” to unmask Igs. Results: Twenty-seven cases of dense deposit disease (DDD) and 14 cases of C3 glomerulonephritis (C3GN) were retrieved. C4d demonstrated a range of staining intensities with negative/traces in only 22% of DDD and 64% of C3GN. Lower-intensity C4d staining (1 to 2+) was mostly concordant with similar amounts of Igs/C1q. Discordant 3+ staining was noted in approximately 50% of cases of DDD and 20% of cases of C3GN. Among them, paraffin immunofluorescence unmasked polyclonal Igs in 2 of 5 cases of DDD and 1 of 3 cases of C3GN. Conclusion: This observational study suggests that the presence of glomerular C4d should not exclude a C3 glomerulopathy. In lower intensities, it appears to represent overlying classical/lectin pathway activation with concordant Ig/C1q deposits. A subset of cases, however, displays intense and discordant C4d staining, which raises the possibility of an associated lectin pathway abnormality, a potential future area of study. Keywords: C3 glomerulonephritis, C3 glomerulopathy, C4d, dense deposit disease, lectin pathway

Studies of nephrotic syndrome show that substitution of calcineurin inhibitors by mycophenolate mofetil (MMF) enables sustained remission and corticosteroid sparing and avoids therapy associated adverse effects. However, controlled studies in patients with steroid resistance are lacking. Here we examined the effect of switching from therapy with tacrolimus to MMF on disease course in an open-label, one-to-one randomized, controlled trial on children (one to 18 years old), recently diagnosed with steroid-resistant nephrotic syndrome, at a referral center in India. Following six months of therapy with tacrolimus, patients with complete or partial remission were randomly assigned such that 29 received MMF while 31 received tacrolimus along with tapering prednisolone on alternate days for 12 months. On intention-to-treat analyses, the proportion of patients with a favorable outcome (sustained remission, infrequent relapses) at one year was significantly lower (44.8%) in the MMF group than in the tacrolimus group (90.3%). The incidence of relapses was significantly higher for patients treated with MMF than tacrolimus (mean difference: 1.05 relapses per person-year). While there was no difference in the proportion of patients with sustained remission, the risk of recurrence of steroid resistance was significantly higher for patients receiving MMF compared to tacrolimus (mean difference: 20.7%). Compared to tacrolimus, patients receiving MMF had a significantly (71%) lower likelihood of a favorable outcome and significantly increased risk of treatment failure (frequent relapses, steroid resistance). Thus, replacing tacrolimus with MMF after six months of tacrolimus therapy for steroid-resistant nephrotic syndrome in children is associated with significant risk of frequent relapses or recurrence of resistance. These findings have implications for guiding the duration of therapy with tacrolimus for steroid-resistant nephrotic syndrome.

While studies show that prolonged initial prednisone therapy reduces the frequency of relapses in nephrotic syndrome, they lack power and have risk of bias. In order to examine the effect of prolonged therapy on frequency of relapses, we conducted a blinded, 1:1 randomized, placebo-controlled trial in 5 academic hospitals in India on 181 patients, 1-12 years old, with a first episode of steroid-sensitive nephrotic syndrome. Following 12 weeks of standard therapy, in random order, 92 patients received tapering prednisolone while 89 received matching-placebo on alternate days for the next 12 weeks. On intention-to-treat analyses, primary outcome of number of relapses at 1 year was 1.26 in the 6-month group and 1.54 in the 3-month group (difference -0.28; 95% confidence interval (CI) -0.75, 0.19). Relative relapse rate for 6- vs. 3-month therapy, adjusted for gender, age, and time to initial remission, was 0.70 (95% CI 0.47-1.10). Similar proportions of patients had sustained remission, frequent relapses, and adverse effects due to steroids. Adjusted hazard ratios for first relapse and frequent relapses with prolonged therapy were 0.57 (95% CI, 0.36-1.07) and 1.01 (95% CI, 0.61-1.67), respectively. Thus, extending initial prednisolone treatment from 3 to 6 months does not influence the course of illness in children with nephrotic syndrome. These findings have implications for guiding the duration of therapy of nephrotic syndrome.

In this prospective study, we measured serum levels of the soluble urokinase receptor (suPAR) in pediatric patients with nephrotic syndrome of various etiologies. Mean levels of suPAR were 3316pg/ml in 99 patients with steroid-resistant focal segmental glomerulosclerosis and 3253pg/ml in 117 patients with biopsy-proven minimal change disease, which were similar to that of 138 patients with steroid-sensitive nephrotic syndrome (3150pg/ml) and 83 healthy controls (3021pg/ml). Similar proportions of patients in each group had suPAR over 3000pg/ml. Compared with controls, suPAR levels were significantly higher in patients with focal segmental glomerulosclerosis (FSGS) and estimated glomerular filtration rate (eGFR) under 30ml/min per 1.73m 2 (6365pg/ml), congenital nephrotic syndrome (4398pg/ml), and other proteinuric diseases with or without eGFR under 30ml/min per 1.73m 2 (5052 and 3875pg/ml, respectively; both significant). There were no changes following therapy and during remission. Levels of suPAR significantly correlated in an inverse manner with eGFR ( r =-0.36) and directly with C-reactive protein ( r =0.20). The urinary suPAR-to-creatinine ratio significantly correlated with proteinuria ( r =0.25) in 151 patients and controls. Using generalized estimating equations approach, serum suPAR significantly correlated with eGFR (coefficient=-13.75), age at sampling (2.72), and C-reactive protein (39.85). Thus, serum suPAR levels in nephrotic syndrome are similar to controls, and do not discriminate between FSGS, minimal change disease, or steroid-responsive illness.

A proportion of patients with steroid-dependent nephrotic syndrome (SDNS) experience frequent relapses despite long-term treatment with steroids, levamisole, or/and cyclophosphamide. We prospectively examined the efficacy of long-term therapy with mycophenolate mofetil (MMF) as a steroid-sparing agent in this group.Nineteen patients with a mean age of 99.1 months (95% confidence interval [CI], 85.3 to 113) who had previously undergone long-term therapy with prednisolone (n = 19), levamisole (n = 16), and cyclophosphamide (n = 15), but had continued to show steroid dependence over many years, were studied. Renal biopsy showed minimal change disease and focal segmental glomerulosclerosis in 10 and 3 patients, respectively. Patients were administered MMF at a mean dose of 29 mg/kg/d (95% CI, 27.4 to 30.7) in 2 divided doses and decreasing doses of alternate-day prednisolone for a mean of 11.8 months (95% CI, 11.4 to 12.2). Relapses were treated with daily prednisolone until remission, with tapering later. They were additionally followed up for a mean of 17 months (95% CI, 15.9 to 18.1).Mean relapse rates decreased from 6.6 (95% CI, 5.4 to 7.7) to 2 episodes/y (95% CI, 1.2 to 2.7) during MMF treatment (P0.0001). Four patients each had 0, 1, 2, and 3 relapses; failure of MMF therapy (3 relapses during treatment) was seen in 3 patients. Treatment with MMF resulted in steroid sparing, with a reduction in mean prednisolone dose from 0.7 (95% CI, 0.6 to 0.8) to 0.3 mg/kg/d (95% CI, 0.2 to 0.4; P0.0001). Fourteen patients showed a 50% or greater reduction in relapse rates; prednisolone therapy could be discontinued for 6 or more months in 8 patients. No significant gastrointestinal or hematologic side effects of MMF treatment were noted. After discontinuation of MMF treatment, 68.4% of patients had an increased frequency of relapses and recurrence of steroid dependence, requiring treatment with other medications.Long-term therapy with MMF results in significant steroid sparing and reduction in relapse rates in patients with SDNS. Therapy with MMF and tapering doses of prednisolone appears to be a promising intervention in children with SDNS.