Sequential administration of paricalcitol followed by IL-17 blockade for progressive refractory IgA nephropathy patients

There is no established treatment for progressive IgA nephropathy refractory to steroids and immunosuppressant drugs (r-IgAN). Interleukin 17 (IL-17) blockade has garnered interest in immune-mediated diseases involving the gut-kidney axis. However, single IL-17A inhibition induced paradoxical effects in patients with Crohn’s disease and some cases of de novo glomerulonephritis, possibly due to the complete Th1 cell response, along with the concomitant downregulation of regulatory T cells (Tregs). Seven r-IgAN patients were treated with at least six months of oral paricalcitol, followed by the addition of subcutaneous anti-IL-17A (secukinumab). After a mean follow-up of 28 months, proteinuria decreased by 71% (95% CI: 56–87), P < 0.001. One patient started dialysis, while the annual eGFR decline in the remaining patients [mean (95% CI)] was reduced by 4.9 mL/min/1.73 m2 (95% CI: 0.1–9.7), P = 0.046. Circulating Th1, Th17, and Treg cells remained stable, but Th2 cells decreased, modifying the Th1/Th2 ratio. Intriguingly, accumulation of circulating Th17.1 cells was observed. This novel sequential therapy appears to optimize renal advantages in patients with r-IgAN and elicit alterations in potentially pathogenic T helper cells.


Patients
This pilot trial treatment was conducted from May 2018 to November 2022.All patients suffered from refractory IgAN (r-IgAN).r-IgAN was defined as proteinuria > 1 g/24 h and a progressive decline in the estimated glomerular filtration rate (eGFR): > 2 mL/min/1.73m 2 /year despite renin-angiotensin system inhibitors, steroids, and immunosuppressive drugs.The eGFR was determined by the EPI-CKD equation (Epidemiology Collaboration Chronic Kidney Disease).The flowchart of the study is displayed in Fig. 1A.The inclusion and exclusion criteria of the patients in the trial treatment are described in Supplementary Methods 1.

Determinations
Hematological indexes were performed by flow cytometry using the automatic analyzer CELL-DYN Sapphire (Abbott Diagnostics®, Abbott Laboratories.Abbott Park, Illinois, USA).Standard methods were used to assess biochemical metabolism parameters.
Determinations were performed at 0, 3, and 9 months and at the end of follow-up.Proinflammatory ratios were calculated using Th1, Th17, and Th17.1 cells as numerators and Treg cells as denominators at month 0 and at the end of the follow-up.The Th1/Th2 ratio was also calculated.T cells were determined by flow cytometry in our immunology laboratory (Supplementary Methods 2).

Drug therapy
The sequential combined therapy consisted of PRC (Zemplar, AbbVie Laboratory) 1 mcg per day for at least 6 months before adding SCK (a fully human IgG1/ k monoclonal antibody that neutralizes IL-17A) that was subcutaneously administered as follows: 300 mg weeks 0, 1, 2, 3, and 4-induction phase-, and monthly for maintenance.The background treatment was maintained at the same doses throughout the study (Fig. 1B).

Statistical analysis
The data were obtained from the patient's electronic medical records and are presented as the mean (95% CI) or median (p25-p75) as needed.The Shapiro-Wilk test was used to determine the normality distribution of the data.T-paired test or Wilcoxon rank test was performed as appropriate to evaluate potential differences.Pearson or Spearman test correlation (one-sided) was used to assess the association between variables.The pre-combined therapy eGFR slope was determined by calculating the difference between the eGFR at the time of renal biopsy and the eGFR at the start of the combined therapy, then dividing this value by the time elapsed (in years) from the renal biopsy to the initiation of PRC + SCK.The post-combined therapy slope was calculated by subtracting the eGFR value at the beginning of PRC + SEC from the eGFR at the end of the follow-up and dividing this value by the number of years from the start of PRC + SEC to the end of the follow-up.The comparison of the two slopes was performed using linear regression based on the principle of least squares.
Statistical analyses were performed using the Statistical Package for the Social Sciences software version 21.0 for Windows.P < 0.05 was considered statistically significant.

Overall response
Six out of seven patients achieved sustained recovery of proteinuria and eGFR after combined therapy during the follow-up period.One patient (patient #3) showed only a transient proteinuria decrease while renal function worsened, so he was considered a nonresponder, and SCK stopped at month 12.This patient started chronic renal replacement therapy two years after the combined treatment failed.Patient #2 exhibited spontaneous recurrence after 9 months of therapy, and patient #5 exhibited coincidental recurrence after 15  www.nature.com/scientificreports/

Proteinuria
Proteinuria decreased significantly at month three, and the same trend was maintained throughout the follow-up (Fig. 2A).At the end of the follow-up period, proteinuria decreased by 71% (95% CI: 56-87) compared to that observed at baseline (P < 0.001).The proteinuria evolution in each of the patients is shown in Fig. 2B.The maximum decrease in proteinuria achieved at some point during the follow-up was [median (p25-p75): 0.5 g/24 h (0.4-0.7)] (Supplementary Result 1 Figure S1).

Hematuria
Hematuria (red blood cells/high power field > five) was observed in patients #4 and #5.It disappeared during combined therapy at month 3.Both patients developed recurrent mild hematuria at month nine and at the end of the follow-up (7 and 9 red blood cells/high power field, respectively) (Fig. 2E).

Biochemical and hematological parameters
Total plasma proteins increased at the end of the follow-up period.No significant changes were observed additionally (Table 2).
Circulating T cells and Th1/Treg, Th17/Treg, Th17.1/Treg, and Th1/Th2 ratios Compared to the baseline, the numbers of Th1, Th17 cells, and T cells did not change by the end of the follow-up (Fig. 4A and B).The Th1/Treg and Th17/Treg ratios remained unchanged (Fig. 4C and D).Th17.1 cells increased by the end of the follow-up (Fig. 4E), while Th2 cells decreased (Fig. 4F).Treg cell numbers did not change by the end of the study (Fig. 4G).The Th2/Treg ratio decreased at the end of the follow-up (Fig. 4H), while the Th1/Th2 and Th17.1/Treg ratios increased (Fig. 4I and J).The changes in Th17 cells negatively correlated with changes in Th2 cells (r = − 0.69, P = 0.041).No association among the changes in other T cells was found (Table 3).

Safety
Patient #7 developed trigeminal herpes zoster infection and needed hospital admission five months after the onset of SCK resolved with intravenous acyclovir.Patient #1 developed esophageal candidiasis, and patient #5 had asymptomatic candiduria that was successfully treated with oral fluconazole.For SARS-Cov-2 infection, patient #5 fully recovered after pneumonia in February 2022, requiring hospital admission (the patient was not previously vaccinated).Patients #2 and #4 had an oligosymptomatic SARS-Cov-2 infection in June 2022 (both correctly vaccinated) and were successfully treated with oral nirmatrelvir-ritonavir and intravenous remdesivir, respectively.These patients did not require hospitalization.

Discussion
This is the first study evaluating the effect of IL-17A blockade on progressive r-IgAN previously treated with PRC.The combined therapy seems highly effective in controlling proteinuria and hematuria and stabilizing renal function, which is associated with changes in circulating Th17.1 and Th2 cells.The presence of only one nonresponder emphasizes that this combined therapy might target a critical pathophysiological pathway, the IL-17A, and the gut-kidney axis.In line with this proposed mechanism, orally administered enteral budesonide, designed to address inflammation within the gut-associated lymphoid system in IgAN patients, has demonstrated significant advantages in managing proteinuria and maintaining eGFR stability 31,32 .To the best of our knowledge, no therapy has proven such an effect in patients with r-IgAN, even without driving 24-h proteinuria below the 1 g threshold 33 .
Our novel approach designed with the intention of downregulating the Th1 inflammatory response, followed by IL-17A blockade and was based on several experimental studies evaluating anti-IL-17 therapy, in which worsening of colitis in patients 21 or glomerulonephritis in animal models 34 was observed in the presence of an active Th1 pro-inflammatory response.More recently, a study described that autonomous activation of IL-17 receptor is responsible for continuous inflammation 35 .This could explain the lack of efficacy in some IL-17associated pathologies when IL-17A inhibition is administered alone.
Proteinuria decreased by 71% with respect to baseline.All but one patient (patient #3) achieved partial remission of proteinuria (< 1 g/24 h) at any time throughout the follow-up, which may improve renal prognosis 36 .Notably, the maximum decrease obtained in proteinuria [median (p25-p75)] was 0.5 g/24 h (0.4-0.7), which suggests the high antiproteinuric effect of the combined therapy in our patients.
The mean eGFR at baseline in our patients was lower than 60 mL/min/1.73m 2 , which is considered an independent high-risk factor for developing end-stage renal disease 37 .Except for patient #3, the annual decline improved significantly to 4.9 mL/min/1.73m 2 (mean), which is clinically meaningful.Furthermore, a 1-year eGFR slope could be considered a surrogate renal endpoint in IgAN patients 38 ; therefore, the combined therapy showed benefits in both proteinuria and eGFR.Patient #3 experienced a rapid progressive eGFR decline and was needed to start dialysis 36 months after the inclusion in the study.Patient #7 continued decreasing eGFR; however, the patient started combined therapy with a low eGFR (32 mL/min/1.73m 2 ) and nephrotic proteinuria (5.1 g/24 h).At the end of the follow-up, his proteinuria was well controlled (0.8 g/24 h); therefore, whether this benefit will slow the decline in kidney function will be evaluated in the coming months.
The benefits of the combined therapy were independent of antihypertensive treatment and body weight since no significant changes in these markers were observed.However, hypotensive drugs had to be reduced in two patients.At the same time, systolic blood pressure tended to decrease at the end of the follow-up period, which suggests a potential benefit of the combined therapy on blood pressure in our patients.The role of  www.nature.com/scientificreports/adaptive immunity (Th1 and Th17 cells) and IL-17 in the pathogenesis of hypertension has been confirmed by its actions on the proximal and distal tubules, in the thick ascending limb, and the epithelial sodium channel in the collecting duct 39 .Notably, the sole use of SCK did not show benefits in blood pressure in patients with psoriasis, psoriatic arthritis, and axial spondyloarthritis 40 .Further investigation is needed to determine whether the decrease in hypotensive drugs in our patients results from the effect of IL-17A blockade on the arteries, renal environment, or both.Following the study hypothesis, combined therapy slightly decreased circulating Th1 cells and stabilized Treg cells during the follow-up.In contrast with our findings, Treg cells decreased in skin lesions from patients with psoriasis treated with SCK 41 and decreased peripheral Treg cells in ankylosing spondylitis patients 42 .During IL-17A blockade, peripheral Treg cells remained stable, potentially enhancing the safety of IL-17 inhibition.We observed that peripheral Th17 cells initially tended to fall but returned to baseline values at the end of the follow-up.We do not have a clue about this finding, but we do humbly speculate that the peripheral number of Th17 cells may not reflect the effect on the kidney of the combined therapy (given the profound changes observed in proteinuria and eGFR) or maybe reflect changes in the Th17 cells phenotype could have produced it.
Unexpectedly, peripheral Th17.1 cells increased during the study.These cells are considered pathogenic in lupus, multiple sclerosis, and sarcoidosis 43 ; specifically, Th17.1 cells display high expression of multidrug resistance protein 1 (MDR1) and low expression of glucocorticoid receptors 44 that confer a limited response to steroids.In IgAN, Th17.1 cells express the chemokines C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 3 (CXCR3), which increase the recruitment of proinflammatory cells and produce tubulointerstitial fibrosis 34,45 , respectively.Therefore, the accumulation of these cells in the blood highlights their potential critical role in r-IgAN pathogenesis.If the elevation of circulating Th17.1 cells results from cellular trafficking from inflamed tissues, as proposed in multiple sclerosis patients after natalizumab treatment 46 , or stems from enhanced differentiation of precursor cells with a concurrent reduction in their pathogenic potential, it necessitates comprehensive investigation.While Th2 cells were not originally the focus of the combined therapy, our research indicates a significant decrease in Th2 cells, with an observed inverse correlation between these changes and Th17 cells, suggesting a bidirectional influence.We speculate that the blockade of IL-17A following SCK administration could induce this effect.According to data from a murine model of atopic dermatitis, IL-17 is instrumental in transforming naive T cells into Th2 cells while concurrently diminishing Th2 chemokine expression and populations of IL-4producing cells in the absence of IL-17A 47 .Additionally, emerging evidence supports the interplay between Th17 and Th2 cells through the transcription factor retinoic acid-related orphan receptor γt (RORγt).For instance, RORγt has a dual role: it not only regulates Th17 cells but also inhibits their conversion into Th2 cells 48 .Vitamin D metabolites are known agonists of RORγt 49 , and paricalcitol (19-nor-1alpha-25-dihydroxyvitamin D2) may influence RORγt.Thus, we consider it feasible that combination therapy could decrease Th2 cells.Therefore, whether a beneficial synergistic effect of combined therapy on Th2 cells exists, or if SCK alone can impact Th2 cells in IgAN, warrants further investigation.
The decrease in Th2 cells could be beneficial and it is supported by the fact that higher circulating levels of Th2 cells have been observed in IgAN patients compared to healthy controls 50 .Additionally, the reversal of the Th1/Th2 cell imbalance, as observed in our patients, might reduce the severity of IgAN 51 .In our patients, the Th2/Treg ratio decreased due to the impact on Th2 cells.While the number of circulating Treg cells remained stable in our study, the potential influence of Treg cells on Th2 cells could theoretically be attributed to improved Treg cell functionality resulting from the combined therapy.The negative correlation between the number and function of Treg and Th2 cells has been well-documented 52 .
The pathogenic role of Th1 and Th2 cells in IgAN is a subject of debate, without a proven predominance one over other.Importantly, Th1 is widely acknowledged as a pathogenic factor in IgA nephropathy, contributing to glomerular sclerosis, exacerbating proteinuria severity, and serving as an indicator for renal function decline.INF-γ is crucial for synergistic interactions with other pathogenic cytokines 53 , and for crescentic formations, highlighting its role in the initial stages of the disease.Nonetheless, the heightened expression of Th2 cytokines is associated with tubular interstitial injury and mesangial cell proliferation, and fibrosis 54 indicating their involvement in chronic renal damage, which may explain the progressive degradation of renal function.In our specific set of severe patients suffering from chronic disease, the impact of Th2 cells seems to exceed that from Th1 cells, but both populations might be relevant when managing IgA nephropathy.Moreover, the dominance between Th1 and Th2 cells may shift as the disease evolves, influencing disease progression and management strategies.
Despite the observed benefits in r-IGAN patients, the question arises as to whether the isolated use of the treatment could have produced the same effect.Contrary to our findings, the solitary use of SCK did not show benefit in proteinuria, as per the study and some clinical cases [22][23][24]27 . Whie Treg cell counts were stabilized in our study, a decrease in Treg cell numbers has been observed in cutaneous tissue and at peripheral levels in ankylosing spondylitis 42 .In light of our hypothesis, this could be potentially detrimental if IL-17 is blocked.Furthermore, in patients with ankylosing spondylitis, the use of SCK did not decrease Th2 cells 55 , paralleling recent findings in psoriatic patients 56 .In contrast, in our study we observed a significant decrease in Th2 cells.
Finally, the combined therapy may play a role in the autonomous activation of IL-17 receptor, IL-17 activates the protein tyrosine phosphatase SHP2 (SHP2) and employs it for the autonomous activation of the IL-17R signal in the absence of IL-17.Additionally, SHP2 is activated by TGF-beta 57 .Significantly, TGF-beta is downregulated by paricalcitol 58 , which could facilitate the inhibition of IL-17R, thereby enhancing the blockade of IL-17.
The combined therapy, in general, was well tolerated.Two patients needed hospital admission, one for trigeminal herpes zoster infection and the other one by SARS-CoV-2 infection, who was not previously vaccinated.In the first patient, although the infection appeared after the onset of SCK, the patient's age, high proteinuria, and basal immunosuppression therapy were risk factors for developing herpes zoster infection.www.nature.com/scientificreports/Candidiasis was observed in two patients, as expected according to the datasheet, and satisfactorily resolved with oral fluconazole.Two patients had oligosyntomatic SARS-CoV-2 infection.Considering the pandemic situation, having contracted SARS-CoV-2 infection in these patients may not be related solely to the combined therapy.
The main strengths of this study include the inclusion of rapid progressive and difficult-to-treat refractory IgAN patients, the evaluation of the IL-17A blockage and the relative long-term sustained response in clinical practice.This subset of patients is commonly excluded from clinical trials and lacks effective alternative therapies.The drawbacks of our study include being a single-center study, having a small sample size, lacking a control group, not being able to phenotype Th cells, nor to assess their renal actions induced by the combined therapy.Due to a small sample size, a low statistical power to detect significant differences before and after treatments should be recognized, so the reported data should be interpreted with caution and not generalized until larger studies validate our results.
In conclusion, the novel sequential combined therapy-first with anti-inflammatory PRC followed by IL-17A blockade-appears effective in managing r-IgAN patients.These initial observations undeniably pave the way for potential therapeutic advancements in tackling challenging progressive refractory cases of this disease.The current data underscores the need for more extensive studies to validate its efficacy and safety.

Figure 2 .
Figure 2. Proteinuria, eGFR, and hematuria evolution during the follow-up.(A) Evolution of median (p75) 24 h collected proteinuria.*P = 0.030; **P = 0.023; ***P = 0.001, respect to M0. (B) 24 h collected proteinuria at each patient included.(C) Evolution of the eGFR during the study.* P < 0.001 respect to RB, Bonferroni adjusted P-value.Data mean ± standart error of the mean.(D) Annual trend in eGFR (mean ± standard error of the mean).Precombined therapy: PRC + SCK (Pre-CT) corresponds to the period between renal biopsy and the onset of SCK, and Postcombined (Post-CT) corresponds to the period from SCK to the end of the follow-up.(E) Evolution of hematuria (red blood cell/high power field, RBC/HPF) during the study.*P = 0.027 with respect to M0. Data: median (p75).M0, the onset of paricalcitol + secukinumab (PRC + SCK).EFU, end of follow-up; RB, renal biopsy; M, month.

Table 3 .
Association between changes in T cells during the study.Δ Stands for the changes between baseline and the end of the follow-up.*P < 0.05.