Opportunities and challenges of Nephrin research (Part 2)

Nephrotic syndrome and Nephrin antibodies

Prior to the identification and sequencing of the NPHS1 gene (Nephrin), scientists had begun to explore the characteristics of antibodies that could bind to the glomerular foot process surface and induce proteinuria. Orikasa et al. [50] immunized BALB/b mice with collagenase-treated Wistar rat glomeruli and produced highly organ-specific and species-specific antibody IgG1, called mAb 5-1-6. In in vitro studies, mAb 5-1-6 was observed to bind to the surface of glomerular foot processes. When this mAb was injected in rats, proteinuria was induced immediately without complement activation. This pioneering study establishes evidence for a strong association between circulating osmotic factor antibodies and the induction of proteinuria in nephrotic syndrome. This approach was later used by researchers to generate similar Nephrin antibodies in animal models to study Nephrin localization within the glomerulus and as a potential drug target to induce proteinuria and nephrotic conditions [42-43,51].

Although animal models have successfully demonstrated the role of anti-Nephrin antibodies in mediating podocyte injury, there have been few reports investigating its role in nephrotic syndrome patients over the past few years. A recent multicenter cohort study by Watts et al [37] has sparked renewed interest in reassessing the relationship between anti-Nephrin antibodies and the development of nephrotic syndrome. Among MCD patients recruited into the study, 18 (29%) tested positive for anti-Nephrin antibodies and 44 (71%) tested negative. Biopsy analysis showed that nephrin colocalizes with punctate IgG deposits in the podocyte SD region. In some biopsies with IgG deposition, there was a redistribution of nephrin away from the SD, indicating the impact of antibodies on nephrin localization and destruction. When patients with active MCD are treated, high levels of circulating anti-Nephrin antibodies are significantly reduced or eliminated, which correlates with significant reductions in proteinuria during treatment. A recent study by Hengel et al. [36] showed that among 539 patients diagnosed with glomerular disease, 46 of 105 (44%) adult MCD patients and 7 of 74 (9%) FSGS patients had elevated anti-Nephrin antibody levels. Additionally, in a cohort of children diagnosed with idiopathic nephrotic syndrome, 94 of 182 (52%) showed elevated levels of anti-Nephrin antibodies. Among patients who did not receive immunosuppressive treatment, the antibody-positive rates in patients with MCD and idiopathic nephrotic syndrome were found to be as high as 69% and 90%. Among study subjects, patients with positive MCD and FSGS showed more severe nephrotic syndrome than those with negative antibody tests. In the mouse model, IgG deposition was observed in the SD area of ​​podocytes, especially in areas where foot process fusion occurred. This observation was also reported by Shirai et al. [38]. During follow-up, it was observed that patients receiving immunosuppressive therapy with glucocorticoids and cyclosporine showed transient remission, whereas patients receiving rituximab (CD20) showed complete and sustained remission in antibody levels [52]. To examine the direct effects of anti-Nephrin antibodies on the interhemispheric diaphragm, phosphoproteome analysis of antibody-injected mice at 3 weeks showed increased phosphorylation of Nephrin at tyrosine residue Y1191. Phosphorylation of this tyrosine residue may lead to actin assembly, cytoskeletal reorganization, and Nephrin endocytosis.
In the review report of Cui and Zhao [53] on the study of Hengel et al. [36], it was proposed that these anti-Nephrin autoantibodies may not be limited to the IgG class, and deposition of other classes such as IgM can also be observed in MCD and FSGS. Although Shirai et al. [38] observed trace deposits of IgM and C3 in some biopsies taken during relapse, this suggested that these deposits did not co-localize with Nephrin. Furthermore, no evidence of IgG deposition was observed after remission, suggesting that circulating anti-Nephrin antibodies are highly likely to be a circulating permeability factor involved in the pathogenesis of relapse after renal transplantation. In a recent case reported by Bressendorff et al. [52], an 84-year-old male patient with a history of multiple medical conditions, including stage 3 chronic kidney disease, was admitted with shortness of breath and edema and was treated and discharged. On readmission, urinalysis revealed progressive acute kidney injury with hypoalbuminemia and proteinuria. Renal biopsy showed that FSGS developed into punctate IgG deposits overlapping with Nephrin, with foot process effacement, but no formation of IgM, IgA, C3, C1q or kappa and lambda light chains was observed, and the patient did not respond to glucocorticoid treatment. Testing showed positive circulating anti-Nephrin antibodies, which gradually decreased after plasma exchange treatment. After seven plasmapheresis treatments, antibody levels were below the limit of detection and similar to those reported in the control population, along with recovery of baseline renal function, which remained stable for a year and a half with no signs of relapse.

Recurrence of FSGS in renal transplantation and Nephrin antibodies


In a multicenter study by Shirai et al. [38], among 22 FSGS kidney transplant recipients (8 patients with genetic mutations and 14 patients with non-hereditary disease), 11 of 14 patients with non-hereditary FSGS developed FSGS recurrence after transplantation, which was significantly different from non-recurrent FSGS (165 U/mL) and hereditary FSGS (113 U/mL), the anti-Nephrin antibody levels in 11 relapsed patients were significantly higher (831 U/mL before transplantation and 1292 U/mL at relapse after transplantation). Elevated antibody levels are suggestive of recurrence of FSGS. Anti-Nephrin antibody levels are also associated with proteinuria levels in post-transplant patients, with higher levels of proteinuria observed in patients with recurrent FSGS. Recently, Batal I et al. [54] evaluated the predictive value of anti-Nephrin antibody levels before renal transplantation for postoperative diffuse podocytopathy (DP) recurrence. They retrospectively analyzed 38 patients with serum samples retained before transplantation. After a median follow-up of 43 months (interquartile range 8-79 months), 21 patients had DP recurrence, and 17 patients had no DP recurrence. Pretransplant anti-Nephrin antibody levels predicted disease recurrence (area under the curve 0.78, P=0.03). When the previously studied 187 U/ml was used as the diagnostic threshold, 8 of 21 relapsed patients (38%) were antibody positive, and all 17 relapse-free patients were antibody negative (P=0.005). Anti-Nephrin antibody levels predicted DP with 100% specificity, 100% positive predictive value, 38% sensitivity, and 57% negative predictive value. It is suggested that patients with high levels of anti-Nephrin antibodies before surgery should pay close attention to the recurrence of kidney disease after surgery (this cohort showed 100% recurrence), and programmatic monitoring of anti-Nephrin antibody levels is required. The results of survival analysis (time-to-event analysis) showed that patients with positive anti-Nephrin antibodies before transplantation had a higher risk of DP recurrence (hazard ratio 4.9, 95% confidence interval 1.25-18.8, P<0.001).

Outlook: Opportunities and Challenges

Unlike other blood circulation permeability factors (suPAR, CD40, etc.), there are currently no studies denying the clinical value of anti-Nephrin antibodies as potential markers and blood circulation permeability factors for FSGS and MCD. However, the reported studies have certain limitations that may need to be considered and addressed in the future.
(1) Ultra-sensitive, standardized quantitative analysis methods are very important to ensure the accuracy and comparability of anti-Nephrin antibody detection.The main limiting factor at present is the lack of commercialized human anti-Nephrin antibodies, and it is unknown whether the antibodies are polyclonal or monoclonal, and whether they are consistent among different patients. Therefore, there is currently a reliance on using positive patient sera to prepare standard curves, which results in inconsistent thresholds across laboratories. For example, in the study by Watts et al. [37], the maximum anti-Nephrin antibody threshold in healthy humans was set at 187 U/mL for a sample dilution of 1:100. In the study by Shirai et al. [38], the anti-Nephrin antibody positivity threshold was defined as the maximum antibody titer (231 U/mL) at a sample dilution of 1:400. In addition, the content of anti-Nephrin antibodies in serum is low, and it is difficult for ordinary ELISA to accurately detect such low-abundance antibodies. Especially when using no antigen coating as a negative control, the probability of the test result (OD difference) being negative is high. In the study of Hengel et al. [36] in the New England Journal, the abundance of anti-Nephrin antibodies in the test sample was increased by using enrichment of IgG antibodies. The development of anti-Nephrin antibody detection methods based on the "enrichment pathway" is a development direction for highly sensitive detection in the future.
(2) Are there other circulating osmotic factors involved in pFSGS?Studies have shown that not all patients diagnosed with pFSGS or MCD test positive for anti-Nephrin antibodies. In the Watts [37] study, only 18 of 62 MCD patients were anti-Nephrin antibody positive. This result suggests that other circulating permeability factors may be involved in the induction of MCD in the remaining 44 patients. In the study by Hengel et al. [36], 94 children with idiopathic nephrotic syndrome were observed to be positive for Nephrin antibodies, while 88 were negative. There were 46 anti-Nephrin antibody positive and 59 antibody negative patients in adult MCD patients, and 7 antibody positive and 67 antibody negative patients in pFSGS patients. These observations suggest that although anti-Nephrin antibody may be considered a novel biomarker and circulating permeability factor for idiopathic nephrotic syndrome factors including MCD and FSGS, it may not be universally applicable to all patients with MCD and FSGS. For example, recent studies reported that Crb2 is an important diaphragm muscle protein, and mice knocking out CRB2 in podocytes developed massive proteinuria at 2 months or immediately after birth, and also showed reduced expression of NPHS1&2, PODXL, and reduced glomerular WT-1 cells [55-56]. Furthermore, mice administered podocyte CRB2 protein were observed to develop anti-CRB2 antibodies against podocyte protein, accompanied by proteinuria and characteristic features of MCD and FSGS [57]. However, reports on the detection of CRB2 antibodies in people with idiopathic nephrotic syndrome remain limited.
(3) Desensitization treatment for renal transplantation in anti-Nephrin antibody-positive recipients.For recipients with anti-Nephrin antibody-positive desensitization treatment regimens, there is a lack of research on postoperative FSGS recurrence. The current study cohort of FSGS recurrence has not distinguished the antibody-positive subcohort. In an early report by Gallon et al. [9], 10 high-risk renal transplant recipients received two preoperative plasma exchanges and five postoperative plasma exchanges, and 7 patients did not experience FSGS recurrence at the end point. In a recent study by Bressendorff et al. [52], a patient who underwent 7 rounds of plasmapheresis recovered without evidence of recurrence, and the lack of recurrence was thought to be a result of combined high-dose glucocorticoid therapy. Carvajal Abreu, K. et al. [58] also pointed out that 25 plasma exchanges within 14 weeks, together with the use of methylprednisolone and prednisolone, are crucial to prevent transplant rejection and recurrence of FSGS. A study by Kim et al. [59] also showed that a renal transplant patient relapsed after failing to respond to calcineurin inhibitors. He underwent 13 courses of plasma exchange therapy and was given rituximab at the same time, and the patient achieved partial recovery. Studies have also shown that in a study of 66 pFSGS recipients, 62% of the 37 recipients who received plasma exchange with or without anti-CD20 monoclonal antibodies had FSGS recurrence, and only 51% of the 27 recipients who did not receive any desensitization treatment relapsed [60]. These studies have shown that pre-desensitization regimens can reduce the recurrence of FSGS, but no large-scale studies have yet confirmed whether preoperative desensitization therapy can affect the recurrence of FSGS, and there are still inconsistent results. It is important to study the desensitization regimen for the subcohort of anti-Nephrin antibody-positive FSGS in the future.

(4) Finally, there are currently no studies examining the interaction between different epitopes of Nephrin and anti-Nephrin antibodies. It is not clear whether multiple epitopes show antibody binding ability, and whether the binding abilities of different epitopes are consistent.It is also not well understood that anti-Nephrin antibodies bind to specific regions of Nephrin to induce Nephrin redistribution and podocyte damage. There are also no studies to determine how blocking Nephrin epitopes prevents interactions with antibodies. Solving these issues is very important to elucidate the pathogenic mechanism of Nephrin antibodies and to develop specific drugs for patients with anti-Nephrin antibody-positive FSGS. The next step requires a comprehensive study of the different domains of Nephrin, the various epitopes that can serve as binding sites for anti-Nephrin antibodies, and potential ligands that can block these epitopes to inhibit binding to anti-Nephrin antibodies.

References
[50] Orikasa, M., Matsui, K., Oite, T., and Shimizu, F., , The Journal of Immunology, 1988, Vol 141(3) p. 807-814.
[51] Kikuchi, H., Kawachi, H., Ito, Y., Matsui, K., Nosaka, H., Saito, A., Orikasa, M., Arakawa, M., and Shimizu, F., Severe proteinuria, sustained for 6 months, induces tubular epithelial cell injury and cell infiltration in rats but not progressive interstitial fibrosis, Nephrology Dialysis Transplantation, 2000, Vol 15(6) p. 799-810.
[52] Bressendorff, I., Nelveg-Kristensen, K. E., Ghasemi, M., Watts, A. J. B., Elversang, J., Keller, K. H., Nielsen, F. C., Szpirt, W., and Weins, A., Antinephrin-Associated Primary Focal Segmental Glomerulosclerosis Successfully Treated With Plasmapheresis, Kidney International Reports, 2024, Vol 9(9) p. 2829-2831.
[53] Cui, Z. and Zhao, M.-h., Anti-nephrin autoantibodies: a paradigm shift in podocytopathies, Nature Reviews Nephrology, 2024, Vol 20(10) p. 639-640.
[54]Batal I, Watts AJB, Gibier JB, Hamroun A, Top I, Provot F, Keller K, Ye X, Fernandez HE, Leal R, Andeen NK, Crew RJ, Dube GK, Vasilescu ER, Ratner LE, Bowman N, Bomback AS, Sanna-Cherchi S, Kiryluk K, Weins A. Pre-transplant anti-nephrin antibodies are specific predictors of recurrent diffuse podocytopathy in the kidney allograft. Kidney Int. 2024 Oct;106(4):749-752. doi: 10.1016/j.kint.2024.07.022.
[55] Tanoue, A., Katayama, K., Ito, Y., Joh, K., Toda, M., Yasuma, T., D’Alessandro-Gabazza, C. N., Kawachi, H., Yan, K., Ito, M., Gabazza, E. C., Tryggvason, K., and Dohi, K., Podocyte-specific Crb2 knockout mice develop focal segmental glomerulosclerosis, Scientific Reports, 2021, Vol 11(1) p. 20556.
[56] Möller-Kerutt, A., Rodriguez-Gatica, J. E., Wacker, K., Bhatia, R., Siebrasse, J. P., Boon, N., Van Marck, V., Boor, P., Kubitscheck, U., Wijnholds, J., Pavenstädt, H., and Weide, T., Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier, J Am Soc Nephrol, 2021, Vol 32(5) p. 1053-1070.
[57] Hada, I., Shimizu, A., Takematsu, H., Nishibori, Y., Kimura, T., Fukutomi, T., Kudo, A., Ito-Nitta, N., Kiuchi, Z., Patrakka, J., Mikami, N., Leclerc, S., Akimoto, Y., Hirayama, Y., Mori, S., Takano, T., and Yan, K., A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2, J Am Soc Nephrol, 2022, Vol 33(11) p. 2008-2025.
[58] Carvajal Abreu, K., Loos, S., Fischer, L., Pape, L., Wiech, T., Kemper, M. J., Tönshoff, B., Oh, J., and Schild, R., Case report: Early onset de novo FSGS in a child after kidney transplantation—a successful treatment, 2023, Vol 11 p.
[59] Kim, Y.-J., Lee, S.-W., Kim, M.-S., Kim, Y.-J., Choi, J.-Y., Cho, J.-H., Kim, C.-D., Kim, Y.-L., Yun, W.-S., Huh, S., Lim, J.-H., and Park, S.-H., Anuria after kidney transplantation diagnosed as early recurrence of focal segmental glomerulosclerosis combined with acute calcineurin inhibitor nephrotoxicity: a case report and literature review, BMC Nephrology, 2024, Vol 25(1) p. 123.
[60]Alasfar S , Matar D , Montgomery RA ,et al. Rituximab and therapeutic plasma exchange in recurrent focal segmental glomerulosclerosis postkidney transplantation[J]. Transplantation， 2018，102(3)：e115-e120. DOI： 10.1097/TP.000000000000200