New-Onset and Relapsed Membranous Nephropathy post SARS-CoV-2 and COVID-19 Vaccination

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak and COVID-19 vaccination, new-onset and relapsed clinical cases of membranous nephropathy (MN) have been reported. However, their clinical characteristics and pathogenesis remained unclear. In this article, we collected five cases of MN associated with SARS-CoV-2 infection and 37 related to COVID-19 vaccination. Of these five cases, four (4/5, 80%) had acute kidney injury (AKI) at disease onset. Phospholipase A2 receptor (PLA2R) in kidney tissue was negative in three (3/5, 60%) patients, and no deposition of virus particles was measured among all patients. Conventional immunosuppressive drugs could induce disease remission. The underlying pathogenesis included the subepithelial deposition of viral antigens and aberrant immune response. New-onset and relapsed MN after COVID-19 vaccination generally occurred within two weeks after the second dose of vaccine. Almost 27% of patients (10/37) suffered from AKI. In total, 11 of 14 cases showed positive for PLA2R, and 20 of 26 (76.9%) presented with an elevated serum phospholipase A2 receptor antibody (PLA2R-Ab), in which 8 cases exceeded 50 RU/mL. Conventional immunosuppressive medications combined with rituximab were found more beneficial to disease remission for relapsed patients. In contrast, new-onset patients responded to conservative treatment. Overall, most patients (24/37, 64.9%) had a favorable prognosis. Cross immunity and enhanced immune response might contribute to explaining the mechanisms of MN post COVID-19 vaccination.


Introduction
The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought significant challenges to human beings. As of 11 September 2022, more than 605 million infected cases have been confirmed globally, and deaths have reached 6.4 million [1]. Current research found that SARS-CoV-2 could affect multiple human organs with a high expression of angiotensinconverting-enzyme 2 (ACE2) receptor, including respiratory tracts, heart, kidney, nervous system, etc. [2].

Treatment and Prognosis
Case reports on new-onset MN post SARS-CoV-2 infection were rare. Only one case [9] achieved spontaneous remission without treatment. Steroids, CTX, and TAC were the principal immunotherapy approaches. Up to now, the published cases of MN associated with SARS-CoV-2 infection were not recommended using rituximab (RTX) ( Table 1, case [1][2][3][4][5]. Previous literature reported that RTX treatment could cause viral reactivation among patients with hepatitis B virus-associated MN [14]. Whether this phenomenon would occur in MN post SARS-CoV-2 infection still needs further clarification. In contrast, the prognosis of elderly patients with chronic kidney disease was relatively dismal compared with those without a medical history of renal involvement. In addition, one patient with a prior diagnosis of MN infected with SARS-CoV-2 following the administration of RTX. He achieved viral elimination within 3 weeks after anti-virus medications, and no serious adverse events occurred ( Further, we conducted a literature search based on PubMed, EMBASE, Google Scholar, and Web of Science electronic database via the following keywords: ("membranous nephropathy" OR "proteinuria" OR "nephrotic syndrome") AND ("SARS-CoV-2" OR "COVID-19" OR "2019-ncov" OR "novel coronavirus" OR "coronavirus") AND ("vaccine" OR "vaccination") to collect the clinical information of new-onset and relapsed MN post COVID-19 vaccination.

New-Onset MN
We collected 20 patients with new-onset MN post COVID-19 vaccination, and the median age of onset was 57  years, of which 12 were males. mRNA vaccines were the leading type (15/21, 71.4%), usually occurring after the second dose of vaccine (10/20, 50.0%), with the most common onset time within two weeks (10/20, 50.0%). Edema and proteinuria were commonly observed in these cases. In total, 6 of 20 cases (30%) showed AKI. There were 7 cases associated with PLA2R, one case [4] with thrombospondin type-1 domain-containing 7A (THSD7A), and the other case [21] was diagnosed as neural epidermal growth factor-like 1 (NELL-1) related MN.
A total of 11 cases were treated with immunosuppressive drugs, 8 of which were given RTX, 4 patients achieved remission within the follow-up period, 1 case showed no response within 2 months, and 3 cases were lost to follow-up. Another 9 cases received conservative measures, 7 cases underwent remission, 1 case showed no response, and 1 case had no follow-up information. The median remission time was 41 (14-180) days. In general, the clinical treatment effect on COVID-19 vaccination-associated MN was worthy of being recognized. Tables 2 and 3 illustrated the detailed data.

Relapsed or Worsening MN
Overall, 17 cases showed worsening edema and proteinuria. Of the majority of enrolled patients, 76.5% (13/17), were males, with the median age of 65 (39-84) years. A total of 15 cases were associated with mRNA vaccines, in which Pfizer-BioNTech accounted for 86.7% (13/15). Two doses of vaccines were more likely to cause disease recurrence. Among these 17 patients, 8 patients relapsed within 2 weeks. In total, 14 of 15 cases (93.3%) were represented as an elevation of PLA2R-Ab. AKI was reported in 4 cases (4/17, 23.5%).
There were 13 cases treated with immunosuppressive medications, of which three cases responded to TAC, 2 to prednisone, and 1 patient using obinutuzumab had unclear prognostic information. A total of 7 cases received RTX, only one elderly patient [29] showed no remission in 4 months. Three cases received conservative treatment, only 1 case [31] showed improvement in proteinuria. The median remission time was 58  days. In all relapsed cases, in 3 patients [29] using immunosuppressive medications during vaccination, edema occurred (Tables 2 and 3).

Treatment and Prognosis
Generally, clinical cases of new-onset and relapsed MN associated with COVID-19 vaccination had an excellent prognosis. Some patients, especially new-onset patients, could achieve remission via conservative management. The median remission time was 30 (14-210) days. In contrast, conventional immunosuppressive drugs combined with RTX were required for relapsed patients, and the median remission time was 60 (21-180) days. It was widely accepted that PLA2R-Ab was a crucial clinical indicator for predicting the prognosis of MN [34]. Among these 20 cases of MN secondary to COVID-19 vaccination with positive expression of PLA2R-Ab, 14 had achieved remission in the follow-up period, and the median remission time was 60 (21-180) days (Table 2).

Subepithelial Deposition of Viral Antigens
Spike protein was the pivotal structure for SARS-CoV-2 to infect host cells through specifically recognizing ACE2 [35] and with the assistance of being cleaved by transmembrane protease serine 2 (TMPRSS2) [36,37]. Notably, both ACE2 and TMPRSS2 were also expressed on podocytes [38]. According to reports in the literature, SARS-CoV-2 viral particles were detected in podocytes of postmortem kidney samples in 26 patients with COVID-19 [39], indicating potential evidence that SARS-CoV-2 could directly invade into podocytes. Nevertheless, the process of viral infection on podocytes might contribute to the subepithelial deposition of viral antigens [40], thus forming in situ antigens to stimulate the production of corresponding antibodies, leading to the deposition of viral immune complexes in glomeruli (Figure 1). the subepithelial deposition of viral antigens [40], thus forming in situ antigens to stimulate the production of corresponding antibodies, leading to the deposition of viral immune complexes in glomeruli (Figure 1).

Massive Release of Cytokines
In COVID-19, viral infection in pulmonary epithelial cells triggered the recruitment of immune effector cells and released massive proinflammatory cytokines and chemokines [41,42], which subsequently advanced T lymphocyte differentiation. T helper (Th) 17 cells generated interleukin (IL)-17A, IL-17F, IL-22, and granulocyte-macrophage colony-stimulating factor, inducing the aggregation of inflammatory cells, such as neutrophils [43]. IL-4 produced by Th2 cells and IL-21 produced by follicular T helper (Tfh) cells contributed to B lymphocyte survival and proliferation as well as generated higher affinity to the IgG4 antibody [44]. Moreover, IL-4, IL-13, and IL-10 could promote the conversion of antibody category to IgG4 [44]. In addition, decreased levels of Th1 cells and regulatory T (Treg) cells secondary to SARS-CoV-2 infection destroyed immune tolerance [45].

Massive Release of Cytokines
In COVID-19, viral infection in pulmonary epithelial cells triggered the recruitment of immune effector cells and released massive proinflammatory cytokines and chemokines [41,42], which subsequently advanced T lymphocyte differentiation. T helper (Th) 17 cells generated interleukin (IL)-17A, IL-17F, IL-22, and granulocyte-macrophage colony-stimulating factor, inducing the aggregation of inflammatory cells, such as neutrophils [43]. IL-4 produced by Th2 cells and IL-21 produced by follicular T helper (Tfh) cells contributed to B lymphocyte survival and proliferation as well as generated higher affinity to the IgG4 antibody [44]. Moreover, IL-4, IL-13, and IL-10 could promote the conversion of antibody category to IgG4 [44]. In addition, decreased levels of Th1 cells and regulatory T (Treg) cells secondary to SARS-CoV-2 infection destroyed immune tolerance [45].
In addition to acting as receptors, ACE2 also presented critical functions as a counterregulatory enzyme to convert angiotensin II (Ang II) into Ang-(1-7), the latter of which performed attenuating inflammation effects [46]. Virus-occupied ACE2 might weaken their intrinsic function, which could enhance inflammatory response, neutrophils accumulation, and vascular permeability, and ultimately result in influenza-like symptoms, even severe acute respiratory distress syndrome among SARS-CoV-2 infected individuals [47], whereas in the kidneys, elevated immune response made it easier to develop glomerular diseases, such as MN (Figure 1).

Speculation about PLA2R Antigen
Accumulating evidence has demonstrated that PLA2R, a pathogenic antigen of MN, was expressed not only in airway epithelial cells [48], neutrophils [49], and pulmonary macrophages [50], but also in podocytes. Once activated by foreign antigens such as SARS-CoV-2, these cells could secret extracellular vesicles containing PLA2R or cause the spatial release of PLA2R by generating extracellular traps, subsequently stimulating B lymphocytes to produce PLA2R-Ab [51]. In addition, the oxidation environment induced by inflammatory cytokines could bring about long-term expression of PLA2R pathogenic epitopes and enhance the capacity of binding to circulating antibodies [51] (Figure 1).

Activation of the Complement System
The previous literature reported that the concentration of anti-SARS-CoV-2 immunoglobulin lacking glycan fucosylation was elevated in COVID-19 patients [52], which could help mannose-binding lectin to combine with aberrant glycans, thereby activating the complement system. The formed C5b-9 membrane attack complex on podocyte membranes participated in mediating the proteolysis of podocyte synaptophysin and NEPH1, resulting in the destruction of podocyte cytoskeleton [53,54] and eventually proteinuria ( Figure 1).

Elevated Expression of Human Leukocyte Antigen
Human leukocyte antigen (HLA) was expressed on the surface of immune cells and acted as presenters of epitopes to CD4 + T cells [55], indicating potent immunoregulatory properties. The present study demonstrated that activation of HLA-DR in circulating monocytes increased instantaneously in patients with SARS-CoV-2 infection [56], which could promote antigenic epitopes presentation to T lymphocytes and destroy immune tolerance (Figure 1).

Cross Immune Response
Some scholars have proven that amino acid sequence similarity between hepatitis B virus surface antigen and multiple sclerosis (MS) autoantigens might be part of the account of MS secondary to hepatitis B vaccination [57]. Recently, Vojdani et al. conducted an investigation aimed at studying the relationship between autoimmune target proteins and SARS-CoV-2 spike protein antibodies, the results of which proved that there were multiple tissue antigens that showed powerful reactions with the SARS-CoV-2 antibodies, such as transglutaminase 3, anti-extractable nuclear antigen, thyroid peroxidase, etc. [58], which indicated the fundamental role of cross-immune response in autoimmune diseases. Therefore, we proposed an underlying mechanism that podocyte surface-specific antigens might share similar amino acid sequences with spike protein or other components of SARS-CoV-2, which would be further supported along with the discovery of more pathogenic antigens on the surface of podocytes (Figure 2).

Subepithelial Deposition of Circulating Immune Complexes
Proverbially, intramuscular vaccine components served as foreign substances to evoke the host's immune response. As antigens, the vaccines promoted subepithelial deposition of circulating immune complexes in renal tissue via combining with native antibodies in vivo, which was plausible for explaining the MN secondary to influenza vaccines [59] (Figure 2).

Enhanced Immune Response
Anti-SARS-CoV-2 neutralizing antibodies were of particular significance in evaluating protective immunity. For 250 patients with past-COVID-19, all moderate-severe patients and more than 80% of mild patients had positive antibodies [60]. Undeniably, vaccination was indeed an important initiative to enhance immune response. Compared with other types of COVID-19 vaccines, mRNA vaccines have been revealed to induce a more potent immune response. The rate of seroconversion was observed to increase five-fold from the baseline after the first dose of mRNA vaccines at two weeks [60]. Two doses of vaccines effectively induced antibody titers to exceed 300 U/mL and without evident decrease at 2 months [60]. Vaccines strengthened virus-specific responses and effectively activated T and B lymphocytes, accompanied by elevated generation of T cell inflammatory cytokines (e.g., interferon γ, tumor necrosis factor α, and IL-2, etc.) and higher levels of

Subepithelial Deposition of Circulating Immune Complexes
Proverbially, intramuscular vaccine components served as foreign substances to evoke the host's immune response. As antigens, the vaccines promoted subepithelial deposition of circulating immune complexes in renal tissue via combining with native antibodies in vivo, which was plausible for explaining the MN secondary to influenza vaccines [59] (Figure 2).

Enhanced Immune Response
Anti-SARS-CoV-2 neutralizing antibodies were of particular significance in evaluating protective immunity. For 250 patients with past-COVID-19, all moderate-severe patients and more than 80% of mild patients had positive antibodies [60]. Undeniably, vaccination was indeed an important initiative to enhance immune response. Compared with other types of COVID-19 vaccines, mRNA vaccines have been revealed to induce a more potent immune response. The rate of seroconversion was observed to increase five-fold from the baseline after the first dose of mRNA vaccines at two weeks [60]. Two doses of vaccines effectively induced antibody titers to exceed 300 U/mL and without evident decrease at 2 months [60]. Vaccines strengthened virus-specific responses and effectively activated T and B lymphocytes, accompanied by elevated generation of T cell inflammatory cytokines (e.g., interferon γ, tumor necrosis factor α, and IL-2, etc.) and higher levels of antibody titers, especially two doses of vaccines, including elderly individuals [61,62]. Due to the dose-dependent characteristics of COVID-19 vaccines, most adverse immune events typically occurred post the second dose [63] (Figure 2), which was consistent with our result. According to the clinical records we collected, only four new-onset patients had available data on antibody titers (case 2, 147.0 U/mL; case5, 32.8 U/mL; case19, 2334 U/mL; case 20, 1500 U/mL, Table 2). Consequently, further studies regarding the detection of antibody titers were required, which might contribute to clarifying the correlation between antibody level and disease onset.

Adjuvants
The application of adjuvants achieved the possibility that small doses of vaccines could stimulate individuals to generate sufficient antibodies. Adjuvants, as antigens, could provide pathogen-associated molecular patterns and be recognized by toll-like receptors on the surface of antigen-presenting cells, activating downstream inflammatory signaling pathways and inducing an enhanced immune response [64]. Inactivated vaccines had decreased immunogenicity and usually required adjuvants, especially in elderly individuals considering immune senescence [65] (Figure 2).

Limitations
This article had several limitations. Most clinical cases were reported from single-case studies, and further research was needed to verify the causal relationship between MN and SARS-CoV-2 infection and COVID-19 vaccination. Moreover, there was the possibility that multiple cases were not reported, which might perplex us concerning the clinical characteristics and treatment response of MN associated with SARS-CoV-2 infection and COVID-19 vaccination. Furthermore, the pathogenesis involved in this review was based on our hypothesis, which demanded additional verification in the future. In addition, insufficient clinical information might lead to errors in data analysis.

Conclusions
In the era of the COVID-19 pandemic and the widespread requirement for vaccines, MN was an unavoidable but uncommon disease. Edema and proteinuria were the leading clinical manifestations. Overall, most cases had a good prognosis. Conservative and conventional immunosuppressive therapies with or without RTX promoted disease remission. Of note, it was not emphasized enough on the detection of antibody titers in currently published cases of MN post COVID-19 vaccination, which would be meaningful for assessing the potential association between antibody levels and disease onset. In addition, whether routine urine testing after vaccination contributed to the timely detection of the disease deserved more attention. In conclusion, further exploration was urgently needed to advance our knowledge of the incidence and recurrence rate, pathogenesis, treatment, and prognosis of MN post SARS-CoV-2 infection and COVID-19 vaccination.
Author Contributions: Q.M. conducted data collection and wrote the manuscript. X.L. conducted data analysis. G.X. was responsible for the idea, funds, and paper revision. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.