Introduction:
Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults, characterized by the deposition of immune complexes along the glomerular basement membrane. The traditional classification of membranous nephropathy (MN) was primarily based on clinical and histopathological features, categorizing the disease as either primary (idiopathic) or secondary to underlying conditions such as infections or autoimmune diseases. However, recent advances in understanding MN have led to a novel classification system centered around the identification of specific podocyte antigens like PLA2R, THSD7A, and NELL1, which allows for more precise disease categorization and tailored therapeutic approaches. This antigen-based classification has revolutionized the diagnosis and management of MN, offering insights into disease prognosis and guiding targeted treatments.
This shift mirrors the evolution seen in the classification of focal segmental glomerulosclerosis, where emphasis has moved from light microscopic patterns to detailed pathogenic and genetic studies, highlighting specific pathogenic pathways.
NELL1 is emerging as a novel antigen in membranous nephropathy, distinguished by its unique association with specific clinical and pathological features that differ from other known antigens like PLA2R and THSD7A. While preliminary findings suggest that NELL1-related membranous nephropathy may present with distinct disease characteristics, comprehensive studies are necessary to fully elucidate its role in disease pathogenesis and patient outcomes.
Recent literature on NELL-1-associated membranous nephropathy has elucidated its unique clinical and pathological spectrum. Studies have highlighted the specific staining patterns and etiological profiles associated with NELL-1, underscoring its role as a significant marker in the subset classification of MN. Further research will help to clarify the diagnostic and therapeutic implications of NELL1, potentially leading to more personalized treatment strategies in nephrology.
There is an unmet need to feed global data on this subject from different geographical locations and ethnicities to uncover the local attributes of NELL-1 pathogenesis as well.
This case series provides valuable insights into the diverse clinical presentations and outcomes associated with NELL1-MN, underscoring the complexity and variability of this condition.
Methods:
Study Design and Population
This is a 3-year retrospective study with patients from Government Medical College Srinagar, and Shifa Medical Centre Srinagar. Both of these hospitals are located in Srinagar, the union territory of Jammu and Kashmir, India. Membranous nephropathy was diagnosed based on a Kidney biopsy. Both hospitals mentioned above send their kidney biopsy samples to Lal Path Labs in New Delhi for analysis. Lal Path Labs, a renowned diagnostic centre, handles the biopsy processing and provides expert pathology reports.
Light microscopy and immunofluorescence were conducted in all cases. However, electron microscopy, which significantly increases the cost of the procedure, was only performed when necessary or when the patient could afford it, as most of these patients were not covered by insurance.
The antigen staining protocol followed by Lal Path Laboratories involves an initial staining of all kidney biopsy samples with the PLA2R antibody. If the PLA2R staining is negative, the samples are tested with the THSD7A antibody. For cases where both PLA2R and THSD7A stains are negative, the laboratory proceeds to stain the samples with the NELL1 immunohistochemical marker. Our study specifically included those cases that were positive for NELL1 staining, while being negative for both PLA2R and THSD7A
All biopsies were meticulously reviewed by an experienced renal pathologist at Lal Path Labs. The immunofluorescence findings were carefully documented, with the intensity of staining for IgG, IgM, IgA, C3, and C1q evaluated on a semiquantitative scale ranging from 1 to 4. In addition to the intensity, the specific location and pattern of the staining were also noted. Immunohistochemical staining was assessed based on the presence or absence of the stain, as well as its distribution pattern, to provide a comprehensive analysis of the biopsy samples.
Definitions
Membranous glomerulonephritis was classified as primary or secondary according to the clinical context, depending on whether the disease is renal limited or has systemic causes.
Microscopic hematuria was defined as at least 5 red cells per high-power field on microscopic examination or positive blood by urine dipstick. Nephrotic syndrome was defined as nephrotic range proteinuria > 3.5 g per 24 hours per 1.73 m2 (in children, >40 mg/m2 /hr or PCR >2000 mg/g [>200 mg/ mmol]) along with hypoalbuminemia and edema4. Advanced renal failure at the presentation of illness was defined as dialysis-dependent renal failure or eGFR < 15 ml/min.
Data source and variables
Clinical and demographic data, laboratory data, and treatment received by the patients were recorded from the patient files. Records were checked for any malignancy, autoimmune disease, or infectious disease either at the time of renal biopsy or within three years of the same. Medication history within three months of the disease was also noted.
Treatment, Follow-up and outcome
The treatment received by the patients in the form of immunosuppression was recorded.
For proteinuria, spot urine protein-to-creatinine ratio or 24-hour urine protein was analyzed interchangeably.
Estimated GFR (eGFR) was calculated using the 2021 Chronic Kidney Disease-Epidemiology Collaboration Creatinine equation. Outcomes studied were complete remission, partial remission, chronic kidney disease, end stage renal disease (ESRD) and death. Complete remission (CR) was defined as the return of serum creatinine to the previous baseline, plus the reduction in proteinuria to <0.5 g/day or 0.5 g/g creatinine by urinary proteinuria: creatinine ratio (uPCR). Partial remission (PR) was defined as stable (±25%) or improved serum creatinine, but not to normal, plus ≥50% reduction in proteinuria to <3 g/day (or 3 g/g uPCR)3. Chronic kidney disease (CKD) was defined as kidney damage or glomerular filtration rate (GFR) <60 mL/min/1.73 m2 for 3 months or more. eGFR was calculated using the modified CKD-EPI equation4. ESRD was defined as a decrement in the patient's kidney function to a level at which either long-term dialysis or kidney transplantation is required to sustain life5.
Remission was classified as either spontaneous, induced by immunosuppressive agents, by treatment of the associated condition or withdrawal of the offending drug.
Results:
Total of 8 patients were studied, out of which 4 were females. Median age at diagnosis was 48 years (range, 26–65). The patients with associated malignancies were relatively older than rest of the cases. The mean age of these patients range from 49 years8 to 63.1 years1 in previously described studies. These cases have been said to have a higher mean age of presentation as compared to PLA2R cases9. Although most of the studies showed an almost an equal male/female ratio1, similar to our cases, Wang etal noted a female predominance of these cases8. At the time of biopsy, median proteinuria was 4.9 (range 2.46g–6.80 g), with 6 (75%) having ≥3.5 g; 6 patients (75%) had an eGFR <60 ml/min per 1.73 m2 . At the time of biopsy, median eGFR was 34 (range 15–127 ml/min per 1.73 m2)
Case wise details of each case are shown in Table1.
CASE 1: A 55-year-old male bicycle mechanic presented to the nephrology outpatient department with complaints of nausea and edema in his feet and legs. He was normotensive, non-diabetic, and had no history of drug intake or recreational drug use. However, he had a significant history of chronic smoking (90 pack years). Upon evaluation, he was found to have normocytic anemia and renal dysfunction, with a serum creatinine level of 4.3 mg/dl and 24-hour urinary protein of 4 g/24 hrs. His serum albumin was low at 2.5 mg/dl.
With a syndromic diagnosis of nephrotic syndrome, a kidney biopsy was performed, revealing thickening and stiffening of the glomerular basement membrane with mild mesangial proliferation. Immunofluorescence showed fine granular positivity predominantly for IgG1, segmentally located in the subepithelial region. Electron microscopy confirmed segmental subepithelial immune deposits. Immunohistochemistry was positive for NELL-1 stain.
A comprehensive investigation panel was conducted to rule out any secondary causes. Serologies for Hepatitis B and C, as well as HIV, were negative. Initially, a chest X-ray was normal, but a non-contrast CT scan revealed a lesion in the left upper lobe of the lung, which biopsy confirmed as squamous cell carcinoma. The patient underwent chemotherapy and radiotherapy for the lung malignancy. However, he dropped out of treatment due to financial constraints and was hospitalized multiple times with superadded bacterial lung infections.
At the last follow-up, there was a decrease in proteinuria and serum creatinine, and the patient had achieved partial remission.
CASE 2: A 26-year-old female, a 3rd-year medical student pursuing her MBBS in Tajikistan, returned home to Kashmir, India, after developing edema in both her feet and legs. She also experienced periorbital puffiness, especially in the mornings upon waking. Two years ago, during her 1st year of medical school in Tajikistan, she had a self-limiting illness characterized by polyarthralgias and an urticarial rash, with high anti-streptolysin O antibody titers. She received steroids for three months, and the condition resolved without recurrence.
In recent months, the patient had been suffering from chronic back pain. An MRI of the lumbosacral spine revealed a diffuse disc bulge at L4-L5, causing thecal sac compression without significant lateral recess narrowing. For her back pain, she was prescribed Neurolife tablets, containing methylcobalamin (1500 mcg), folic acid (1.5 mg), alpha-lipoic acid (100 mg), pyridoxine (3 mg), and vitamin D3 (1000 U). After taking 25 tablets, amounting to a cumulative dose of 2500 mg of alpha-lipoic acid, she developed lower limb edema and facial puffiness, leading to discontinuation of the medication.
Evaluation revealed she had nephrotic syndrome (24-hour urinary protein: 5.4 g/d, serum albumin: 2.5 g/dl) and mild renal dysfunction (serum creatinine: 0.59 mg/dl). She had no history of NSAID or other medication use. Hepatitis and HIV serologies were negative, ANA was normal, and serum complements were within normal limits. A kidney biopsy showed thickening and stiffening of the glomeruli with mild mesangial proliferation. Immunofluorescence revealed fine granular positivity predominantly for IgG1, segmental and located in the subepithelial region. Immunohistochemistry was positive for NELL-1 stain.
Alpha-lipoic acid was discontinued, and she was not given any immunosuppression. On follow-up, her proteinuria decreased, and she remains in complete remission.
CASE 3: A 65 year old male presented with nephrotic syndrome, mild renal dysfunction and moderate anemia. Kidney biopsy was done which showed membranous nephropathy along a focal segmental glomerulosclerosis in one of the glomerulus. Immunofluorescence showed granular deposits of IgG1 and complement C3 along the capillary wall. On investigating the patient had prostatic carcinoma, Gleasons grade group 2. He underwent treatment for malignancy along with immunosupression. The combination of steroid therapy (prednisone 30 mg/d) and tacrolimus(2 mg/d) was prescribed. Low dose tacrolimus was given due to renal dysfunction at presentation and due to the severe nephrosis portending an additional thrombotic risk in addition to the underlying malignancy. The patient is on follow-up and has achieved complete remission.
CASE 4: Our patient 4 was a 48 year old female who presented with nephrotic syndrome. Her kidney biopsy showed pure membranous picture with co-dominant, segmental staining for IgG1 and IgG4, and positive NELL-1 on immunohistochemistry. Electron microscopy showed segmental subepithelial electron dense immune deposits. On thorough search no secondary cause was found. She was kept on immunosupression and after 17 months of follow-up she was in partial remission.
CASE 5: This was a 51 year old female presenting with nephrotic syndrome and moderate renal dysfunction. Rest all other investigations were normal. She was also not on any medication. Her kidney biopsy showed membranous nephropathy. There were no secondary features on biopsy. Immunofluorescence showed fine granular positivity predominantly for IgG1 in the subepithelial location which was diffuse in nature. Immunohistochemistry was negative for PLA2R and TSHDA and was positive for NELL-1. Workup done couldnt identify any primary cause. In view of nephrotic syndrome with moderate renal dysfunction, this patient recieved modified ponticelli regimen and achieved partial remission.
CASE 6: A 45 year old male presented with proteinuria and mild renal dysfunction. His renal biopsy showed NELL-1 associated membranous nephropathy. Immunofluorescence showed segmental positivity predominantly for IgG1. No secondary cause could be elucidated even after thorough screening. This patient was given angiotenin receptor blocker therapy (telmisartan 40 mg / day) and is in complete remission.
CASE 7: This was a 48 year old male presenting with proteinuria and moderate renal dysfunction. He had a history of chronic knee pain (osteoarthritis) for which he was taking herbal medication. His renal biopsy showed membranous picture along with diffuse, subepithelial fine granular codominant IgG1 and IgG2 deposits on immunofluorescence. Immunohistochemistry showed NELL-1 positivity. The patient was advised to stop taking the herbal medicines and prescribed steroids. Steroids were given to tackle both pain in the knees and ensure early recovery of renal dysfunction. He followed after 7 months and was in complete remission.
CASE 8: This 32 years female who works in dubai, UAE developed nephrotic syndrome in 2023 and kidney biopsy done showed NELL-1 membranous nephropathy which was attributed to high blood mercury levels (34.90 µg/L, with upper limit of normal being < 5.0 µg/L). Her facial creams were stopped but the nephrotic syndrome persisted and she developed anasarca. She was give 3 pulses of iv methylprednisolone and the kept on oral steroids. She was given two doses of intravenous rituximab 1 g each , 15 days apart in february 2024. With no improvement tacrolimus was also added which didnt do any better, meanwhile triple immunosupression was prescribed again with no improvement even after 3 months of treatment. She consulted in srinagar , jammu and kashmir, india with no improvement. Upon reviewing she was found to again have high blood mercury levels (73 µg/L), inspite of stopping her skin whitening cream. Her brother living with her in dubai was also tested for mercury levels thinking about common source of mercury, but the level was normal (1.51 µg/L). The persistent mercury antigenemia was found to be the the cause for her ongoing treatment resistant membranous nephropathy. Since the immunoupression would only decrease the level of inflammation at the glomeruli level with no effect at the blood mercury level, the immunosupression was not able to control the disease. She was advised to stop all skin creams and body sprays. After doing so she achieved complete remission as on august 2024
Discussion
NELL1-associated membranous nephropathy (NELL1-MN) has emerged as a distinct clinical and pathological entity within the spectrum of membranous nephropathy (MN).
Various comparison of our results with other remarkable studies are listed in table 2.
In our study, eight patients were diagnosed with NELL1-MN, with a median age of 48 years at diagnosis, younger than some other studies (Caza et al. and Tesar et al. reported older median ages). Our study reported a balanced male/female ratio, while Wang et al. noted a female predominance. The predominance of proteinuria and renal dysfunction at presentation in our study is consistent with the characteristics of MN. Median proteinuria in our study was 4.9 g/day, with 75% of patients exhibiting nephrotic-range proteinuria (≥3.5 g/day); consistent with high levels of proteinuria seen in other studies like Sato et al. A significant proportion of patients (75%) had an eGFR of less than 60 ml/min per 1.73 m², aligning with findings from Tesar et al. and Caza et al.
The hallmark histopathological features of NELL1-associated membranous nephropathy (NELL1-MN) in our cohort include notable thickening of the glomerular basement membrane, accompanied by mild mesangial proliferation. Immunofluorescence predominantly revealed fine granular IgG1 staining, which was a consistent to findings by Caza et al., Sethi et al., and Wang et al. Importantly, immunohistochemistry confirmed the diagnosis by demonstrating positive NELL1 staining in every instance. Electron microscopy further supported these findings, revealing segmental subepithelial immune deposits that align with previously documented descriptions of NELL1-MN. The segmental glomerular capillary membrane staining observed on immunofluorescence (in five out of eight patients) closely corresponds with the findings of other studies. Notably, Caza et al. highlighted the distinct immunofluorescence staining patterns among various types of membranous nephropathy. Their study found that 48.3% of NELL1-MN cases exhibited segmental staining of the glomerular capillary loops, a feature not observed in cases of PLA2R-, THSD7A-, or EXT1/EXT2-positive MN. This difference is further emphasized by the IgG subclass distribution, with a predominance of IgG1 staining in NELL1-MN, a pattern that has been consistently reported in the literature. Therefore, in the diagnostic evaluation of membranous nephropathy, the presence of segmental capillary membrane staining on immunofluorescence, particularly with a predominant IgG1 subclass, should prompt consideration of NELL1-MN as a differential diagnosis, distinguishing it from other MN subtypes such as those associated with PLA2R, THSD7A, or EXT1/EXT2.
The association between NELL1-MN and malignancies, as observed in two of our cases (squamous cell carcinoma and prostatic carcinoma), supports the notion that NELL1-MN may be a paraneoplastic syndrome. The presence of NELL1 as a potential target antigen in these cases suggests a link between tumor antigens and the development of MN, reinforcing the need for thorough malignancy screening in patients diagnosed with NELL1-MN . In both of our patients with associated malignancies, the diagnosis of malignancy was made upon screening for associated tumors after diagnosis of NELL-1 MN. One patient had squamous cell carcinoma of the lung and another one had advanced prostatic carcinoma with bony metastasis at diagnosis. Initially in the pilot study by Sethi et al2, tumors were not detected in any of the cases. Also in a study conducted by Wang etal, tumors were not detected in any of the 15 cases with NELL-1 MN at the time of diagnosis8. However as more studies came in light it was seen that NELL1-associated MN is commonly associated with malignancy2. Infact in a recent study by Caza etal9, in terms of prevalence, NELL-1 was the most common antigen associated with malignancy in MN (33% as compared to 11% in THSD7A and only 4% for PLA2R). It is impressed upon that all cases with NELL-1 positive MN should be thoroughly screened for associated malignancies and even followed up for the same.
Attention must be exercised when considering the use of over-the-counter medicines, as they may pose significant risks that outweigh potential benefits. For instance, one patient, a young female, had been consuming alpha-lipoic acid for six months prior to presentation. Another case involved a 48-year-old male who had been taking Ayurvedic preparations for osteoarthritis. Both patients achieved complete remission following the cessation of the implicated therapies, supplemented by supportive care.
Interestingly, one patient’s condition was attributed to high blood mercury levels, possibly from the use of skin-whitening creams. The persistence of high mercury levels despite cessation of cream use was thought to be the reason for her treatment resistant nephrotic syndrome. This scenario highlights the intricate challenges in managing secondary causes in NELL1-associated membranous nephropathy (NELL1-MN). While immunosuppressive therapy may address the autoimmune aspects of membranous nephropathy, its efficacy can be undermined by ongoing antigen exposure. The continued presence of the primary antigen impedes effective disease control, suggesting that in cases of treatment resistance, it is crucial to reassess potential antigen exposures before classifying the disease as refractory. This case also raises the possibility of environmental or exogenous factors playing a role in the pathogenesis of NELL1-MN, a hypothesis that merits further exploration.
The treatment outcomes were mostly favourable but with adoption of varied approaches, reflecting the challenges in managing NELL1-MN. The remission rate in our study was high, with 5 complete remissions (CR) and 3 partial remissions (PR), which is comparable to the remission outcomes in Caza et al. and Tesar et al. Drug-induced cases in your study had favorable outcomes upon withdrawal of the offending agents. In these cases stopping the offending drug will do the trick and prolonged immunosuppression can be avoided in these cases. Patients with malignancy-associated NELL1-MN showed partial or complete remission following cancer treatment and immunosuppressive therapy, suggesting that addressing the underlying malignancy is crucial for disease control. However, the case with persistent high blood mercury levels highlights that in some instances, removing the source of antigenemia is essential for achieving remission, beyond conventional immunosuppressive strategies .
There were several limitations of this study. First and foremost is the limited number of cases available. Secondly, there have been reports of dual PLA2R and NELL-1 positive MN8. We applied NELL-1 staining only on PLA2R negative cases and as such the dual positive cases may have been missed. Third is the retrospective nature of the study with its associated limitations. Nevertheless, it is a first such study from this region and it may be a small step to the long journey of exploring this upcoming disease with fair understanding of the interplay of key local factors.
Conclusions:
This case series adds to the growing body of evidence that NELL1-MN is a heterogeneous condition with diverse clinical and pathological features. The strong association with malignancies and other secondary causes highlights the need for comprehensive evaluation in these patients. While immunosuppressive therapy remains a cornerstone of treatment, addressing underlying etiologies is crucial for optimal outcomes. Further studies are needed to elucidate the pathophysiological mechanisms underlying NELL1-MN and to develop targeted therapeutic strategies for this unique subset of MN.
I have no potential conflict of interest to disclose.
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