Introduction
Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerulonephritis in the world [
1]. As the leading cause of end-stage renal disease (ESRD), approximately 10–60% of patients with IgAN progress to kidney failure within 10–20 years [
1,
2]. Mesangial cell proliferation and IgA1-predominant mesangial deposits are pathological hallmarks of IgAN. Previous studies have suggested that podocyte injury also occurs in IgAN and is associated with the pathogenesis of Gd-IgA1. Podocyte injury is usually associated with significant proteinuria, manifested as foot process effacement, which is considered to be a key factor leading to progression and poor prognosis in IgAN [
3‐
5].
Podocytes are terminally differentiated and have a poor proliferative capacity. Mitochondrial dysfunction is a major contributor to podocyte injury and death [
6,
7]. Various mitochondrial dysfunction pathways have been identified as the main molecular mechanisms of podocyte injury, such as elevated mitochondrial ROS production [
8], imbalanced mitochondrial dynamics [
9], and decreased mitochondrial biogenesis [
10,
11].
Mitofusin2 (Mfn2) was initially identified as a dynamin-like protein involved in fusion of the outer mitochondrial membrane (OMM) that participates in mitochondrial fusion and contributes to the maintenance of the mitochondrial network [
12]. Moreover, Mfn2 is involved in the clearance of damaged mitochondria, serves as a mitochondrial receptor for Parkin (E3 ubiquitin ligase), and facilitates the recruitment of Parkin to the impaired mitochondria, which participates in mitophagy [
13‐
15]. Our previous study showed that Mfn2 deficiency participates in podocyte injury in a focal segmental glomerulosclerosis (FSGS) animal model by inhibiting Pink1/Parkin-associated mitophagy. In diabetic kidney disease (DKD), Cao et al. [
16] reported that Mfn2 regulates the morphology and functions of mitochondria-associated ER membranes (MAMs) and mitochondria by inhibiting the PERK pathway and exerts anti-apoptotic effects on podocytes. Whether Mfn2 participates in podocyte injury and is related to clinical and pathological characteristics in IgA nephropathy has not been reported to date. Therefore, this study aimed to explore the relationship between Mfn2 expression and podocyte injury and further elucidate its potential predictive value for IgAN prognosis.
Methods
Patients
Patients aged ≥ 18 years with biopsy-proven IgAN in the Hangzhou Hospital of Traditional Chinese Medicine between April 2022 and August 2022 were enrolled, and secondary causes of IgAN, such as liver or inflammatory bowel diseases, other autoimmune disorders, infections, and Henoch–Schönlein purpura, were excluded. Clinical data, including sex, age, proteinuria, serum creatinine (Scr), blood urea nitrogen (BUN), serum albumin (ALB), blood pressure, and serum IgA, were collected at the time of biopsy.
Histopathology
Renal histological lesions were graded based on the MEST-C score [
17]. M0/M1 was defined as ≤ / > 50% of glomeruli exhibiting mesangial hypercellularity, E0/E1 as the absence/presence of endocapillary hypercellularity, S0/S1 as the absence/presence of segmental glomerulosclerosis, T0/T1/T2 as tubular atrophy/interstitial fibrosis ≤ 25–50% > 50%, and C0/C1/C2 as absence/ < 25%/ ≥ 25% of crescent lesions.
The immunofluorescence samples were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies specific for human IgG, IgM, IgA, C3, C4, and C1q (1:50, DAKO, Glostrup,Denmark). The degree of immunofluorescence was scored on a scale of 0–4 (score 0, negative; score 1, + ; score 2, + + ; score 3, + + + ; score 4, + + + +).
Immunofluorescence staining
Frozen tissues were embedded in OCT, cut into 5 μm sections, and then stored at – 20 ℃. Rabbit anti-human Mfn2 antibody (1:100; Cat No. M6319, Sigma-Aldrich), rabbit anti-human nephrin monoclonal antibody (1:100; ab50339, Abcam), rat anti-human collagen IV alpha 5 monoclonal antibody (1:100; C-452, Cosmo Bio), rabbit anti-human Parkin antibody(1:100; 14060-1-AP, Proteintech) were reacted with renal tissue at 4 °C overnight. AF488-conjugated donkey anti-rabbit IgG antibody (1:500; A-21206, Invitrogen), and FITC conjugated donkey anti-rat IgG antibody (1:500, A-18740, Invitrogen) were incubated for half an hour at 37 ℃. Sections were observed using a fluorescence microscope (Nikon 80i; Nikon, Tokyo, Japan).
Electron microscopy
The renal biopsy specimens were fixed with osmic acid and glutaraldehyde, dehydrated, and embedded in EPON™ resin. Sections with a thickness of 1 mm were cut and stained with uranyl acetate and lead citrate. Thin sections were examined using a JEOL-1400 electron microscope (JEOL, Tokyo, Japan).
The degree of podocyte effacement was graded on a scale of 1–5 with 1 = podocyte effacement < 25%, 2 = 25–50%, 3 = 50–75%, 4 = 75–95%, and 5 = ≥ 95%.
The number of mitochondria (M) and the area of podocytes (A) were assessed using ImageJ software. The ration M/A was used to evaluate the number of mitochondria per area and the length-to-width ratio of mitochondria was used to evaluate mitochondrial morphology. All pathological parameters were assessed and measured by two independent pathologists.
Apoptosis assay
Tissue sections were stained with an In Situ Cell Death Detection Kit, Fluorescein, (Roche Cat No. 11684795910), and the slices were exposed to freshly prepared permeabilization solution for 2 min on ice (0.1% Triton X-100, 0.1% sodium citrate). After washing with PBS, the samples were resuspended in 50 µL of TUNEL (terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) reaction mixture and incubated for 60 min in a dark, humidified environment. The samples were then washed with PBS, and the slides were examined using a fluorescence microscope (Nikon 80i; Nikon, Tokyo, Japan).
Statistical analysis
Statistical analyses were performed using SPSS (version 23.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). Fisher's exact test or the χ2 test was used to compare qualitative data, and the Wilcoxon rank-sum test was used for continuous variables. Spearman’s correlation test was used to assess the strength of the association between Mfn2 levels and clinical or pathological variables. Logistic regression analysis was performed to ascertain the relationship between Mfn2 expression and podocyte effacement. Statistical significance was set at P < 0.05.
Discussion
In our study, we found that nearly 75.44% of patients with IgAN had Mfn2 expression in the kidney. Through immunofluorescence co-staining of Mfn2 with Col IV α5 and nephrin, we found that Mfn2 was mainly expressed in podocytes, and the intensity of Mfn2 decreased consistently with nephrin expression. TUNEL staining revealed an increase in podocyte apoptosis in the Mfn2-negative group. These findings indicate that Mfn2 expression is associated with podocyte injury in IgAN patients. In patients with DKD, Cao et al. similarly found a dramatic reduction in Mfn2 expression in DKD patients compared to the expression in healthy individuals, along with increased podocyte apoptosis, as detected by TUNEL and decreased synaptopodin expression [
16].
As a mitochondrial membrane protein, Mfn2 participates in mitochondrial fusion and contributes to the maintenance and functioning of the mitochondrial network [
20]. Additionally, Mfn2 is also involved in the regulation of mitophagy [
13]. Mfn2 deletion has been reported to suppress mitophagy in mouse embryonic fibroblasts, cardiomyocytes, and macrophages. [
13‐
15] Cao et al. reported that high glucose (HG)-induced podocyte mitochondrial dysfunction, MAMs reduction, and increased apoptosis in vitro were accompanied by downregulation of Mfn2 [
15]. Jiang et al. found that palmitic acid (PA)-induced podocyte injury was accompanied by downregulation of Mfn2 and inhibition of mitophagy [
21]. Our previous in vitro and animal model studies have indicated that overexpression of Mfn2 can inhibit puromycin aminonucleoside (PAN)-induced podocyte apoptosis. In the present study, we made similar findings: patients lacking Mfn2 expression had more serious podocyte injury and mitophagy. Electron microscopy showed that the degree of podocyte effacement was more pronounced in the Mfn2-negative group. By observing the number and the morphological structure of mitochondria in podocytes, we discovered that, compared with the positive group, the number of mitochondria per unit area was higher in the Mfn2-negative group, while the length-to-width ratio of mitochondria was smaller, indicating that there were more damaged mitochondria in podocytes in the Mfn2-negative group. Lack of Mfn2 inhibits mitophagy, which results in the accumulation of damaged mitochondria and excessive ROS production in cells, ultimately promoting podocyte apoptosis, which causes podocyte effacement and impairs the selective filtration of GBM, thereby leading to proteinuria.
Podocyte injury plays an important role in IgAN. Podocyte dedifferentiation is associated with IgAN [
3,
22,
23]. Podocytopenia (loss of podocytes) has been reported to correlate with proteinuria and renal outcomes in IgAN patients [
24,
25]. Moreover, mitochondria play a key role in maintaining the function and structure of podocytes. In our study, we found that 75% of IgAN were Mfn2-positive and 24% were negative. The negative patients had higher levels of proteinuria and Scr, higher S/T scores, and a higher degree of podocyte effacement and M/A value, indicating that Mfn2 expression negatively correlates with the severity of IgAN. Furthermore, TUNEL staining demonstrated that negative Mfn2 expression was associated with severe podocyte apoptosis, and the expression of Mfn2 was an independent risk factor for the severity of podocyte effacement, thus indicating that a reduced Mfn2 results in inhibition of mitophagy and the accumulation of damaged mitochondria and excessive ROS production in the cytoplasm. Ultimately, it promotes podocyte apoptosis and exacerbates IgAN progression.
Conclusion
In IgAN, Mfn2 is mainly expressed in podocytes and is negatively correlated with proteinuria and renal function. A lack of Mfn2 in podocytes indicates severe podocyte injury and a high degree of podocyte effacement. Thus, Mfn2 could be a useful indicator of the severity and poor prognosis of IgAN.
This article still has several shortcomings. Firstly, it is a single-center study with a limited sample size. Additionally, due to a short-term follow-up, the treatment and outcome data have not been collected. Secondly, this article does not include a study of other mitochondrial morphology-related proteins such as mitofusin 1 (Mfn1), optic atrophy 1 (OPA1), and dynamin-related protein 1 (Drp1).
Further studies are necessary to enhance the understanding of the correlation between Mfn2 and the outcome in IgAN patients, as well as to investigate other mitochondrial fusion and fission proteins.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.