, Introduction
Kidney serves as the main excretory organ of circulating oxalate. Deposition of oxalate crystals in the kidney parenchyma may lead to a condition known as oxalate nephropathy, which may clinically manifest as acute kidney injury (AKI), acute kidney disease (AKD), and/or chronic kidney disease (CKD) [1]. The prognosis of oxalate nephropathy is generally poor, with nearly half of patients requiring renal replacement therapy because of a lack of proven efficient therapy [2]. Recently, Waikar et al. [3] demonstrated that higher versus lower 24-hour urinary oxalate excretion was independently associated with a 32% increased risk of CKD progression and a 37% higher risk of kidney failure in the CRIC (Chronic Renal Insufficiency Cohort) study, highlighting the role of oxalate nephrotoxic in a broader population of kidney diseases.
The pathogenesis of oxalate-induced kidney injury is not entirely clear now. Tubulointerstitial injury and fibrosis with extensive oxalate crystal deposition in kidney tissue is the characteristic pathological feature of oxalate nephropathy [4]. Nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is the most widely studied inflammasome currently, which has been implicated in the pathogenesis of oxalate crystal-induced kidney damage by in vitro and animal studies [5–11]. Moreover, oxalate crystals may migrate into the renal interstitium, where they become surrounded, engulfed, and eventually cleared by tissue macrophages [5]. The tissue macrophages present heterogeneity with different phenotypes, which are determined by the microenvironment and may play different roles in the process of tissue inflammation, regeneration, and fibrosis. It has been reported that NLRP3 inflammasome can also promote the shift of the renal macrophage phenotype in hyperoxaluria animal models [11].
Herein, we performed a pilot study of NLRP3 inflammasome activation and macrophage phenotype distribution in kidney specimens of patients with biopsy-proven acute oxalate nephropathy (AON), aiming to address the gap between studies from in vitro and animal models and human live tissues and reveal its potential clinicopathological significance in the disease.
Methods
The study protocol was approved by the Ethics Committee of Peking University First Hospital (No. 2017[1333]) and performed in accordance with the Declaration of Helsinki. Informed consent was obtained from each patient at kidney biopsy.
1. Study patients
Twelve patients with renal biopsy-proven AON diagnosed between January 2016 and October 2022 at Peking University First Hospital were enrolled in the present retrospective cross-sectional study. The inclusion criteria were as follows: 1, presented with AKI (defined as an increase in serum creatinine to ≥1.5 times baseline, which is known to occur within the prior 7 days) or AKD (defined as >50% increase of serum creatinine within 3 months) clinically; 2, extensive oxalate crystals deposition in kidney with tubular injury, obstruction, interstitial inflammatory cells infiltration and fibrosis; and 3, exclusion of other nephropathies apart from nonspecific microvascular (nephrosclerosis) and diabetic nephropathy. Estimated glomerular filtration rate (eGFR)-matched 24 patients with acute tubulointerstitial nephritis (ATIN) not related to oxalate according to propensity score match were enrolled as disease controls. The causes for ATIN were as follows: 13 cases were considered drug-induced ATIN, one case was attributed to infection-related ATIN, and the other 10 cases were diagnosed as idiopathic ATIN. Moreover, 10 renal specimens were obtained from donor kidneys for transplantation as normal control (NC) (Supplementary Table 1, available online).
2. Clinical and laboratory data collection
Demographic (age, sex), medical history (hypertension, diabetes mellitus [DM], prior CKD history), biological data (body mass index, systolic blood pressure, diastolic blood pressure), and medication information including renin-angiotensin system inhibitors, antibiotics, nonsteroidal anti-inflammatory drugs, and Chinese herbs were collected from electronic medical charts. Laboratory data of serum (serum creatinine, electrolytes) and urinalysis (hematuria, leukocyturia, N-acetyl-beta-D-glucosaminidase, albumin creatinine ratio, urine protein, and alpha-1-microglobulin) at the time of renal biopsy were also collected. eGFR was calculated using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation [12]. The therapy given during the hospital stay including dialysis and corticosteroid was also recorded.
3. Renal histopathology evaluation
The kidney biopsy specimens were routinely stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and periodic acid-silver methenamine. The tubular and interstitial lesions were semi-quantitatively scored according to the Banff working classification criteria with a few modifications (Supplementary Table 2, available online) [13,14]. Calcium oxalate crystals were detected under polarized light. Moreover, oxalate crystal density was calculated and expressed as the number of CaOx crystals over the total parenchyma area at 200× magnification per area (mm2). The evaluation was performed by two independent pathologists.
4. Immunohistochemistry staining and quantification
In brief, 4-μm-thick sections of formalin-fixed and paraffin-embedded kidney tissues were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed with high-pressure treatment following the instructions provided for each primary antibody (Supplementary Table 3, available online). Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 20 minutes. After further blocking with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 minutes, sections were incubated with primary antibodies overnight at 4 °C. After rinsing with PBS, the sections were incubated with a biotinylated secondary antibody (Zhongshan Golden Bridge Biotechnology) for 35 minutes at 37 °C and developed with 3,3’ diaminobenzidine as the chromogen.
Stained samples were analyzed by Image Pro Plus software (version 6.0; Media Cybernetics) as the average optical density (integrated optical density/relative area) [15]. At least 20 fields of tubulointerstitial vision per renal section at ×400 were observed blindly for quantitative assessments of immunohistochemical staining.
5. Statistical analysis
Continuous variables are expressed as the mean ± standard deviation or median (interquartile range). The Student t test, analysis of variance with post hoc Bonferroni correction, Mann-Whitney test, or Kruskal-Wallis test were used for the comparisons among groups as the data indicated. Categorical variables were expressed as proportions and compared using the chi-square test or Fisher exact test. Correlations between two variables were evaluated by the Pearson correlation test as the data indicated. A two-sided p-value of <0.05 was considered statistically significant. All analyses were performed using IBM SPSS version 27 (IBM Corp.).
Results
1. General data
The baseline data of 12 patients with AON is presented in Table 1. The majority was male, with a mean age of 56.9 ± 9.0 years. The proportion of DM (10 of 12, 83.3%) was extensively high in patients with AON as compared with ATIN patients (5 of 24, 20.8%) (Table 1). Six diabetic patients with AON were found with thickening of glomerular basement membrane under electronic microscope examination, indicating the concomitant existence of early diabetes nephropathy (DN), while none of the diabetic patients with ATIN had DN findings. Eight cases (66.7%) with AON received corticosteroid treatment, which was similar to that (75.0%) of patients with ATIN. After a comparable follow-up period, patients with AON presented with higher serum creatinine levels (155.5 vs. 112.0 μmol/L, p = 0.02) and borderline lower eGFR level (42.5 vs. 59.8 mL/min/1.73 m2, p = 0.05) at the last follow-up as compared with patients with ATIN, respectively.
2. Pathological data
Fan- or plate-shaped, birefringent crystals of oxalate crystals were predominantly identified within the tubular lumens and/or embedded within the cytoplasm of renal tubular epithelial cells. Ultrastructural examination by electron microscopy revealed the oxalate crystals as fissure-like, electron-lucent entities. These crystalline deposits are implicated in the pathogenesis of acute tubular injury, characterized by the loss of the brush border, epithelial flattening, and denudation of the underlying basement membrane (Fig. 1A–C). In 50% of the cohort (six out of 12 cases), a subset of the crystals was observed to transgress the confines of the tubular lumina, extending into the interstitial compartment and eliciting a localized inflammatory response (Fig. 1D). Notably, glomeruli oxalate deposition was absent in the subjects examined.
Quantitative analysis revealed a mean density of oxalate crystal deposition in the AON cohort of 3.0 ± 1.9 deposits/mm2, with 5% to 20% of tubules exhibiting signs of crystal accumulation. No notable differences in oxalate crystal deposition within intact tubules versus those in the context of acute tubular injury were observed. A positive correlation was established between the extent of oxalate deposition and Sirius Red staining, indicative of fibrosis (rp = 0.668, p = 0.02). The assessment of tubulointerstitial injury, as per the Banff criteria, yielded similar scores of most indices for patients with AON and ATIN as summarized in Table 2. However, there was a significant increase in the disruption of tubular basement membrane (TBM) integrity and a more pronounced inflammatory response in the AON samples (both p < 0.05), as illustrated in (Fig. 1E, F).
Due to the high presence of DM in the AON cohort, further assessment among patients with DM was made, who were categorized as ATIN with DM, AON with DM alone, and AON with DN. As shown in Supplementary Table 4 (available online), three groups presented similar degrees of tubulointerstitial changes in most indices per the Banff criteria. However, the AON subgroups, either with DM alone or with DN, manifested a heightened incidence of TBM rupture and a more conspicuous inflammatory response (both p < 0.05) with a similar degree as compared with diabetic ATINs.
3. NLRP3 inflammasome expression in renal tissues
The expression patterns of NLRP3 and caspase-1 were detected in consecutive section staining as shown in Fig. 2. Almost no positive staining of NLRP3 could be observed in NC with occasional weak staining for caspase-1 observed. In contrast, positive staining of both NLRP3 and caspase-1 could be detected in tubules and some interstitial cells with nearly consistent distribution patterns in specimens of other two groups. The mean optical densities of NLRP3 in AON specimens were significantly higher than those in NC (0.0158 ± 0.0115 vs. 0.0028 ± 0.0026, p < 0.001), and ATIN (0.0158 ± 0.0115 vs. 0.0076 ± 0.0040, p = 0.004), respectively (Fig. 3). Similarly, the mean optical densities of caspase-1 in AON specimens were also significantly higher than those in NC (0.0136 ± 0.0061 vs. 0.0025 ± 0.0019, p < 0.001), and ATIN (0.0136 ± 0.0061 vs. 0.0058 ± 0.0026, p < 0.001), respectively (Fig. 3).
Positive staining of gasdermin D (GSDMD) could be detected in a few tubules in AON specimens, which were also positive for NLRP3 and caspase-1 staining as shown by consecutive staining (Fig. 2). The median optical densities of GSDMD in AON specimens were significantly higher than those in NC (0.0003 [0.0002–0.0012] vs. 0.00002 [0.000001–0.00025], p = 0.008), and ATIN (0.0003 [0.0002–0.0012] vs. 0.00005 [0.00001–0.00015], p = 0.02), respectively (Fig. 3).
The expression of osteopontin (OPN) was increased in tubules in AON kidneys than that in NC (0.0057 ± 0.0028 vs. 0.0003 ± 0.0003, p < 0.001) and ATIN group with no statistical significance found (0.0057 ± 0.0028 vs. 0.0036 ± 0.0026, p = 0.05) (Fig. 3). A positive correlation was found between the expression of NLRP3 with OPN (rp = 0.793, p = 0.002) in AON patients, respectively.
4. Distribution of macrophage subtype in renal tissues
The presence of both M1 macrophages (human leukocyte antigen-DR [HLA-DR] positivity) and M2 macrophages (cluster of differentiation 163 [CD163] positivity) was observed in AON specimens (Fig. 4). The mean optical densities of HLA-DR in AON specimens were comparable to that of ATIN (0.006 ± 0.002 vs. 0.007 ± 0.002, p > 0.05). The mean optical densities of CD163 in AON specimens were comparable to that of ATIN (0.007 ± 0.002 vs. 0.008 ± 0.004, p > 0.05) as well. No significant association between NLRP3 expression with HLA-DR (rp = 0.001, p = 0.998) and CD163 expression (rp = –0.053, p = 0.89) was found in AON specimens, respectively.
The patients of AON were further divided into two groups according to the ratio of M1/M2 (HLA-DR+/CD163+). Five patients (41.7%) were found with greater CD163+ M2 macrophage presence with M1/M2 (HLA-DR+/CD163+) ratio <1. As shown in Table 3, patients with an M1/M2 ratio <1 presented with less interstitial edema (40.0% vs. 100%, p = 0.02), more TBM disruption (100% vs. 28.6%, p = 0.01), more severe tubular atrophy (p = 0.03), and prominent renal fibrosis (p = 0.01). These patients also had a lower eGFR (24.9 ± 10.6 mL/min/1.73 m2 vs. 55.1 ± 21.2 mL/min/1.73 m2, p = 0.02) and a higher ratio of remnant CKD (5 of 5 [100%] vs. 3 of 7 [42.9%], p = 0.04) at the end of follow-up period as compared with patients with an M1/M2 ratio >1. Only one patient had end-stage kidney disease with eGFR <15 mL/min/1.73 m2 at the last follow-up. Kaplan-Meier survival curve analysis did not show significant differences between groups of patients with different M1/M2 ratios (p = 0.32) (Supplementary Fig. 1, available online).
Discussion
This pilot study investigated the activation of NLRP3 inflammasome pathway and distribution of M1 and M2 macrophages in kidney tissues and their potential clinicopathological significance based on a cohort of well-defined patients with AON.
Oxalate nephropathy is a relatively rare but potentially devastating disease that can cause irreversible impairment of kidney function, either due to sudden onset of oxalate deposition leading to AKI or long-standing gradual crystal formation and deposition inducing CKD. Standard treatments for oxalate nephropathy, such as hydration, alkali therapy, and pyridoxine, often yield unsatisfactory results. Corticosteroids are sometimes used empirically in the management of oxalate nephropathy due to the presence of interstitial inflammation as shown in the present patient cohort, but their effectiveness has not been confirmed. Therefore, a better understanding of the mechanisms underlying oxalate nephropathy is needed to develop effective treatment strategies.
In the current study, we observed activation of the NLRP3 inflammasome pathway with increased expression of NLRP3 and caspase-1 in renal tissues of AON patients as compared with NCs and eGFR-matched ATIN patients. Inflammasomes are high-molecular-weight complexes that reside in the cytosol of cells and are part of the innate immune system [16]. Several studies have suggested the pivotal role of NLRP3 inflammasome in crystalline nephropathy, including oxalate nephropathy. It has been demonstrated that oxalate may induce NLRP3 activation in renal tubular epithelial cells, macrophages, and dendritic cells in animal models of AKI and CKD. Upon activation, NLRP3 can cleave pro-caspase-1 to produce activated caspase-1, another essential component of the NLRP3 inflammasome. This can subsequently cleave the biologically inactive precursors of interleukin (IL)-1β and IL-18 to generate their mature inflammatory counterparts. In this study, we found prominent increased expression of NLRP3 and caspase-1 with a consistent distribution pattern revealed by serial section staining in kidney tissues of AON patients as compared with NCs, supporting activation of NLRP3 inflammasome in human live tissues of AON. NLRP3 inflammasome activation has been reported in tubulointerstitial nephritis as well. Similar scores for most indices of tubulointerstitial injuries as per the Banff criteria were found in the AON and ATIN specimens in the present study. However, oxalate crystals had been found inducing more TBM rupture with a subsequent conspicuous localized inflammatory response in the present study as compared with the ATIN specimens. Based on these observations, higher expression of NLRP3 and caspase-1 in AON specimens as compared to ATIN tissues, at least partly provided evidence for NLRP3 inflammasome activation upon oxalate stimulation in human live tissues.
Pyroptosis is a recently identified type of programmed cell death. Recent studies showed that caspase-1 can cleave GSDMD to form an amino-terminal fragment, which oligomerizes and generates pores on the cell membrane to induce pyroptosis. Most previous studies have focused on immunocytes and identified GSDMD as a key protein in caspase-1-mediated pyroptosis in mouse bone marrow macrophages. Recently, Liu et al. [17] found altered phenotype and protein expression of pyroptosis in oxalate nephropathy model in vivo and in human proximal tubule HK2 cells in vitro upon stimulation of oxalate crystal. Accordingly, we detected increased GSDMD expression in tubular cells of AON patients in the present study, supporting the role of NLRP3 inflammasome activation in inducing tubular cell pyroptosis upon oxalate stimulation.
The adhesion of crystals to epithelial cells is an essential step for the onset of crystal-induced kidney damage. OPN has been extensively studied as an adhesion molecule. Asselman et al. [18] observed damage to tubular cells and over-expression of OPN in the kidneys of rats with oxalate crystal deposition. Qin et al. [19] demonstrated that OPN increased calcium oxalate crystal aggregation and adherence, while Tsuji et al. [20] found that inhibition of OPN expression in hyperoxaluric rats inhibited the deposition of oxalate crystals. These results highlight the pivotal role of OPN as an adhesion molecule in enhancing the adhesion of oxalate crystal to renal tubular epithelial cells. Furthermore, a recent study showed that oxalate crystal-induced upregulation of NLRP3 inflammasome could mediate the expression of OPN via the p38 mitogen-activated protein kinase signaling pathway in NRK-52E cells, which subsequently changed the adhesion of oxalate crystal to NRK-52E cells [21]. In this study, we found that the expression of OPN was significantly elevated in the tubules of kidneys of AON patients than NCs and associated with NLRP3 expression. Collectively, these results supported that NLRP3 activation may favor crystal adhesion via increased expression of crystal-binding molecules on tubular epithelial cells in human kidney tissues upon insult from oxalate crystal.
We also detected the presence of M1 and M2 macrophages in AON kidney tissues in the present study. Oxalate crystal may be translocated into the interstitium and thus attract/activate monocytes and macrophages as observed in previous studies and the current study. In general, activated tissue monocytes can differentiate into either M1 or M2 macrophages, depending on the stimuli, and may trans-differentiate [22]. Previous research has shown human monocytes exposed to calcium oxalate crystals in vitro could differentiate into M1 macrophages and increase production of the pro-inflammatory cytokines [23]. Conversely, M2 macrophages were found to suppress calcium oxalate crystal formation and deposition by phagocytosing oxalate crystals [24,25]. In the present study, we observed that a higher presence of CD163+ M2 macrophage was correlated with more chronic tubulointerstitial lesions in AON patients. In addition, these patients with a higher presence of CD163+ M2 macrophage found at the time of biopsy seemed to have worse kidney function recovery during follow-up with a tendency towards CKD remanence as compared to those with less presence of CD163+ M2 macrophage who had comparable baseline kidney functions. Therefore, CD163+ M2 macrophage might be involved in the development of interstitial fibrosis with subsequent kidney function decline under oxalate nephropathy. Recent research has focused on identifying regulators that alter the macrophage phenotypes aiming to develop novel remedies. Being a cross-sectional observational study, we could not evaluate M1/M2 trans-differentiation during the disease course in the present study. However, there has already study reporting that NLRP3 inhibition in hyperoxaluric mice could protect against calcium oxalate deposition and CKD via a shift in the phenotype of renal macrophages, promoting anti-inflammatory rather than pro-inflammatory and pro-fibrotic responses [11].
There are some important limitations that should be noted. First, as a relatively rare disease entity, the sample size of AON in this study was small, which weakened the statistical power. Second, being a cross-sectional study, the association observed could not be used directly to assess causality. Third, given the retrospective nature of the study, the data of follow-up was limited. Although we observed some difference in kidney function at the last follow-up between AON patients with different M1/M2 ratios, we lack enough hard-end events for the kidney to address the role of M2 macrophage predominance in renal tissues. Nevertheless, the results of this pilot study did provide evidence from real-world patients for the speculative pathophysiological mechanisms for oxalate-induced kidney injury derived from animal and in vitro studies. Upon verification, it may help to increase our understanding of oxalate nephropathy and provide clues for developing proper treatment strategies in future studies.
In conclusion, oxalate crystal-induced kidney damage is a potentially devastating condition that can lead to irreversible kidney function decline. NLRP3 inflammasome pathway was activated in kidney tissues of patients with oxalate nephropathy, and the predominance of M2 macrophage presence was correlated with the chronicity of tubulointerstitial pathological lesions, which need further exploration.
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