Association Between TRPC6 Channel and NLRP3 Inflammasome Activities in Kidney Tissue of Children With Henoch-Schönlein Purpura Nephritis


 BackgroundDuring the development of Henoch-Schönlein purpura nephritis (HSPN), the activation and overexpression of transient receptor potential cation channel protein 6 (TRPC6) and the activation of Nod-like receptor protein 3 (NLRP3) inflammasome play a very important role. However, whether the expression of TRPC6 in children with HSPN is related to the activation of NLRP3 inflammasome has not been reported.MethodsWe obtained kidney biopsy specimens of 33 children with HSPN and 6 controls with renal trauma. Immunohistochemistry was used to detect the expression of TRPC6, NLRP3, ASC, caspase-1, IL-1β, IL-18. Student’s t-test was used to analyze the difference between the HSPN and control group. Pearson correlation test was used to estimate the correlation between TRPC6 and NLRP3 inflammasome among HSPN children. A bootstrap approach was performed for mediation effects of NLRP3 inflammasome on the association between TRPC6 and HSPN.ResultsThe expressions of TRPC6, NLRP3, ASC, Caspase-1, IL-1β, IL-18 in kidney tissues of children with HSPN were significantly higher than those in the control group (all P values <0.05). Significant correlations of TRPC6 with NLRP3, ASC, and IL-18 were observed. NLRP3 and ASC were also related to the levels of IL-1β and IL-18. Significant mediation effects (0.08, 95%CI = 0.04-0.15) of ASC on the association between TRPC6 and HSPN was revealed, with explaining 58.25% of the total effects.Conclusion﻿TRPC6 may cause the release of IL-1β and IL-18 by activating the NLRP3 inflammasome, especially the expression of ASC, thereby damaging the kidneys of children with HSP.


Abstract Background
During the development of Henoch-Schönlein purpura nephritis (HSPN), the activation and overexpression of transient receptor potential cation channel protein 6 (TRPC6) and the activation of Nod-like receptor protein 3 (NLRP3) in ammasome play a very important role. However, whether the expression of TRPC6 in children with HSPN is related to the activation of NLRP3 in ammasome has not been reported.

Methods
We obtained kidney biopsy specimens of 33 children with HSPN and 6 controls with renal trauma.
Student's t-test was used to analyze the difference between the HSPN and control group. Pearson correlation test was used to estimate the correlation between TRPC6 and NLRP3 in ammasome among HSPN children. A bootstrap approach was performed for mediation effects of NLRP3 in ammasome on the association between TRPC6 and HSPN.

Results
The expressions of TRPC6, NLRP3, ASC, Caspase-1, IL-1β, IL-18 in kidney tissues of children with HSPN were signi cantly higher than those in the control group (all P values <0.05). Signi cant correlations of TRPC6 with NLRP3, ASC, and IL-18 were observed. NLRP3 and ASC were also related to the levels of IL-1β and IL-18. Signi cant mediation effects (0.08, 95%CI = 0.04-0.15) of ASC on the association between TRPC6 and HSPN was revealed, with explaining 58.25% of the total effects. Conclusion TRPC6 may cause the release of IL-1β and IL-18 by activating the NLRP3 in ammasome, especially the expression of ASC, thereby damaging the kidneys of children with HSP.

Background
Henoch-Schönlein purpura nephritis (HSPN) is a common clinical pediatric disease, mainly with gross or microscopic hematuria, repeated skin purpura, edema, and increased blood pressure as clinical manifestations 1 . HSPN is the most common and serious complication of Henoch-Schönlein purpura (HSP), and the main factor affecting the long-term prognosis of HSP children. It has been reported that 6 to 24 per 100 000 children younger than 17 years will develop HSP 2 . However, up to half of HSP children will develop HSPN, 90% of which will occur within 6 months of onset 3,4 . In China, HSPN has been the second-most common secondary glomerular disease 5 . If not treated in time, it can lead to chronic renal insu ciency in children, which will have a serious impact on the health and quality of children, even on their whole life span. Therefore, it is important to clarify the pathogenesis and the clinical manifestations and to ascertain the most appropriate treatment of HSPN.
NOD-like receptor protein 3 (NLRP3) in ammasome is one of the most famous in ammasomes in mammals, and it plays a very important role in innate immunity. The activation of NLRP3 in ammasome is closely related to a variety of in ammation-related diseases, such as type 2 diabetes, atherosclerosis, Alzheimer's disease, and other diseases 6 . Studies have shown that in high glucose or diabetes, the activation of the NLRP3 in ammasome of kidney tissue is the key upstream mechanism of podocyte in ammatory injury 7,8 . Research 9 pointed out that ion e ux (K + , Cl -), reactive oxygen species (ROS), or mitochondrial DNA (mtDNA) oxidation and NLRP3 post-translational modi cation (ubiquitination, phosphorylation) can regulate the activation of NLRP3 in ammasome. In addition to bacterial, viral, and fungal infections, more and more endogenous danger signals, such as mitochondrial ROS 10 and potassium e ux 11 , maybe activators of NLRP3 in ammasome. The key role of NLRP3 in ammasome in kidney in ammation has been con rmed in a variety of kidney disease models, including I/R injury 12, 13 , folic acid nephrotoxicity 14 , caused by rhabdomyolysis AKI 15 , and contrast-induced AKI 16 . However, few studies have explored its role in podocyte damage in immune-related kidney diseases. Transient receptor potential (TRP) channels are a large class of plasma membrane proteins, including TRPC, TRPM, etc.
Typical TRPC6 is a non-selective Ca 2+ permeable cation channel expressed in many different cell types.
Renal tissue TRPC6 channel protein is mainly expressed in the foot process slit membrane 17 , but also in mesangial cells 18 . The TRPC6 channel is associated with the progression of several forms of kidney disease, such as primary and secondary FSGS (familial forms of focal segmental glomerulosclerosis), glomerulosclerosis during autoimmune glomerulonephritis, and type 1 diabetes 19 . Previous research by our research group found that RAS activation in the kidney may be involved in the occurrence and development of kidney damage in children with HSP 20 . Experiments with nephropathy model rats also indicate that the increased expression of AGT in the kidney is related to the increased production of angiotensin (Ang ) in the kidney, which promotes the development of glomerular injury 21 . Studies have shown that Ang can quickly activate the TRPC6 channel of podocytes and mediate calcium in ux, and at the same time cause a signi cant upregulation of TRPC6 activity and expression 22,23 . Experimental evidence showed that calcium channel proteins (e.g., TRPM1, TRPM2) can mediate NLRP3 activation and cause cell damage in other tissues 24,25 . Extracellular Ca 2+ in ux or Ca 2+ release from the intracellular calcium pool to the cytoplasm and cause mitochondrial dysfunction is a common pathway for the activation of NLPRP3 in ammasome by multiple activators 26 . Therefore, we believe that TRPC6 and NLRP3 in ammasome play an important role in the occurrence and development of HSPN.
This study was to investigate the changes in the expression of TRPC6 and NLRP3 in ammasome in kidney tissues of children with HSPN and their relationship.

Participants and specimens
From January 2018 to December 2019, 33 children with HSPN were executed a renal biopsy, when they hospitalized in the Department of Nephrology of the First A liated Hospital of Anhui Medical University. 19 males and 14 females were included with an average age of 10 years. All cases met the diagnostic criteria of the Evidence-Based Guidelines for the Diagnosis and Treatment of Henoch-Schönlein purpura Nephritis (2016) by the Nephrology Group of the Pediatric Branch of the Chinese Medical Association 27 . The control group specimens were derived from normal tissues from kidney trauma or tumor surgery. The renal biopsy specimens and those control group specimens were further analyzed in the laboratory. The study was approved by the Medical Research Ethics Committee of the First A liated Hospital of Anhui Medical University, informed consent was obtained from all guardians of individual participants included in the study.

TRPC6 and NLRP3 in ammasome related indicators detection
Immunohistochemistry (IHC), the wax block was cut into 4um thick tissue sections, dewaxed the tissue sections, and rehydrated, and then repaired antigen, incubated with 3% hydrogen peroxide for 15 minutes, blocked with 10% goat serum for 30 minutes at room temperature. After incubation with the primary antibody(anti-IL-1β, anti-IL-18, anti-NLRP3, and anti-Caspase-1 antibodies, Wan Biotechnology Company, 1:200; anti-ASC antibody, Wan Biotechnology Company, 1:150; anti-TRPC6 antibody, Wuhan Sanying Biological Company, 1:200) overnight at 4°C, washed with phosphate buffered saline (PBS) 3 times, incubated with secondary antibody at room temperature for 50 minutes, and washed with PBS for 3 times. Added the 3,3-diaminobenzidine (DAB) chromogenic solution to develop color, put the slice into hematoxylin counterstain for 2 minutes, then dehydrated, transparentized, and sealed, and nally observed under the microscope.

Statistical analysis
In this study, Image-Pro Plus 6.0(IPP) software was used to analyze the integrated optical density (IOD) to quantify the expression of each indicator. SPSS 22.0 software was used for statistical analyses. All data were presented as means ± standard deviation (SD). Differences between the two groups were analyzed using Student's t-test. Correlation between TRPC6 and NLRP3 expression levels was analyzed using the Pearson correlation test. To investigate whether NLRP3 in ammasome activation simultaneously mediates the HSPN development, a test of multiple mediations using the multiple-step mediator model was used 28 . Namely, the PROCESS plugin was installed into the IBM SPSS 22.0 software. The model number was 4, and bootstrap samples were 5000. Items "Bias Corrected" and "Compare indirect effects" were opted. P values <0.05 were considered signi cant.

Comparison of TRPC6 expression and NLRP3 in ammasome related indicators
In the normal control group, little NLRP3, ASC, Caspase-1, IL-1β, IL-18 were observed. There was a little TRPC6 expression in mesangial cells and podocytes in the control group. The expression of NLRP3, ASC, Caspase-1, IL-1β, IL-18, TRPC6 was signi cantly higher than that of the control group (P<0.001). The positive products of NLRP3, ASC, Caspase-1, IL-1β, IL-18, and TRPC6 were all brownish yellow ne particles observed under the microscope, and they were mainly located in the cytoplasm of glomerular cells, and the renal tubular cells were also expressed in large amounts (Table 1 and Figure 1).

Correlation between TRPC6 and NLRP3 in ammasome related indicators in the HSPN group
In the HSPN group, the expression of TRPC6 was moderately related to the expression of NLRP3, ASC, and IL-18. Low to moderate correlation of NLRP3 with IL-1β and IL-18 was revealed. The expression level of ASC was moderately associated with the expression of IL-1β and IL-18 ( Figure 2).

Multiple mediation analysis results
A separately run individual set of regressions indicated signi cant direct effects of TRPC6 on NLRP3 and ASC, effects of ASC on IL-1β and IL-18, effects of IL-1o on IL-18. However, only the mediation path from TRPC6 to ASC, and from ASC to HSPN remained signi cant. A 95% con dence interval from 0.04 to 0.15 for the indirect effect (0.08) was revealed, explaining 58.25% of the total effect. All other mediation paths became insigni cant (Figure 3).

Discussion
HSPN as the most serious complication of HSP, because the speci c pathophysiology of its pathogenesis is not yet clear, its incidence has not decreased over time 29 . The results of this study showed that the expressions of TRPC6 and NLRP3 in ammasome in kidney tissues of children with HSPN were signi cantly higher than those in the normal control group (P<0.05). The increased expression of TRPC6 in kidney tissues of HSPN children was correlated with the increased expression of NLRP3, ASC, and IL-18. The expression of IL-1β and IL-18 was correlated with the expression of NLRP3, ASC. It indicated that the increased expression of TRPC6 and the activation of NLRP3 in ammasome do participate in the process of HSPN kidney injury.
TRPC6 is one of the newly discovered podocytes slit diaphragm proteins (SD). It interacts with a variety of SD molecules such as Podocin, Nephrin, and CD2AP to form a signaling complex to maintain the structural and functional integrity of glomerular podocytes 30,31 . TRPC6 expression is increased in a variety of acquired proteinuria glomerular diseases, which may be caused by regulating calcium in ux 32,33 . However, it is not very clear how TRPC6 is speci cally involved in the pathogenesis of podocyte injury.
Studies have shown that the upregulation of TRPC6 can be detected in various glomerular diseases such as minimal change nephropathy, membranous nephropathy, and FSGS 34 . Another study has found that TRPC6 is found in some secondary glomerular diseases such as diabetic nephropathy (DN) 35 . It also signi cantly increased the expression of glomerular podocyte TRPC6 in children with nephrotic syndrome 36 . Combined with the results of this study, it is not di cult to see that TRPC6 plays an important role in the progression of glomerular disease, and taking measures against it will greatly help the diagnosis and prevention of kidney disease. NLRP3 in ammasome is one of the members of the intracellular in ammatory protein complex family, which is composed of NLRP3 protein, adaptor ASC (an apoptosis-related speck-like protein containing CARD domain) and Caspase-1 precursor 9 . In normal cells, these protein complexes exist in an inactive form. After activation of the in ammatory body, the precursor protein of Caspase-1 is cleaved into functional Caspase-1. Its main function is to convert the inactive and intracellular pro-in ammatory cytokines pro-IL-1β and pro-IL-18 It is converted into active IL-1β and IL-18 and released to the outside of the cell, causing local tissue in ammatory damage or systemic in ammatory response 37,38 . Zhang used confocal microscopy to observe the expression of NLRP3 and ASC in glomerular podocytes in vitro 39 . At the same time, studies have found that the amount of NLRP3 protein and its mRNA will be signi cantly increased in kidney diseases, such as IgA nephropathy 40 35,44 . The NLRP3 in ammasome is one of the intracellular in ammasomes. When the body senses an external stimulus, the stimulus signal enters the cell, activates NLRP3, recruits ASC, and Pro-Caspase1 to form the NLRP3 in ammasome, generates activated Caspase-1, cleaves the in ammatory factors precursor and activated in ammatory factors IL-1β and IL18 are produced, and cell e ux leads to in ammation. It is currently believed that various NLRP3 in ammasome stimulants can cause mitochondrial dysfunction, leading to the release of reactive oxygen species ROS and mtDNA, thereby activating the NLRP3 in ammasome. Ca 2+ released into the cytoplasm under endoplasmic reticulum stress can be taken up by the mitochondrial unidirectional transporter, which in turn reduces the normal negative mitochondrial transmembrane potential, activates the mitochondrial outer membrane voltage-gated channel, promotes related metabolites and ion transport and produce ROS, activates the NLRP3 in ammasome. When the voltage on the outer membrane of the mitochondria is knocked down, the generation of ROS is reduced, and the activation of the NLRP3 in ammasome is impaired 45 . A report 46 believed that oxidized mtDNA released by dysfunctional mitochondria is also associated with the activation of NLRP3 in ammasome. They found that the α7-nicotinic acetylcholine receptor signaling pathway can inhibit the activation of the NLRP3 in ammasome by blocking the release of mtDNA. Recently, more and more researchers have begun to pay attention to the role of Ca 2+ in the activation of NLRP3 in ammasome. Extracellular Ca 2+ in ux or Ca 2+ release from the intracellular calcium pool to the cytoplasm and cause mitochondrial dysfunction is a common pathway for multiple activators to activate the NLRP3 in ammasome 26 . Based on the results of this study, it is reasonable to infer that: TRPC6 channel-mediated calcium in ux may release ROS and mtDNA through mitochondrial damage and participate in the activation of NLRP3 in ammatory bodies, producing free IL-1β and IL-18, and participating in HSPN kidney damage process (Figure 4).
To our knowledge, this is the rst time to investigate the relationship between expression levels of TRPC6 and NLRP3 in ammasome in kidney tissue of children with HSPN. Some limitations should be considered. First, the sample size of this study recruiting 33 cases and 6 controls was small, with the statistical power is limited. Second, demographic characteristics as well as other covariates were not abstracted. Other indicators including Ang and ROS expression levels were not detected because of limited samples and funding. Finally, only the statistical association between TRPC6 and NLRP3 was observed. The speci c mechanism pathway should be further explored by animal experiments and in vitro cell experiments. This study also did not delve into the effects of changes in the molecular structure of TRPC6 and NLRP3 in ammasome on the disease. The main objective was to propose a hypothesis based on ndings of this study, to provide the guidelines for our subsequent cell experimental research.

Conclusion
In summary, TRPC6 and NLRP3 in ammasome play an important role in the pathogenesis of kidney disease in children with HSPN. TRPC6 may cause the release of IL-1β and IL-18 by activating the NLRP3 in ammasome, especially the expression of ASC, thereby damaging the kidneys of children with HSP.
These new ndings were conducive to further study the mechanism of kidney injury in children with HSPN, the speci c interaction mechanism between TRPC6 and NLRP3 in ammasome in kidney tissues, and then as a molecular target for the targeted treatment of HSPN.

Consent for publication
Our data have consent for publication from that person, or in the case of children, their parent or legal guardian.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Expression of TRPC6 and NLRP3 in ammasome related indicators in kidney tissues of two groups A: Correlation between TRPC6 and NLRP3 in ammasome related indicators among the HSPN group (*P <0.05) Figure 2 Correlation between TRPC6 and NLRP3 in ammasome related indicators among the HSPN group (*P <0.05)  Hypothesis diagram of TRPC6 and NLRP3 in ammasome activation pathway Hypothesis diagram of TRPC6 and NLRP3 in ammasome activation pathway