Podocyte injury is a hallmark of glomerular diseases; however, the underlying mechanisms remain unclear. B7-1 is increased in injured podocytes, but its intrinsic role is controversial. The clinical data here revealed the intimate correlation of urinary B7-1 with severity of glomerular injury. Through transcriptomic and biological assays in B7-1 transgenic and adriamycin nephropathy models, we identified B7-1 is a key mediator in podocyte injury and glomerulosclerosis through a series of signal transmission to β-catenin. Using LC-MS/MS, Hsp90ab1, a conserved molecular chaperone, was distinguished to be an anchor for transmitting signals from B7-1 to β-catenin. Molecular docking and subsequent mutant analysis further identified the residue K69 in the N terminal domain of Hsp90ab1 was the key binding site for B7-1 to activate LRP5/β-catenin pathway. The interaction and biological functions of B7-1-Hsp90ab1-LRP5 complex were further demonstrated in vitro and in vivo. We also found B7-1 is a novel downstream target of β-catenin. Our results indicate an intercrossed network of B7-1, which collectively induces podocyte injury and glomerulosclerosis. Our study provides an important clue to improve the therapeutic strategies to target B7-1.
Glomerular disease is the major cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) . Podocyte is the main component of the glomerular filter barrier. With high energy consumption and the inability to proliferate, podocytes are vulnerable to stimuli such as oxidative stress and immune attack [2,3,4,5]. Injured podocyte can go through dedifferentiation, apoptosis, and also senescence [4, 6, 7], which play key roles in the pathogenesis of diabetic nephropathy (DN), lupus nephritis (LN), IgA nephropathy (IgAN), membranous nephropathy (MN), minimal change disease (MCD), and other glomerular diseases [8,9,10,11,12]. However, the underlying mechanisms are not elucidated.
Recent reports found podocyte may also function as a non-hematopoietic antigen-presenting cell (APC) to be involved in glomerular nephritis [5, 13]. Like other APC cells such as dendritic cells or macrophages, podocytes express MHC class II molecules [13, 14] and importantly, the co-stimulatory molecule B7-1 [15, 16], also known as CD80, to initiate T cell activation and inflammation . Commonly, APC cells present antigens to T lymphocytes to trigger inflammation [18,19,20]. B7-1 plays an important role in this process . Whereas, as non-hematopoietic APC, podocyte expresses B7-1 to possibly result into self-injury . However, the intrinsic role of B7-1 in podocyte injury and glomerular diseases remains largely unknown.
Podocyte B7-1 was firstly identified in 2004 , after that it was inconsistently detected in the studies of clinical nephropathy [22,23,24]. In addition, the therapeutic effects of abatacept, a CTLA4-immunoglobulin fusion protein (CTLA4-Ig) which blocks the interaction between B7-1 (APC) and CD28 (T cell), are still in uncertainty in glomerular diseases . CTLA4-Ig has already been investigated in primary and recurrent focal segmental glomerulosclerosis (FSGS), DN, and MCD [26,27,28,29]. Although there is a remarkable remission of proteinuria among some patients during medication ; however, relapse occurs after withdrawal of treatment . It suggests the immune response is not the only reason for B7-1-mediated glomerular diseases. Indeed, B7-1 could inactivate integrin α3β1  and disrupts NEPH1 signaling  to induce podocyte injury and promote albuminuria. These results suggest that B7-1 could solely mediate podocyte injury independent of immune involvement. However, the underlying mechanisms have not been completely clarified.
Wnt/β-catenin is a developmental signaling, but highly reactivated in CKD . The low-density lipid receptor family members LRP5/6 are the key components of Wnt receptor complexes . The phosphorylation of LRP5/6 leads to Wnt signal transmission to β-catenin, resulting into β-catenin’s translocation into nuclei to activate targeted genes such as RAS systems . Our previous report found β-catenin crucially contributes to podocyte injury through ubiquitinated degradation of WT1 , a key transcription factor for podocyte differentiation [33, 36]. Hence, B7-1 and β-catenin, could possibly have an intimate correlation in podocyte injury. But this should be determined in detail.
In this study, we identified β-catenin is an important mediator in B7-1 signaling. Furthermore, Hsp90ab1 mediates the communication between B7-1 and LRP5/β-catenin signaling in podocyte injury. Our study provides the important mechanisms of podocyte injury, and improves the understanding of strategies targeting B7-1.
Podocyte B7-1 is upregulated in a variety of glomerular diseases and accompanied by β-catenin activation
We first performed in situ RNA hybridization for B7-1 using RNAscope technology, a more specific and sensitive method compared with immunological assessment [37,38,39]. Compared to the negative signals in healthy control, B7-1 RNA was increased in patients with class III active LN, accompanied by β-catenin increasing (Fig. 1A). B7-1 was also upregulated in MCD, IgAN, FSGS, DN, and MN (Supplementary Fig. S1). B7-1 was highly colocalized with α-actinin-4, a podocyte marker, and β-catenin (Fig. 1B, Supplementary Fig. S1). We also observed that β-catenin was largely colocalized with α-actinin-4 (Fig. 1C, Supplementary Fig. S1). These suggest the intimate correlation between B7-1 and β-catenin in podocyte injury. The specificity of B7-1 RNAscope was testified by a negative probe control (Supplementary Fig. S1).
We next analyzed the correlation of urinary B7-1 with estimated glomerular filtration rate (eGFR), albumin/urine creatinine ratio (ACR), a marker of glomerular proteinuria, and β-2-microglobulin (β2-MG), a marker of tubular proteinuria, in clinical cohort of patients with glomerular diseases (Supplementary Table S1). As shown in Fig. 1D, urinary B7-1 levels gradually increased following the progression of CKD. Urinary B7-1 was negatively associated with eGFR, and positively associated with ACR (Fig. 1E, F), but showed no correlation with urinary β2-MG (Fig. 1G).
We further assessed B7-1 and β-catenin in db/db mice, a model of type 2 diabetes mellitus with hyperactive expression of β-catenin in podocytes . As shown, B7-1 was strongly upregulated in glomerulus in db/db mice, along with β-catenin increasing in podocytes and podocyte foot process fusion (Fig. 1H). The costaining showed B7-1 was primarily upregulated in podocytes (Fig. 1I). Western blotting analysis also revealed that B7-1 upregulation was concomitant with β-catenin activation (Fig. 1J, K). The specificity of antibodies was testified by isotype IgG antibody (Supplementary Fig. S1).
To further identify the important role of B7-1 in podocyte injury, we then isolated glomeruli and tubules from mice with adriamycin (ADR) nephropathy, a FSGS model. Interestingly, we found B7-1 mRNA levels were upregulated in glomeruli in a time-dependent manner (Fig. 1L). Notably, B7-1 was significantly increased in glomeruli at 7 days after ADR treatment, a critical time point for podocyte injury [40, 41]. Whereas, B7-1 was only slightly increased in tubules at the late stage (Fig. 1L). B7-1 upregulation was concomitant with the decrease in Nephrin, a podocyte slit diagram marker, and increase in Desmin, a podocyte injury marker (Fig. 1M). Furthermore, compared to extremely weak expression in isolated control glomeruli (Supplementary Fig. S1), B7-1 was upregulated in ADR-treated glomeruli, and perfectly colocalized with β-catenin and Podocalyxin, another podocyte marker (Fig. 1N). We also performed costaining of B7-1 and Endomucin (EMCN), an endothelial cell marker, in the kidney section from ADR mice. We found that there was a very small part of colocalizaton of B7-1 with EMCN (Supplementary Fig. S1). All these results suggest B7-1 is highly involved in podocyte injury, and this possibly was mediated by β-catenin signaling.
Podocyte-specific transgene of B7-1 in mice sufficiently induces podocyte injury and is associated with β-catenin
We generated podocyte-specific B7-1 transgenic (Tg) mice using PiggyBac-induced DNA (a plasmid with NPHS2 promoter-triggered B7-1-3xFlag) microinjection (Fig. 2A). Successful construction of Tg mice or wild type (WT) was identified by PCR (Fig. 2B, C) and Flag staining (Fig. 2D). We analyzed the expression of B7-1 and Flag-tag in Tg mice at different ages. As shown, B7-1 and Flag were both gradually increased with age (Fig. 2E–G, Supplementary Fig. S2). There was no difference in body weight and blood urea nitrogen between WT and Tg mice at the same age, while there was a significant increase in serum creatinine in Tg mice at 6 months of age compared to WT controls (Supplementary Fig. S2).
Albuminuria excretion and glomerular injury of mesangial expansion were then assessed. As shown in Fig. 2H, I, compared to extremely little changes in WT mice at different ages, Tg mice exhibited significantly increased albuminuria and glomerular injury at 3 months old, and those further elevated at six months old. In addition, the expression of Nephrin, Synaptopodin, and Podocalyxin, the epithelial markers of podocyte, showed the decreasing trend with age in Tg mice (Fig. 2J, K). Similarly, as shown in Fig. 2L and Supplementary Fig. S2, podocyte foot processes were irregularly shaped and showed a mild fusion in 3-month-old Tg mice, while there was more disarrangement and partial fusion of podocyte foot processes in 6-month-old Tg mice. Furthermore, Nephrin decreased at 3 months of age in Tg mice, and it was evident at 6 month’s. Periodic acid-Schiff (PAS) staining also showed a slight increase in mesangial expansion and matrix deposition in 3-month-old Tg mice when compared to the 2-month-old Tg mice, and a moderate expansion of mesangium in Tg mice at 6-month old. Whereas, the podocyte epithelial markers, B7-1, active β-catenin, β-catenin, and its ultrastructure remained unchanged in WT mice at 2 to 6 months of age (Supplementary Fig. S2). All these results indicated there would be an amplified transmission process in B7-1 signaling, which mediates the development of podocyte injury.
To explore the underlying mechanisms, we performed transcriptomic analysis. Heatmap and gene ontology (GO) enrichment analysis of the differentially expressed transcripts showed cell adhesion and differentiation, actin cytoskeleton organization, and GBM development were downregulated in Tg mice; whereas the activation of T cell immune response were upregulated (Fig. 2M, Supplementary Fig. S2). As WT1 is a podocyte-sepcific transcription factor, which plays a crucial role in glomerular differentiation and podocyte function, we observed its expression. As shown in Fig. 2M and Supplementary Fig. S2, WT1 was downregulated in Tg mice, accompanied by decrease in a series of podocyte epithelial markers such as Nephrin, Podocin and Synaptopodin. We next performed Gene set enrichment analysis (GSEA). As shown in Fig. 2N, β-catenin pathway, a key player in podocyte injury and glomerular sclerosis [33, 40], was enriched to be activated in Tg mice when compared to WT mice at 6-month-old age.
We then performed co-staining of Flag-B7-1 and β-catenin. Compared to WT mice (Supplementary Fig. S2), they were highly co-expressed in Tg mice (Fig. 2O). We also observed the nuclear expression of β-catenin in Tg mice (Fig. 2O), suggesting its activation. Western blotting analysis of active β-catenin and β-catenin further demonstrated their upregulation in Tg mice (Fig. 2P–R). We then performed the costaining of β-catenin and Podocalyxin, and found they were largely colocalized in Tg mice (Fig. 2S). These data suggest that B7-1 plays a key role in podocyte injury via β-catenin activation.
Knockdown of B7-1 protects against podocyte injury and glomerular damage through inhibiting β-catenin
To knock down B7-1 in ADR mice, we injected an shRNA vector encoding the interference sequence for B7-1 (pLVX-shB7-1) by a hydrodynamic approach . The interference efficiency of B7-1 was first testified in several organs. It showed that B7-1 was successfully interferred in kidney and liver (Supplementary Fig. S3). PAS staining showed that B7-1 knockdown strongly alleviated glomerular injury (Fig. 3B, C). The leakage of albuminuria was also significantly decreased by B7-1 knockdown (Fig. 3D).
We next performed costaining. As shown in Fig. 3E, B7–1 was upregulated in ADR mice and colocalized with α-actinin-4. We then assessed the relationship of B7-1 and β-catenin in isolated glomeruli from three groups of mice. As shown in Fig. 3F–H, B7-1 mRNA and protein were upregulated in ADR-glom, but inhibited by B7-1 knockdown. Furthermore, active β-catenin and β-catenin expression showed the same trend as B7-1 (Fig. 3G–I), suggesting the intimate correlation between them.
We then assessed podocyte injury and fibrosis. As shown in Fig. 3I–L, the expression of Nephrin, Zo-1, and Synaptopodin were decreased in ADR mice, but largely restored by B7-1 knockdown. TEM analysis also revealed that B7-1 knockdown could effectively maintain the integrity of podocyte foot processes (Fig. 3O). Furthermore, B7-1 knockdown in ADR mice strongly blocked the expression of collagen IV and Fibronectin, the fibrogenesis markers (Fig. 3K, M–O). We also knocked down B7-1 in 5/6 nephrectomy mice, a model of chronic renal failure with podocyte injury , and found the similar results (Supplementary Fig. S4). These results further suggest B7-1 plays an important role in podocyte injury through β-catenin signaling.
B7-1 mediates podocyte injury through activating β-catenin signaling in vitro and in glomerular mini-organ culture
We further assessed the role of B7-1 in podocyte injury in cultured mouse podocyte cell line (MPC5) and glomerular mini-organ culture. MPC5 cells were firstly transfected with B7-1 siRNA and co-treated with ADR. Transcriptomic and heatmap analysis showed ADR downregulated epithelial cell differentiation and triggered the activation of Wnt/β-catenin signaling, along with the increase in cell apoptosis (Fig. 4A, Supplementary Fig. S8). GSEA further clarified β-catenin pathway was highly related with B7-1 signaling (Fig. 4B). Western blotting analysis (Fig. 4C, D) showed active β-catenin was upregulated in ADR-treated cells, but greatly inhibited by B7-1 knockdown, which was accompanied by the restoration of Nephrin and decrease in Desmin, a podocyte injury marker.
MPC5 cells were then transfected with B7-1 expressing plasmid. Ectopic B7-1 induced the increase in active β-catenin, and promoted its translocation into the nuclei (Fig. 4E). Concomitantly, B7-1 overexpression triggered the actin skeleton rearrangement, loss of epithelial properties, and epithelial to mesenchymal transition (EMT) in podocytes, as assessed by F-actin, Zo-1 and Fibronectin staining (Fig. 4F), and western blotting analysis of other podocyte epithelial or EMT markers (Fig. 4G, Supplementary Fig. S5). Furthermore, pretreatment with ICG-001, an inhibitor of β-catenin activation, could greatly inhibit B7-induced those effects (Fig. 4H, Supplementary Fig. S5).
We further testify the role of B7-1 in rat glomerular mini-organ culture (Fig. 4I). As shown in Fig. 4J, K, and Supplementary Fig. S5, transduction of lentivirus expressing B7-1 gene triggered the decrease in Podocin, Synaptopodin, Zo-1, Podocalyxin, and WT1, but increased the expression of active β-catenin and Fibronectin. However, ICG-001 could greatly inhibit these effects.
To deeply identify the function of B7-1 in podocyte and glomerular injury, we established B7-1flox/flox mice (Supplementary Fig. S5). To specifically knockout B7-1 in podocytes, we isolated glomeruli from B7-1flox/flox mice and transduced them with adenovirus encoding NPHS2-driving Cre recombinase (Fig. 4L, M). After that, the glomerular mini-organ culture was treated with ADR. As shown in Fig. 4M–O, and Supplementary Fig. S5, podocyte specific knockout of B7-1 could significantly restore the expression of Nephrin, decreased the expression of active β-catenin, Desmin, and Fibronectin. These data further suggest B7-1 plays a key role in podocyte injury via β-catenin signaling.
Hsp90ab1 plays a key role in signal transmission from B7-1 to the LRP5/β-catenin pathway
We tried to find the protein binding to B7-1. Through immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS), we found nearly 20 proteins binding to B7-1. We then performed GO enrichment analyses using B7-1-specific-binding partners (Fig. 5B). As shown, B7-1 was primarily involved in immune-related responses and cell homeostasis-related pathways. Notably, in B7-1-specific-binding partners, heat shock protein 90 alpha, class B member 1 (Hsp90ab1), a conserved molecular chaperone, contributed to most of these enriched pathways . STRING analysis of the protein-protein interaction network with β-catenin indicated that B7-1 linked to β-catenin through interacting with Hsp90ab1 (Fig. 5C). To verify it, we conducted co-immunoprecipitation (Co-IP) experiments in B7-1-overexpressed cells and podocyte-specific B7-1 Tg mice (Fig. 5D, E). As shown, B7-1 could perfectly bind with Hsp90ab1. MPC5 cells were pretreated with 17-allylaminogeldanammycin (17-AAG, a type of Hsp90 inhibitor targeting the N-terminal domain (NTD)), or transfected with siRNA to Hsp90ab1 NTD domain, an important region involving in modulating chaperone function. As shown in Fig. 5F, G, the interruption of Hsp90ab1 significantly inhibited the activation of β-catenin and the expression of its target PAI-1 in B7-1-overexpressed podocytes. We then assessed the expression of Hsp90ab1 in Tg mice. As shown in Fig. 5H, I, the expression of Hsp90ab1 was significantly induced in B7-1-Tg mice. The co-staining of Hsp90ab1 with Flag (the tag of podocyte B7-1) showed that Hsp90ab1 was increased in Tg mice and largely co-localized with B7-1 (Fig. 5J), compared with the weak expression in WT mice (Supplementary Fig. S2).
We then performed molecular docking of the conserved B7-1 protein sequence (containing IgC and IgV domains) and the full-length protein of Hsp90ab1 (highly homologous in evolution) using Discovery Studio. The best-predicted conformations are shown in Fig. 6A. BLAST analysis was performed to determine the conservative binding sites between Hsp90ab1 and B7-1 protein sequences. The results showed that the residues L65, K69, R168, and D170 in NTD domain of Hsp90ab1 could serve as the key sites binding with B7-1 (Fig. 6B). Subsequently, these 4 residues in Hsp90ab1 were mutated to alanine individually to construct mutated plasmids. An immunoprecipitation (IP) assay was performed in 293 T cells. As shown in Fig. 6C, D, compared with the full binding activity of wild-type Hsp90ab1 (Full) with B7-1, the Hsp90ab1 with a K69 residue mutation (▴2) significantly decreased its binding activity with B7-1. The other residues mutations (▴1, 3, 4) showed no effects. These suggested that K69 residue in Hsp90ab1 NTD domain might be the key binding site interacting with B7-1. To verify its role, MPC5 cells were first transfected with siRNA to silence the endogenous Hsp90ab1, and then transfected with B7-1 expressing plasmid and wild-type or K69-mutated Hsp90ab1 expressing plasmid. As shown in Fig. 6E, F, compared to wild-type (Full), a mutation at K69 residue (▴2) in Hsp90ab1 significantly repressed B7-1-induced expression of active β-catenin. Furthermore, the expression of Zo-1 and Podocalyxin was significantly restored by K69 mutation (▴2) in Hsp90ab1. Similar results were observed when Zo-1 was assessed by immunofluorescence (Fig. 6G).
It was reported that Hsp90ab1 could interact with LRP5 , an indispensable coreceptor of the Wnt/β-catenin signaling pathway . Therefore, we investigated the role of LRP5 in the B7-1/Hsp90ab1-induced β-catenin pathway. Considering that no LRP5 crystal structures have been published, we built the structure of the LRP5 protein from homologous models according to its conserved sequences. We then conducted docking analysis of B7-1, LRP5 and the functional dimer of Hsp90ab1, and determined the optimal conformation. They showed a perfect binding (Fig. 6H). The binding of B7-1, LRP5, and Hsp90ab1 was then demonstrated in B7-1-overexpressed MPC5 cells and podocyte-specific B7-1 Tg mice (Fig. 6I, J). The upregulation of LRP5 was also testified in Tg mice (Fig. 6K, L), and further identified to be colocalized with B7-1 in podocytes (Fig. 6M). The upregulation of LRP5 was also demonstrated in clinical nephropathy, and verified to be located in podocytes (Supplementary Fig. S7). In MPC5 cells, interference of LRP5 using siRNA transfection significantly blocked B7-1-induced activation of β-catenin and restored the expression of Synaptopodin (Fig. 6N, O). These results suggest that B7-1 could induce the activation of the LRP5/β-catenin pathway, and HspP90ab1, especially the K69 residue, plays a key role in signal transmission.
Consistent with the previous report , we also observed integrin β1 signaling was disrupted by B7-1 overexpression (Supplementary Fig. S8). Interestingly, our results further demonstrated Hsp90ab1 could also mediate the interaction between integrin β1 and B7-1, suggesting that Hsp90ab1 might also contribute to integrin activation and actin cytoskeleton reorganization (Supplementary Fig. S8).
Hsp90ab1 contributes to B7-1-induced podocyte injury and glomerular damage
To further demonstrate the role of Hsp90ab1 in B7-1 signaling, we treated podocyte-specific B7-1 transgenic (Tg) mice with adeno-associated virus (AAV) expressing the interference sequence of Hsp90ab1(AAV9-shHsp90ab1) using in situ injection to kidney (Fig. 7A). The 6-month-old Tg mice were injected with AAV9-shHsp90ab1 or control virus for three months (Fig. 7A). As shown in Fig. 7B, Hsp90ab1was increased in Tg mice at nine months old compared to WT mice, but this was blocked by AAV9-shHsp90ab1 injection. Furthermore, knockdown of Hsp90ab1 in Tg mice also inhibited the excretion of albuminuria (Fig. 7D). Western blotting analysis and immunostaining showed Hsp90ab1 knockdown inhibited the expression of LRP5, active β-catenin and β-catenin, and B7-1 (Fig. 7E–I). The expression of B7-1 in podocytes was also confirmed by costaining with α-actinin-4 (Fig. 7J).
We next analyzed glomerular injury. As shown in Fig. 7K, L, glomerular mesangium expansion was upregulated in Tg mice, but this was inhibited by Hsp90ab1 knockdown. Similarly, TEM analysis, immunostaining, and western blotting further identified podocyte injury and glomerular fibrotic changes were increased in Tg mice, but blocked by Hsp90ab1 knockdown (Fig. 7K–P).
Hsp90ab1 mediates B7-1-induced podocyte injury and glomerular damage in ADR mice
To further clarify the role of Hsp90ab1 in B7-1 signaling, ADR-treated mice were intraperitoneally injected with 17-AAG (Fig. 8A). As shown, 17-AAG dose-dependently decreased the excretion of urinary albumin (Fig. 8B), and significantly inhibited the expression of Hsp90ab1, LRP5, active β-catenin, and B7-1 (Fig. 8C, Supplementary Fig. S6). Their expression was also tested by immunostaining (Fig. 8D). The colocalization of B7-1 with α-actinin-4 was also detected in ADR mice (Supplementary Fig. S6). Furthermore, we found 17-AAG could significantly restore the expression of Nephrin, Synaptopodin and Podocalyxin, and protect the ultrastructure of podocyte foot processes (Fig. 8G and Supplementary Fig. S6).
We further checked the effects of Hsp90ab1 interference in the long-term model of ADR mice. Knockdown of Hsp90ab1 was performed by injection of shRNA to Hsp90ab1 (pLVX-shHsp90ab1) using hydrodynamic approach (Fig. 8H, I). As shown in Fig. 8J, K, Hsp90ab1 knockdown could interrupt the upregulation of B7-1, β-catenin, active β-catenin, and LRP5 in ADR mice. Furthermore, Hsp90ab1 knockdown greatly restored the expression of Nephrin and Podocalyxin, and the normal ultrastructure of podocyte foot processes, but decreased the expression of Fibronectin and glomerular mesangium expansion (Fig. 8L–P). Hsp90ab1 knockdown also decreased the leakage of albuminuria (Fig. 8P). These results further suggest Hsp90ab1 mediates B7-1-induced podocyte injury and glomerular damage.
B7-1 is a downstream target of β-catenin and facilitates a two-way network with β-catenin
MPC5 cells were pre-treated with ICG-001 and then transfected with B7-1 expressing plasmid. As shown, ICG-001 reduced B7-1 expression (Fig. 9A, B), suggesting β-catenin plays a key role in B7-1 signaling. Typically, β-catenin forms a complex with members of the T cell factors/lymphoid enhancer binding factor (TCFs/LEF) family, including TCF1/3/4 and LEF1, to regulate downstream gene expression . We found there are several potential binding sites in the B7-1 promoter regions for binding with TCFs/LEF (Fig. 9C). Chromatin immunoprecipitation (ChIP) assay showed ectopic expression of β-catenin promoted TCF1 and TCF4, but not LEF1 to bind to the TCFs/LEF binding consensus sequence regions in B7-1 gene promoter (Fig. 9D) and upregulated B7-1 mRNA levels (Fig. 9E), suggesting direct stimulatory effects of β-catenin on B7-1. Ectopic β-catenin-induced B7-1 was significantly reduced by ICG-001 treatment (Fig. 9F, G). Similarly, overexpression of Wnt1 or exogenous Wnt3a also increased the mRNA and protein expression of B7-1 (Fig. 9E–K), which was inhibited by ICG-001 treatment (Fig. 9H–K). We also cultured HUVEC and RMC cells and transfected them with β-catenin expression plasmid. Although there was a slight increase in B7-1 mRNA in β-catenin-overexpressed HUVEC cells, it was not statistically significant. As for RMC cells, there was no changes (Supplementary Fig. S9). These results further suggest that β-catenin-induced B7-1 could possibly be podocyte-specific.
Collectively, our results suggest that the B7-1/β-catenin pathway appears to create a two-way network through Hsp90ab1, and plays a key role in the pathogenesis of podocyte injury (Fig. 9L). B7-1 recruits Hsp90ab1 to form a complex together with LRP5 at the cell membrane. Hsp90ab1 serves as a chaperon protein for B7-1 and LRP5. The residue K69 in the Hsp90ab1 N terminal domain is the key binding site for B7-1, which facilitates the signal transmission from B7-1 to LRP5/β-catenin signaling activation. β-catenin translocates into nuclei to promote B7-1 expression through TCFs/LEF transcription factors. These form a reciprocal activation feedback loop, and Hsp90ab1, especially residue K69, plays a key role in this circuit.
To find a therapeutic strategy for glomerular diseases , a better understanding of its underlying mechanisms is required. Among all etiology factors, podocyte injury and the inflammatory response play critical roles in all kinds of glomerular diseases [46,47,48,49].
Podocytes are terminally differentiated epithelial cells that constitute the filtration barrier. Podocytes are divided into cell body, primary process, and foot process, and express slit diaphragm proteins including Nephrin, NEPH1, and FAT to mechanically intercept albumin molecules, and also contribute to the negative charge of the filtration barrier . Podocytes can also secrete matrix components for the formation of GBM and growth factors for the survival of endothelial cells . Interestingly, recent studies have shown podocytes could also serve as non-hematopoietic APCs [5, 13], playing a role in immune responses and inflammation in glomerular diseases . Hence, podocytes play a “gated key lock” role in glomerular diseases. Podocytes could present antigens to activate T lymphocytes and a series of immune responses through MHC II molecules and co-stimulatory protein B7-1 [13, 31]. The immune response contributes to the deterioration in various glomerular diseases such as LN, IgAN and MN [53,54,55]. Of note, the immunosuppressive therapies have achieved some efficacies, however, many patients are facing frequent relapse, temporary remission with reappearance of symptoms, and resistance to the immunosuppressive drugs [30, 56]. All of these evidences suggest that when podocytes acquire the characteristics of APC cells, this feature maybe trigger self-damage except activating immune response.
B7-1 is a co-stimulatory modulator, which primarily expresses in NK cells and macrophages. B7-1 binds with CD28 in T cells to induce their differentiation and activation . Interestingly, B7-1 is also expressed in podocytes and plays a role in T cell polarization in kidneys [5, 31]. Besides the immune response, B7-1 can also promote cell injury by disrupting integrin and NEPH1 in podocytes [31, 32]. However, the role of B7-1 in podocyte injury remains controversial in the past few years, because of its inconsistent staining results in human kidney biopsies [22,23,24]. Therefore, an innovative technology should be adopted in clinical samples and a clinical cohort study in glomerular diseases should be conducted. Notably, B7-1 protein could be hardly seen in long-term preserved tissues and maybe just appear in a small part of patients . The other puzzle is the unreliable treatment effects of abatacept . After withdrawal of it, relapse commonly occurs . The reason maybe lies that abatacept could only block the binding of B7-1 with CD28, it could not inhibit B7-1 expression. While the intrinsic role of B7-1 in podocyte’s self-injury should not be neglected. Thus, it is necessary to construct a podocyte-specific B7-1 transgenic model to assess its role in detail. Except that, one more specific and sensitive technology for B7-1 detection in human kidney tissues is also required.
RNAscope technology is an advanced technic for detecting weak expression, compared with immunological assessment . Using RNAscope, B7-1 was found nearly in all types of glomerular diseases. This is inconsistent with previous studies, as they did not find B7-1 antigen protein expression in DN or others [23, 24]. We thought the reason possibly lies that antigens decay quickly, while genes persist. Furthermore, we found B7-1 is primarily expressed in podocytes, although previous literatures showed B7-1 also exists in endothelial and tubular cells [16, 59, 60]. Several proofs proved our findings. First, we found B7-1 was highly colocalized with α-actinin-4, a podocyte marker. We also performed the co-staining of B7-1 with EMCN, an endothelial cell marker, and found that there was only a very small part of co-localization. Second, to compare B7-1 expression in glomerulus and tubules, we isolated them from ADR mice, and found glomerular B7-1 was highly upregulated in a time-dependent manner, while tubular B7-1 only showed slightly increase in the late stage. Third, to testify whether there is a relationship between urinary B7-1 with tubular injury, we tested urinary β2-MG, a marker for tubular cell injury-derived proteinuria. In our clinical cohort, we found urinary B7-1 had no correlation with it. While urinary B7-1 is highly positively correlated with ACR, an indicator for glomerular proteinuria. Therefore, all these proofs suggest that podocytes are the main source for B7-1 production. Of interest, our investigation focuses in newly diagnostic patients, and identify that urinary B7-1 significantly increased from CKD G1 stage. This implicates urinary B7-1 might serve as a diagnostic marker in glomerular diseases. While the exact prognostic significance of urinary B7-1, in the remission or relapse of the disease, requires a prospective study in a large cohort, which is beyond the scope of this study.
Using podocyte-specific B7-1 transgenic mice, isolated glomerular mini-organ culture from B7-1flox/flox mice and rat, CKD mice models, and clinical samples, etc., we proved that B7-1 itself is sufficient to induce podocyte injury. This finding strongly clarifies the role of B7-1 in podocyte injury. We also discovered β-catenin meditates B7-1 pathway. This was proved by multiple methods such as RNA sequencing and pharmacological inhibition. More importantly, we identified Hsp90ab1 is a key mediator for the signal transmission of B7-1 to β-catenin. We have also discerned its active binding site with B7-1. As a chaperone protein, Hsp90ab1 functions as “an accommodation house” for B7-1 and LRP5, and crucially facilitates the signal transmissions. Finally, we identified β-catenin triggers the transcription and translation of B7-1, suggesting B7-1 is an upstream factor for β-catenin through Hsp90 mediation, but also a downstream of β-catenin. Hence, we provided a new theory for podocyte injury, i.e. B7-1 mediates a vicious cycle through Hsp90ab1-mediated communication with the β-catenin pathway in podocytes. To targeted inhibit any node of the signal path would block the whole process. From the sequencing, we also found B7-1 involves in T cell immune response and podocyte cell apoptosis, these results also deserve detailed analysis in the future.
In our study, we found Hsp90ab1/β-catenin signaling is a novel and major downstream effector of B7-1. The previous findings reported the role of integrin signaling in B7-1-induced podocyte injury . We found that Hsp90ab1 is also involved in that. Both β-catenin and integrin signaling pathways may function in parallel or collectively. This could be assessed in the future. Nevertheless, we found that β-catenin plays a key role in B7-1-induced podocyte injury through Hsp90ab1. Our study further enriches the understanding of β-catenin pathway. Except of its classical induction through Wnt(s) , β-catenin could be activated by multiple factors such as Hsp90ab1. Consistently, we found that β-catenin could be activated by CB2/Src recently .
Our study identified Hsp90ab1 plays a key role in the signal transmission of B7-1 to β-catenin and podocyte injury. Thus, combining an Hsp90 inhibitor with abatacept in refractory nephrotic syndrome may achieve better therapeutic effects in patients. This deserves extensive further investigation. We also found β-catenin is both upstream and downstream effector of B7-1 signals. Hence, managing patients with β-catenin blocker should be also advantageous. Although more studies are needed, our study provides an important indication to help to resolve the ambiguous issues of B7-1 in podocyte injury and glomerular diseases.
The detailed methods are presented in Supplementary Material.
Human urine samples and kidney biopsies
Human urine samples and the fresh frozen kidney sections were collected from patients with newly diagnosis of primary glomerular disease. The control tissues were derived from paracancerous tissues of patients who were performed radical nephrectomy. The demographic and clinical data are presented in Supplementary Table S1.
The in situ hybridization was performed using the RNAscope® Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostic, Inc), and the detection probe against human B7-1 (421471, Advanced Cell Diagnositc Inc.), and negative control probe DapB (421471, Advanced Cell Diagnositc Inc.). According to the manufacturer’s, the fresh frozen kidney sections (6 μm) were fixed, dehydrated and pre-treated with hydrogen peroxide. For ISH staining, sections were then digested with protease III for 18 mins at room temperature, hybridized with target probes, amplified, and labeled with fluorophore Opal 570 at 40°C in the HybEZTM Oven. For co-staining with antibodies, we applied the RNA-protein Co-Detection Ancillary kit (323180, Advanced Cell Diagnostic, Inc) and pre-incubated with the primary antibodies before digestion. Detailed methods were shown in Supplementary Methods.
All mice were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China) or Cyagen (Cyagen Biosciences Inc, China). All animal studies were approved by the Animal Experimentation Ethic Committee at the Nanfang Hospital, and were performed in compliance with the Guidelines for the Care and Use of Laboratory Animal.
Generation of podocyte-specific B7-1 transgenic mice
The podocyte-specific B7-1 transgenic mice (Tg mice) were generated in C57BL/6 background by using PiggyBac transposon system. Genotyping was confirmed by PCR analysis in tail samples from mice at three weeks of age. The F0, F1 and F2 generations were all produced in Cyagen (Cyagen Biosciences Inc, China). In this study, the F1 generations were adopted. Tg and their control mice (WT) were sacrificed at indicated time. Tg mice at 6-month old were treated with adeno-associated virus 9 (AAV9) for interference of Hsp90ab1. Briefly, AAV9 carrying Hsp90ab1 interference sequences (AAV9-shHsp90ab1, 1 × 1012 copies/ml) or the negative control were established by HanBio company (Shanghai, China). Either AAV9-shHsp90ab1 or AAV9-NC was administered at 60 μl of volumes per mouse by in situ injections (six locations) to the cortex region. Mice were scarified and kidney tissues were harvested three months after AAV treatment.
Generation of B7-1flox/flox mice
The B7-1flox/flox mice were generated in C57BL/6 background by CRISPR/Cas9 system and produced in Cyagen (Cyagen Biosciences Inc, China). Genotyping was then confirmed by PCR analysis in tail samples from mice at three weeks of age.
Adriamycin (ADR)-induced nephropathy in mice
Male Balb/c mice (eight weeks of age, weighed 20–25 g) were administered ADR (11.5 mg/kg) by intravenous injection through tail vein. Urine samples were collected weekly to assess for albuminuria. Mice were euthanized at indicated time. Saline injection was applied to control mice. For gene interference of B7-1 or Hsp90ab1, hydrodynamic-based gene delivery approach was applied. Some mice were treated with 17-AAG at 5 or 10 mg/kg every other day. The detailed experimental designs are presented in Figures.
5/6 nephrectomy (5/6NX) model
For 5/6NX models, male CD-1 mice, weighing 23–25 g, were subjected to two surgical resections of two thirds of the left kidney and the whole right kidney, or sham operation, as previously described . Two weeks after the first operation (week 2), the 5/6NX mice were randomly divided into indicated groups.
Isolation of glomeruli and tubules
Glomeruli from mice were isolated as described . Briefly, mice were sacrificed and perfused with Dynabead M-450 (00388551, Invitrogen). The kidneys were digested in collagenase IV (1 mg/ml, Invitrogen) and pressed through a 100-μm cell strainer (BD Falcon, Bedford, MA), and then glomeruli were gathered using a magnetic concentrator, and the remaining fluids were centrifuged for collecting the tubules.
For isolation of rat glomeruli , SD rats were sacrificed in a sterile environment. Kidneys were harvested, decapsulated, minced, and then smashed down with a plunger through three sieves sequentially (200-100-60 mesh opening sizes). After washing and centrifugation, the glomeruli were resuspended in RPMI1640 with 10% FBS medium and plated on noncoated six-well plates for treatment with lentivirus expressing B7-1 (Genechem, Shanghai, China).
Cell culture and treatment
The conditionally immortalized mouse podocyte cell line (MPC5) was cultured and maintained as described previously . MPC5 cells were synchronized into quiescence by growing in serum-free medium, and then treated with 0.25 μg/ml of ADR or 100 ng/ml of recombinant Wnt3a protein for 24 h. Some cells were pre-treated with ICG-001 (5 μmol/L) or 17-AAG (1 μmol/L) for 1 h before indicated treatment. The plasmid or siRNA transfection was carried out using Lipofectamine 2000 reagent (11668-019, Invitrogen). 293 T cells, human umbilical vein endothelial cell (HUVEC) and rat mesangial cell (RMC) were also cultured by routine procedures.
Histology and immunohistochemical staining
Paraffin-embedded mouse kidney sections (3 μm) were prepared and performed immunohistochemical staining using routine protocols. The primary antibodies were indicated in Supplementary Table S2. Some sections were stained with periodic acid-Schiff (PAS) (BA4080A, BASO). Images were photographed by Olympus BX53 microscope with EMCCD camera. At least ten glomeruli per mice in one section were analyzed to quantify positive area through the Image Pro plus software V6.0 (Media Cybernetics, Inc., Rockville, USA).
The kidney cryo-sections and cover slips of cultured cells were fixed with 4% PFA for 15 min at room temperature, following incubating primary antibodies (Supplementary Table S2) overnight at 4 °C. The mouse isotype control IgG was also used to check the specificity of antibody. The glomeruli sediments were embedded in OCT and sectioned at 5 μm thickness, and then fixed in 4% PFA for 30 min at room temperature, following incubation with antibodies.
For immunofluorescence staining of B7-1, the kidney frozen sections were prepared from kidney tissues stored at −80 °C within one month. For F-actin staining, MPC5 cells were fixed with 4% PFA and performed staining according to manufacturer’s instructions (40734ES75; Yeasen, Shanghai, China). All images were taken by confocal microscopy (Leica TCS SP2 AOBS; Leica Microsystems, Buffalo Grove, IL) or Olympus DP80 microscope with EMCCD camera (Olympus, Tokyo, Japan).
Western blot analysis and coimmunoprecipitation
Western blot analysis was performed by routine procedures. In brief, tissues and cell pellets were lysed in lysis buffer containing protease inhibitors, and thoroughly homogenized to lysate by Lu Ka Sample Grinder (LUKYM24). Proteins were separated by 8% or 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with antibodies as indicated (Supplementary Table S2) overnight at 4 °C, and visualized with ECL. The coimmunoprecipitation procedure was as following: Protein lysates were immunoprecipitated overnight at 4 °C with indicated antibodies and protein A/G plus agarose (sc-2003; Santa Cruz Biotechnology). The precipitated complexes were washed for five times and boiled in SDS sample buffer followed by immunoblotting. All of the coimmunoprecipitation experiments were repeated at least three times. Original western blots for all relevant figures are shown in “Supplementary File 1—Full unedited gels”.
Protein-binding sites prediction
The molecular structures of B7-1, Hsp90ab1 and integrin β1 were obtained from the RCSB PDB database, and the structure of LRP5 was established based on the homology modeling technique in Discovery Studio 2019. Sequence analysis showed a high conservation between human and mouse sequences of B7-1 and Hsp90ab1 protein. Molecular docking was performed using ZDOCK and RDOCK program (in Discovery studio 2019), and the optimal binding conformation was analyzed.
The gene ontology (GO) function annotation of genes from RNA-seq were based on the GO database (http://geneontology.org), and the functional enrichment analyses of DEGs were performed using Clusterprofiler in R through Fisher’s Exact Test (P < 0.05). Heatmaps of relative gene expression from RNA-seq were generated based on FPKM values using TBtool software . For GSEA analysis, the involved gene sets were all derived from the Molecular Signatures Database of GSEA web interface. The GO-based pathway analysis and enrichment of the differential proteins was performed on Metascape (http://metascape.org) as described . The protein-protein interaction network was established using STRING database (http://string-db.org).
Statistical analyses were performed using SPSS 20.0 (SPSS Inc. Chicago, IL). All data were presented as means ± SEM. Two group comparisons were made using unpaired Student’s t test. Multiple group comparisons were assessed using one-way ANOVA. Correlation between urinary B7-1 and albumin/urine creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), β-2-microglobulin (β2-MG) was determined using Spearman (nonparametric) analysis. P < 0.05 was considered statistically significant.
Transcriptomic data produced in this study are available at NCBI with accession number PRJNA765221 and PRJNA765122. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD28856. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.
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The authors thank Dr. Nan Jia for his assistance in pathology consultation. The authors also thank Cyagen Biosciences Inc (Guangzhou, China) for providing podocyte-specific B7-1 transgenic mice and B7-1flox/flox mice.
This work was supported by National Key R&D Program of China (2020YFC2005000), National Natural Science Foundation of China Grant 82070707, 91949114; and the project of Innovation team of chronic kidney disease with integrated traditional Chinese and Western Medicine (2019KCXTD014); and Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2019J013, 2021J001) and the Presidential Foundation of Nanfang Hospital (Grant No. 2019Z006), and Guangdong Provincial Clinical Research Center for Kidney Disease (No. 2020B1111170013).
The authors declare no competing interests.
All human studies were performed with informed patient consent and were approved by the Institutional Ethics Committee at Nanfang Hospital (NFEC-2019-209). All animal studies were approved by the Animal Experimentation Ethic Committee at the Nanfang Hospital, and were performed in compliance with the Guidelines for the Care and Use of Laboratory Animal.
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Li, J., Niu, J., Min, W. et al. B7-1 mediates podocyte injury and glomerulosclerosis through communication with Hsp90ab1-LRP5-β-catenin pathway. Cell Death Differ (2022). https://doi.org/10.1038/s41418-022-01026-8