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Research ArticleNephrology
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10.1172/jci.insight.175220
1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
Find articles by Khan, Y. in: JCI | PubMed | Google Scholar
1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
Find articles by Baldwin, W. in: JCI | PubMed | Google Scholar
1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
Find articles by Dvorina, N. in: JCI | PubMed | Google Scholar
1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
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Cravedi, P.
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1Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
2Nephrology, Dialysis and Kidney Transplant Unit, IRCCS Azienda Ospedaliero- University of Bologna, Italy.
3Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
4Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
5Unità Operativa Nefrologia, Azienda-Ospedaliero University of Parma, Department of Medicine and Syrgery, University of Parma, Italy.
6Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Address correspondence to: Paolo Cravedi or G. Luca Gusella, Icahn School of Medicine at Mount Sinai, 1 Levy Place, 10029 New York, New York, USA. Phone: 212.241.3349; Email: paolo.cravedi@mssm.edu (PC). Or to: Phone: 212.241.9597; Email: luca.gusella@mssm.edu (GLG).
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
Find articles by Gusella, G. in: JCI | PubMed | Google Scholar
Authorship note: SB and MY are co–first authors. PC and GLG are co–senior authors.
Published June 24, 2024 - More info
Published in Volume 9, Issue 12 on June 24, 2024
AbstractPatients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1–/– C3–/– mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1–/– C3–/– mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1–/– C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.
Graphical Abstract
Introduction
Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal genetic disorder caused mainly by mutations of the PKD1 or PKD2 gene, which code for polycystin-1 (PC1) or polycystin-2 (PC2), respectively (1, 2). The disease is characterized by the development and growth of tubular cysts that undermine the renal architecture, leading to renal failure in 50% of patients by the age of 60 (3). ADPKD kidneys are in a state of chronic injury and inflammation due to progressive cyst expansion and tissue remodeling (4).
Complement components have been described in the urine and cystic fluid of ADPKD patients, suggesting that complement system activation may play a role in ADPKD pathogenesis (5–8). The complement system comprises over 30 circulating and membrane-bound proteins, activated as a proteolytic cascade through 3 different pathways: classical, lectin, and alternative. Complement split products act as opsonins, chemoattractants, and serve as modulators of innate immune cell function (8). More recently, complement has been shown to also modulate adaptive immune cell responses and nonimmune processes such as cell survival, proliferation, and metabolism (8).
Complement activation is tightly regulated to prevent bystander damage to self-cells (9). This regulation is accomplished through the expression of membrane-bound and soluble complement-regulating proteins. Among these, decay-accelerating factor (DAF or CD55) is a glycophosphatidylinositol-anchored, membrane-bound complement regulator that accelerates the decay of cell surface–assembled C3 convertases, thus limiting the downstream complement activation and formation of cleavage products, including C3a and C5a (10). Notably, DAF functions intrinsically, restricting complement activation only on the surface of the cells on which it is expressed. Data by our group and others have shown a critical role for DAF in limiting complement activation in numerous immunological processes and in glomerular diseases (11–13).
Other complement regulators include the surface-expressed membrane cofactor protein (MCP or CD46 in humans and Crry in mice) that has both decay-accelerating function and cofactor activity (8), CD59 that inhibits formation of the membrane attack complex (MAC or C5b-9), CR1, and the soluble complement factor H (CfH) (8).
Whether and how PKD1 dysregulation affects complement activation, and how the complement system regulates cystogenesis remain unclear. Herein, we assessed the relationship between complement activation and cyst formation using both in vitro and in vivo Pkd1-deficient murine systems.
ResultsComplement and complement regulators in Pkd1–/– kidneys. To test the role of complement in PKD progression, we used a slowly progressing conditional model of renal cystogenesis in which the Pkd1 gene can be ablated by the expression of Cre recombinase under the control of the doxycycline-inducible (Dox-inducible) promoter (Pax8rtTA;TetO-Cre;Pkd1fl/fl mice; hereafter referred to as Pkd1–/–) (Figure 1A). At 16 weeks of age, Pkd1–/– mice show overt cystogenesis (14). To determine the presence of complement, we evaluated C3b deposition in kidneys at 16 weeks of age. While barely detectable in WT kidney tubules, C3b deposition was evident in renal tubular epithelial cells from Pkd1–/– mice (Figure 1, B and C).
Figure 1Pkd1–/– kidneys show signs of complement activation. (A) WT (n = 10), Pkd1–/– C3+/+ (n = 9), and Pkd1–/– C3–/– (n = 14) animals were fed doxycycline-supplemented (DOX-supplemented) chow for 4 weeks, starting at 4 weeks of age. Kidneys were collected at weeks 16 of age. Immunofluorescence detection and corresponding quantification (MFI) of (B and C) C3b, (D and E) DAF, (F and G) CD59, and (H and I) MAC. ***P < 0.001, ****P < 0.0001 by unpaired, 2-tailed t test. Scale bar: 100 μm (applies to all images).
Complement cascade activation is tightly controlled by specific soluble and membrane-bound inhibitors, such as DAF and CD59 (8). Compared with WT controls, we found a significant reduction in DAF protein expression in Pkd1–/– kidneys (Figure 1, D and E), suggesting that spontaneous activation of the alternative complement pathway in ADPKD may be triggered by DAF downregulation. Similarly, the expression of CD59 was reduced in Pkd1–/– kidneys (Figure 1, F and G) concomitantly with the accumulation of MAC in tubular membranes (Figure 1, H and I). Overall, these data suggest that downregulation of complement regulators DAF and CD59 in Pkd1–/– kidneys leads to the activation of the complement cascade and to MAC deposition.
Increased expression of complement genes and downregulation of complement regulators in Pkd1–/– kidneys. The main source of circulating complement components is the liver, but other tissues, including the kidney, can produce complement proteins (8). Therefore, to determine whether Pkd1–/– kidneys represent a source of complement, we assessed the expression of complement genes by quantitative real-time PCR (RT-PCR). The results showed that while the genes for all tested complement effector components were expressed in WT kidneys, C3, C5, and complement factor B (CfB) mRNA expression was significantly higher in Pkd1–/– kidneys (Figure 2A). The expression of complement receptors C3ar and C5ar1 was also significantly increased in Pkd1–/– kidneys (Figure 2A). While no differences were observed in the mRNA levels of CfH between Pkd1–/– and WT kidneys, the expression of Daf and CD59 genes was significantly reduced in Pkd1–/– kidneys (Figure 2A), indicating that the parallel reduction in these factors observed on kidney sections (Figure 1, D–G) was at least in part due to transcriptional downregulation. mRNA expression of Crry, another complement regulator, was also reduced in Pkd1–/– kidneys (Figure 2A).
Figure 2Pkd1–/– tubular cells are a source of complement components. Complement-related gene expression in (A) WT (n = 4) and Pkd1–/– (n = 4) kidneys and in (B) WT and Pkd1KO tubular cell lines. Each dot represents (A) 1 animal or (B) the average of separate experiments. (C) Representative plots (left) and data quantification (right) of DAF and CD59 expression in WT and Pkd1KO epithelial cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, 2-tailed t test (A and B) or 2-way ANOVA with Tukey’s multiple-comparison test (C). NS, not significant.
Pkd1KO tubular cells recapitulate the complement-related gene expression observed in Pkd1–/– kidneys. To determine the relationship between PC1 expression and complement regulation in renal tubular cells, we used a WT renal collecting duct cell line and the derived Pkd1KO cells, which reproduce the deletion of the Pkd1–/– model (see ref. 15 for further details).
We observed that complement gene expression in Pkd1KO cells largely recapitulated the data generated in Pkd1–/– kidneys (Figure 2B), suggesting that the effect on complement genes expression directly results from the loss of Pkd1 in epithelial cells. Compared with controls, Pkd1KO cells also showed a significant downregulation of CfH (Figure 2B), but this difference was not present between Pkd1–/– and WT kidneys (Figure 2A). We confirmed that the downregulation of Daf and CD59 mRNAs in Pkd1KO cells corresponded to a significant decrease in these complement regulator factors at the protein level (Figure 2C).
Overall, these data suggest that the increased production of complement components and reduced expression of complement regulators could be responsible for the local complement activation in Pkd1–/– kidneys.
C3 gene deletion delays renal cystogenesis. To test the causative role of complement activation in ADPKD progression, we crossed Pkd1–/– mice with C3–/– mice (both strains are on a B6 background) to generate Pkd1–/– C3–/– animals. In these mice, lack of complement component C3 prevents the formation of C3 convertase and downstream generation of C3a, C5a, and MAC. Under normal conditions, C3–/– mice do not present any specific phenotype, but exhibit increased mortality after infection by group B streptococci (16). We fed Pkd1–/– C3–/– mice and Pkd1–/– C3+/+ controls Dox-supplemented food following the experimental design in Figure 1A. At 16 weeks of age, Pkd1–/– C3+/+ kidneys appeared overtly cystic, while cystogenesis in Pkd1–/– C3–/– kidneys was significantly reduced (Figure 3A). Consistent with the decrease in cyst formation, Pkd1–/– C3–/–mice showed lower kidneys/body weight ratios and normal blood urea nitrogen (BUN) values (Figure 3, B–D). No significant sex-specific differences were observed. The significantly lower number of Ki-67+ cells in Pkd1–/– C3–/– compared with control Pkd1–/– C3+/+ kidneys indicates reduced cell proliferation in the double knockout (Figure 3, E and F).
Figure 3Deletion of C3 reduces the severity of cystic Pkd1–/– phenotype. (A) Representative PAS staining of kidneys from Pkd1–/– C3+/+ (n = 9) and Pkd1–/– C3–/– (n = 14) mice. (B) Cystic index, (C) kidneys/body weight ratio, and (D) BUN in WT (n = 10), Pkd1–/– C3+/+, and Pkd1–/– C3–/– mice. Female and male mice are indicated by empty and full circles, respectively. **P < 0.01; ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test. NS, not significant. (E) Immunofluorescence detection and (F) quantification of Ki-67+ (red) and DBA+ (green) cells in the same mice as in A; # indicates cysts. **P < 0.01 by unpaired, 2-tailed t test. Scale bars: 50 μm.
Overall, these data demonstrate that the co-deletion of C3 in Pkd1–/– mice reduces cystogenesis and preserves renal function.
C3 deletion associates with reduced inflammation and immune infiltrates in Pkd1–/– kidneys. ADPKD is associated with progressive intrarenal inflammation, a process that is thought to promote cystogenesis (17, 18). When we analyzed the levels of proinflammatory cytokines in whole kidneys, we found that Pkd1–/– C3–/– mice expressed significantly lower levels of Tnfa, Ccl2, Il6, and Il1b mRNAs than Pkd1–/– C3+/+ controls (Figure 4, A–D).
Figure 4C3 deletion reduces inflammatory cytokine expression in Pkd1–/– kidneys. Quantitative RT-PCR for the detection of (A) Tnfa, (B) Ccl2, (C) Il6, and (D) Il1b gene expression in kidneys from WT (n = 3), C3–/– (n = 5), Pkd1–/– C3+/+ (n = 7), and Pkd1–/– C3–/– (n = 6) mice. Expression is reported as fold change relative to WT control average. **P < 0.01; ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.
In agreement with reduced inflammatory cytokine expression, we found that Pkd1–/– C3–/– kidneys have significantly lower F4/80+ macrophage infiltrates than Pkd1–/– C3+/+ controls (Figure 5, A and B). Quantitative RT-PCR analyses of Pkd1–/– C3–/– kidneys showed lower Cd11b mRNA expression, supporting a reduction in macrophage recruitment/expansion in the kidney in the absence of complement activation (Figure 5C), and a decrease in the dendritic cell marker Cd11c mRNA (Figure 5D), indicating that complement may also affect other myeloid cell expansion.
Figure 5C3 deletion reduces immune infiltrates in Pkd1–/– kidneys. (A) Immunofluorescent staining and (B) relative quantification of F4/80 in kidneys from Pkd1–/– C3+/+ (n = 7) and Pkd1–/– C3–/– (n = 6) mice. Quantitative RT-PCR assessment of the expression of immune cell gene markers (C) Cd11b, (D) Cd11c, (E) Cd8, (F) Cd4, and (G) Foxp3 in Pkd1–/– C3+/+ (n = 7) and Pkd1–/– C3–/– (n = 6) kidneys. Expression is reported as fold change relative to WT control average. (H) Representative pictures of T cell infiltrates (CD8+) in the same kidneys, with magnified area of the inserts (bottom). *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired, 2-tailed t test. Scale bars: 100 μm (A), 250 μm (H, top), and 50 μm (H, bottom).
Immune infiltrate analyses also showed a reduction in transcripts for CD4+ and CD8+ T cells and Foxp3+ regulatory T cells (Figure 5, E–G). Immunohistochemical staining confirmed lower infiltrates for CD8+ T cells (Figure 5H), suggesting that in the absence of C3 the recruitment of immune infiltrates in the kidney is overall decreased.
C5aR1 activation mediates Pkd1KO cell proliferation. Epithelial cell hyperproliferation supports the expansion of renal cysts and is a hallmark of ADPKD. Pkd1KO cells released higher levels of C5a (Figure 6A), which has been shown to stimulate the growth of other cells, including leukocytes (8). Therefore, we hypothesized that local complement activation in Pkd1–/– kidneys drives tubular cell proliferation and cyst growth. To test this hypothesis, we measured cell growth following treatment of WT and Pkd1KO cells with C3aR or C5aR1 antagonists. While C3aR antagonism had no significant effect on Pkd1KO cell proliferation,the C5aR1 antagonist significantly rescued the proliferative phenotype of the Pkd1KO cells, confirming the role of local complement activation in supporting the growth of Pkd1-deficient cells (Figure 6B).
Figure 6C5aR1 antagonist and DAF overexpression reduce Pkd1KO cell proliferation. (A) Levels of C5a (pg/mL) in supernatant obtained from cultured WT and Pkd1KO tubular cells. (B) WT and Pkd1KO tubular cell proliferation in the presence or absence of C3aR (10 μM) or C5aR1 (10 μM) antagonist. (C) WT, Pkd1KO, and Daf-transgenic WT/VEAVP-Daf and Pkd1KO/VEAVP-Daf tubular cell proliferation. Cell proliferation after 72 hours of culture is represented as percentage of change in cell number relative to number of cells at time 0. *P < 0.05; **P < 0.01 by unpaired, 2-tailed t test (A) or 2-way ANOVA with Tukey’s multiple-comparison test (B and C). NS, not significant.
DAF overexpression rescues abnormal Pkd1KO cell proliferation. Because DAF downregulation is a mechanism of local complement activation through the alternative pathway (8), we examined whether increased complement activation and C5a/C5aR1–induced tubular cell proliferation were driven by DAF downregulation in Pkd1-deficient cells. We therefore measured the proliferation of Pkd1KO cells following overexpression of DAF.
WT and Pkd1KO cells were transduced with the VEAVP-Daf or VIN16-Daf (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.175220DS1) lentivector that allows the constitutive or inducible expression of DAF, respectively. Proliferation of Pkd1KO/VEAVP-Daf cells was reduced to the control WT/VEAVP-Daf levels, suggesting that DAF overexpression inhibits the hyperproliferation of Pkd1KO cells by preventing activation of locally produced complement (Figure 6C).
CTT overexpression rescues DAF expression and abnormal Pkd1KO cell proliferation. The proteolytically cleaved cytoplasmic C-terminal tail of the PC1 protein (PC1-CTT) has been shown to interact with different transcription factors and to be involved in cell proliferation (19–22). To directly test the hypothesis that PC1-CTT loss is responsible for DAF downregulation, WT and Pkd1KO cells were transduced with a lentivector constitutively expressing PC1-CTT (Figure 7A). We found that the expression of PC1-CTT in Pkd1KO cells restored the levels of DAF protein expression (Figure 7, B–D), thus establishing a direct link between PC1-CTT and control of Daf expression. Importantly, PC1-CTT expression in Pkd1KO cells normalized their proliferation to WT control levels (Figure 7E), in further agreement with a critical role of DAF downregulation and complement activation in Pkd1KO cell proliferation.
Figure 7PC1-CTT overexpression rescues DAF expression and abnormal Pkd1KO cell proliferation. (A) Western blot of PC1-CTT-S (using anti–S-tag antibody) in lentivirally transduced WT/PC1-CTT and Pkd1KO/PC1-CTT cells. Actin expression was measured as loading control. DAF protein expression measured by (B) flow cytometry and (C) immunofluorescence (original magnification, ×200) was reconstituted in Pkd1KO cells after the exogenous expression of PC1-CTT. (D) Quantification of DAF signal by immunofluorescence from 2 independent experiments (gray and black symbols): each data point represents the mean fluorescence per cell from 1 field; n is the total number of cells analyzed in each group. (E) Proliferation of WT and Pkd1KO cells with or without PC1-CTT. Cell proliferation is represented as percentage change in cell number after 72 hours of culture relative to the number of cells at time 0. ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test. NS, not significant.
DiscussionEvidence of complement activation has been reported in murine models of ADPKD and in affected patients (5, 23, 24) and is supported by single-cell RNA sequencing data (25), but the role of complement in cyst growth and the mechanisms underlying this process were unclear. Herein, we showed that genetic deletion of C3 in a murine ADPKD model ameliorates the cystic phenotype, suggesting that complement activation plays an important role in the pathogenesis of ADPKD. Our findings also indicate that Pkd1 deletion is associated with an upregulation of complement genes and a downregulation of complement regulator genes in renal tubular cells.
The increased expression of complement-related genes (C3, C5, CfB) and C3b deposition in conditional Pkd1–/– kidneys and in a Pkd1KO tubular epithelial cell line (15) support the concept that epithelial cells have a role in renal complement production. We further showed that C5ar1 is significantly upregulated in Pkd1–/– tubular cells, thus suggesting the possibility of a paracrine or autocrine stimulatory mechanism. In cancer cells (26, 27), the activation of complement can promote cell growth, which is characteristically abnormally sustained in cystic cells. Our data indicate that the growth of Pkd1KO cells is normalized by a specific C5aR1 antagonist, indicating C5a/C5aR1 signaling as a main contributor to the overproliferative phenotype of mutant cells, likely enabled by the significantly higher expression of C5aR1 on these cells. Of note, the fact that C5a was found in the supernatants of WT and, to a higher extent, of Pkd1KO cells indicates that these cells possess the machinery to produce and activate complement products.
Critical to this mechanism is our finding of the reduced expression of the membrane-bound complement regulators DAF and CD59, which associates with unleashed activation of the complement cascade. The pathogenic role of complement regulator downregulation is corroborated by the normalization of Pkd1KO cell growth following DAF overexpression, which further emphasizes the potential role of complement signaling in cystic cells. This effect suggests a mechanistic link between PC1 and DAF expression, as the overexpression of PC1-CTT in Pkd1KO cells normalizes DAF expression.
These conclusions are supported by our in vivo data showing that genetic deletion of C3 significantly slows cyst development in Pkd1–/– mice. C3 is a critical component of the complement cascade, and its deletion prevents formation of C3a, C5a, and deposition of MAC on cell membranes. Our data confirm and extend prior evidence that rosmarinic acid, a nonspecific inhibitor of the complement system, improves disease severity in murine ADPKD models (24). Therefore, these findings indicate that the deposition of complement split products in ADPKD kidneys and the presence of complement components in cystic fluid (5, 23,
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