Inhibition of the inflammasome ameliorates orthologous polycystic kidney disease

Significance

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited kidney disorder and leads to kidney failure in most affected persons. No effective treatments exist. Characterized by cyst formation and kidney enlargement, inflammation has emerged as a crucial component, yet little information on specific pathways exists. In a mouse model, we identified inflammasomes, part of the innate immune system, as a major driver of disease progression. Disrupting inflammasomes, either genetically or with a drug, reduced inflammation and cyst growth, and preserved kidney function. For the first time, we uncovered a distinct pool of inflammasomes within kidney tubule cells. These findings identify the inflammasome as a critical contributor to ADPKD and suggest its inhibition as a promising therapeutic strategy.

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease. Limited treatment options lead to renal failure in the vast majority of affected individuals. Novel therapeutic approaches are needed. Recent evidence has identified inflammation as an important driver of ADPKD. We analyzed transcriptional profiles in an orthologous Pkd1 mouse model and found a strong upregulation of the inflammasome pathway. To investigate the role of inflammasomes on cyst formation and kidney function, we modulated inflammasome activity through genetic targeting of the essential inflammasome component Pycard/Asc or treatment with the inflammasome inhibitor MCC950. Genetic deletion of Pycard/Asc in Pkd1 mutant mice significantly reduced cyst formation, and kidney function was improved. Reductions were seen in inflammation, fibrosis, and urinary excretion of IL-18. Analogous results were obtained through tubule-specific inactivation of Pycard/Asc or treatment of Pkd1 mutant mice with the inflammasome inhibitor MCC950. These findings demonstrate that inflammasomes act as drivers of disease severity in an orthologous mouse model of ADPKD. We pinpoint a separate, epithelial pool of inflammasomes in the diseased kidney and identify inflammasome inhibition as a promising strategy for the treatment of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is a renal ciliopathy and one of the most prevalent genetic diseases in humans with an incidence of 1:1,000 (1–6). Most affected individuals carry monoallelic loss-of-function mutations in one of the 2 PKD genes, PKD1 or PKD2, with mutations in a small number of other genes reported (7–10). The disease is recessive at the cellular level: loss of heterozygosity in individual tubular cells or reduction of polycystin dose through other factors in an otherwise normal tubule causes sporadic cyst formation (11–13). Clinically, cysts increase in number and size, which results in the enlargement of kidneys to several times their normal size (14). Kidney enlargement is followed by a progressive decrease in renal function, leading to kidney failure in the majority of affected persons (15). Other organs may be involved, most notably the liver, however, liver failure usually does not occur and liver transplantation is only performed in selected individuals, mostly because of organomegaly (16, 17). While rare, ruptures of intracranial aneurysms occur and tend to cluster in families (18).

Treatment of ADPKD consists of blood pressure control, which slows renal growth and glomerular filtration rate (GFR) decline (19). Tolvaptan, a vasopressin receptor antagonist, has been approved for therapy of ADPKD (20, 21). However, its widespread use is limited due to polyuria, on average 7 L per day (22). Studies with SGLT2-inhibitors, which demonstrated benefits in diabetic, as well as nondiabetic, kidney disease, excluded patients with ADPKD, thus precluding their use. Furthermore, SGLT2-inhibitors increase vasopressin levels and have not been successful in orthologous mouse models (23, 24).

The lack of effective treatment options makes renal replacement therapy a certain outcome for the vast majority of affected patients. Kidney failure occurs as early as in the late twenties (15). Persons with a combined kidney volume of >1,5 l loose GFR at a rate of 5 mL/min/y (14). Half of the individuals in the highest risk group (Mayo Class 1E) will have experienced kidney failure by age 45. However, even in aggressive cases, there is a potential treatment window of many years, providing a rationale to identify therapeutic targets.

Inflammation is a prominent feature in ADPKD (25–28): miRNAs associated with inflammation were among the most deregulated in ADPKD (29); cytokines are concentrated in cyst fluid (30, 31) and patient urine, and correlate with disease severity (32–38); the macrophage chemoattractant protein CCL2 is highly abundant in the renal parenchyma of ADPKD kidneys and macrophages have been shown to promote cyst growth (39–42). Indeed, kidney-specific inactivation of CCL2 ameliorates cystogenesis in a Pkd1 mouse model (43, 44).

Lately, observations have emerged implicating a role for inflammasomes in ADPKD: As part of the innate immune system and activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), inflammasomes are macromolecular complexes consisting of multimeric pattern recognition receptors, the adaptor protein ASC, encoded by the Pycard gene, and an effector kinase of the caspase family (45, 46). Upon assembly of the NLRP3 inflammasome, active caspase 1 cleaves pro-IL-1β and pro-IL-18 into their mature forms, which are then secreted. The NLRP3 inflammasome is known to play a role in gout and oxalate-induced renal disease (47–52). IL-1β was increased in sera of patients with ADPKD compared to a group of patients with other forms of chronic kidney disease (53). IL-1β was also upregulated in ADPKD renal tissue, and genetic deletion in a Pkd1 mouse model led to a milder phenotype (54). IL-18 is expressed in cyst lining cells in rats and was detected at markedly elevated levels in human cyst fluid as well as the urine of a subset of ADPKD patients (31). A study in an orthologous murine model of ADPKD found a modest, sex-specific effect of genetic Casp1 silencing, with decreased cyst formation in female, but not in male mice (55). Here, we show that inflammation, and specifically inflammasome-associated transcripts, are upregulated early in Pkd1 inactivated mouse kidneys. Using a loss-of-function approach, we show that inflammasomes contribute to ADPKD severity and decline in renal function and that this pathophysiology is amenable to therapeutic targeting. Tubule-specific inactivation of the inflammasome demonstrates the existence of separate pools of inflammasomes in the kidney.

Results

Pkd1 Deletion in Mouse Kidneys Increases Expression of Inflammasome-Associated Transcripts.

We performed a transcriptional screen in mice with a time-dependent targeted deletion of Pkd1 (SI Appendix, Fig. S1 A and C). Pkd1^flox/flox.Pax8rtTA.TetO-Cre mice (Pkd1 ko) underwent tetracycline-induced tubular deletion of Pkd1 during postnatal week 5 and 6. At 10 wk, the kidneys were analyzed by RNAseq and the transcripts compared to kidneys from noncystic Pkd1^flox/flox.TetO-Cre control animals lacking Pax8rtTA (wt). The principal component analysis (PCA) is shown in Fig. 1A. Overall, we found 3849 up- and 2226 down-regulated genes (Fig. 1B). Gene set enrichment analysis revealed a down-regulation of morphogenesis and developmental as well as epithelial and junctional pathways (Fig. 1C). Among the upregulated gene sets of Gene Ontology: Biological Process, 29 of the top 50 were connected to inflammation (Fig. 1D). To get more insight into the inflammatory processes in PKD, the upregulated Hallmark gene set Inflammatory Response was investigated at the level of its individual genes (Fig. 2A and SI Appendix, Fig. S1B). This revealed increased expression of Ccl2 among others, which is in keeping with the previously published literature (39, 43). Interestingly, among the 10 most significantly upregulated genes were Il-1b and Il-18 (Fig. 2A), suggesting that the inflammasome may be implicated in PKD. Indeed, analysis of the GO set for inflammasome complex components revealed a significant upregulation of Pycard, Casp1, Casp4, and Casp12 (Fig. 2 B and C). Taken together, transcriptional profiles of early PKD in a mouse model of induced deletion of Pkd1 show a strong inflammatory signature, which are in line with previous reports. This includes strong upregulation of inflammasome-associated transcripts which supports a role of inflammasomes in ADPKD.

Fig. 1.

Pkd1 deletion in mouse kidneys increases expression of transcripts associated with inflammation. (A) Principal component analysis (PCA) of the kidney transcriptome from Pkd1 ko and wild type 10-wk-old male mice (n = 3 mice per genotype, each dot represents an individual mouse). (B) Number of differentially expressed genes. Red: 3849 upregulated genes; Blue: 2226 downregulated genes. (adjusted P-value < 0.05, and log2FC > 1 or log2FC < −1) (C) Gene set enrichment analysis results of the GO:BP (biological processes) terms, showing the top 50 downregulated terms (by adjusted P-value) in Pkd1 ko in comparison to wt. (D) Gene set enrichment analysis results of the GO:BP (biological processes) terms, showing the top 50 up-regulated terms (by adjusted P-value) in Pkd1 ko. Terms in red represent those associated with inflammatory processes.

Fig. 2.

Pkd1 deletion in mouse kidneys increases expression of inflammasome-associated transcripts. (A) Volcano plot for the Hallmark: Inflammatory Response genes, showing the differential gene expression analysis of Pkd1 ko versus wt. Red indicates upregulated genes and blue indicates downregulated genes. Each dot represents one gene. Il-1$\beta$ and Il-18 are highlighted in blue, Ccl2 is highlighted in yellow. (B) Heatmap of genes of the GO:CC term Inflammasome Complex, comparing wild-type control and Pkd1 ko mice. Color code represents the Z-score of gene expression. Significant genes are marked with a black square (Left column). (C) Volcano plot for the genes of the GO:CC term Inflammasome Complex, comparing Pkd1 ko to wild-type control mice. Red indicates upregulated genes. Each dot represents one gene.

Effect of Genetic Deletion of the Inflammasome Component Pycard/Asc on the ADPKD Phenotype.

To directly address the question whether the inflammasome is a driver of cystogenesis, we chose a genetic approach. For this purpose, we crossed Pkd1 targeted mice with animals carrying a deletion of Pycard (the gene encoding Asc) to create Pycard/Asc^−/−.Pkd1^flox/flox.Pax8rtTA.LC1-Cre mice. After induction with tetracycline during weeks 5 and 6, the animals were killed after week 14 (Fig. 3A). Total body weight was similar in all groups (SI Appendix, Fig. S2A). Compared to Pkd1 ko mice, the Asc-deleted Pkd1 ko mice showed a significant decrease in kidney-to-bodyweight ratio (Fig. 3B and SI Appendix, Fig. S2B). Interestingly, kidneys from Pkd1 mutant mice with a monoallelic deletion of Asc (SI Appendix, Figs. S2B, S7 and S9, and Fig. 3B, group 3), while larger than mice with a biallelic deletion (SI Appendix, Figs. S2B, S7 and S8, and Fig. 3B, group 1), were significantly smaller than Pkd1 mutant mice without a deletion of Asc (SI Appendix, Figs. S2B, S7 and S8, and Fig. 3B, group 2), suggesting a possible dosage effect of Asc. Histology revealed that the cystic area was strongly reduced in Pkd1 mutant mice with Asc deficiency (Fig. 3D and SI Appendix, Fig. S8). Accordingly, the cystic index was significantly lower in the biallelic Asc -deficient Pkd1 mutant mice compared to animals with only the Pkd1 mutation (Fig. 3C). Renal function, as measured by serum creatinine and BUN, was lower in the biallelic Asc-deficient animals (Fig. 3 E and F). Urine analysis by ELISA demonstrated a significant decrease of IL-18 in the urine of Asc-targeted Pkd1 ko mice compared to Pkd1 ko only animals (Fig. 4A and SI Appendix, Fig. S2 C and D). Quantitative analysis by qRT-PCR from kidney tissue showed decreased expression of Il-1b and Il-18 in the biallelic Asc-targeted vs. Pkd1-only-targeted mice (Fig. 4 B and C). We found significantly fewer macrophages in Asc-targeted mice, the effect was stronger with a biallelic vs. a monoallelic mutation (Fig. 4 D and E). Ccl2 expression was significantly lower in the biallelic targeted Asc mice, compared to Pkd1 only targeted kidneys (Fig. 4F).

Fig. 3.

Genetic silencing of Pycard/Asc ameliorates the ADPKD phenotype. (A) Protocol timeline. (B) Kidney weight (kw)‐to‐body weight (bw) ratio in 14-wk-old mice with Asc ko or Asc wt allele, with Pkd1 ko or wt background. Asc ko designates biallelic and Asc het a monoallelic null allele. (C) Cystic index of 14-wk-old mice with the indicated genotypes. (D) Representative images of PAS staining of kidneys from 14-wk-old mice with the indicated genotypes. (Scale bar, 50 µm.) See also SI Appendix, Fig. S7 for macroscopic images and SI Appendix, Figs. S8 and S9 for low magnification views. (E and F) Serum creatinine (E) and BUN (F) of 14-wk-old mice with the indicated genotypes. B, C, E, F. Each dot represents one individual mouse. Two-way ANOVA followed by Tukey–Kramer test: *P < 0.05, **P < 0.01, ***P < 0.001, ns: not statistically different.

Fig. 4.

Genetic silencing of Pycard/Asc reduces chemokine expression and macrophage recruitment in ADPKD. (A) Urine IL-18 to urine creatinine ratio in 14-wk-old mice with the indicated genotypes. (B and C) mRNA expression of Il-18 (B) and Il-1ß (C) in kidneys from 14-wk-old mice, with the indicated genotypes. (D) Representative images of DAB immunohistochemical staining of F4/80 positive cells in kidneys from 14-wk-old mice with the indicated genotypes. (Scale bar, 50 µm.) (E) Proportion of F4/80 positive cells in kidneys from 14-wk-old mice with the indicated genotypes. (F) mRNA expression of Ccl2 in kidneys from 14-wk-old mice with the indicated genotypes. A–C, E, and F. Each dot represents one individual mouse. Two-way ANOVA followed by Tukey–Kramer test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not statistically different. a.u.: arbitrary units.

In summary, genetic ablation of the central inflammasome adaptor protein Asc resulted in a strong amelioration of cystic renal disease and better renal function in the inducible Pkd1-targeted mouse model.

Effect of Tubule-Specific Deletion of the Inflammasome Component Pycard/Asc on the ADPKD Phenotype.

IL-18 is excreted in higher amounts in the urine of ADPKD patients, suggesting the possibility that inflammasomes are active in renal tubular cells. To test this hypothesis, we employed a floxed allele of Pycard/Asc to inactivate Asc together with Pkd1 in renal tubular epithelial cells while leaving the inflammasome in immune cells intact (Fig. 5A). After 14 wk, the Asc-targeted Pkd1 ko mice had a significantly lower kidney/bodyweight ratio compared to Pkd1 only ko animals (Fig. 5B and SI Appendix, Fig. S3A). There was a strong, while not significant, trend to a decreased cystic index (Fig. 5 C and D). Kidney function, as determined by serum creatinine and BUN, was significantly better in the tubular Asc-targeted group and the normalized urinary excretion of IL-18 was significantly lower (Fig. 5 E–G and SI Appendix, Fig. S3 B and C). The number of mononuclear cells was significantly reduced in tubular Asc targeted kidneys (Fig. 5H, SI Appendix, Fig. S3G). No significant differences were observed in the total kidney expression of Ccl2, Il-18, and Il-1β (SI Appendix, Fig. S3 D–F). These findings reveal the disease relevance of renal tubular inflammasomes in ADPKD. Yet, the effect of tubular inflammasome silencing on disease severity is less when compared to Pkd1 ko animals with a complete Asc ko.

Fig. 5.

Tubular-specific deletion of Pycard/Asc ameliorates the ADPKD phenotype. (A) Protocol timeline. (B) Kidney weight (kw)‐to‐body weight (bw) ratio in 14-wk-old mice with Asc flox/flox (fl/fl) or Asc wt allele, with Pkd1 ko or wt background. Asc fl/fl designates biallelic tubular specific Asc conditional knock-out. (C) Cystic index of 14-wk-old mice with the indicated genotypes. (D) Representative images of PAS staining of kidneys from 14-wk-old mice with the indicated genotypes. (Scale bar, 50 µm.) See also SI Appendix, Fig. S7 for macroscopic images and SI Appendix, Figs. S8 and S9 for low magnification views. (E and F) Serum creatinine (E) and BUN (F) of 14-wk-old mice with the indicated genotypes. (G) Urine IL-18 to urine creatinine ratio in 14-wk-old mice with the indicated genotypes. (H) Proportion of F4/80 positive cells in kidneys from 14-wk-old mice with the indicated genotypes. B, C, and E–H. Each dot represents one individual mouse. Two-way ANOVA followed by Tukey–Kramer test: *P < 0.05, ***P < 0.001. ns: not statistically different.

Pharmacologic Inflammasome Inhibition Ameliorates the ADPKD Phenotype.

To address whether cystogenesis can be ameliorated by a pharmacological approach, we developed a treatment protocol with MCC950, an inhibitor of inflammasome activity (Fig. 6A) (56, 57). Pkd1 ko mice underwent intraperitoneal injections every other day of MCC950 or vehicle. Noncystic mice served as controls (wt). No significant changes were observed in body weight until killing at 14 wk (SI Appendix, Fig. S4 A and B). The kidneys of MCC950-treated Pkd1 ko animals were smaller than those of vehicle-treated Pkd1 ko mice (SI Appendix, Figs. S4C and S6). Correspondingly, we found lower kidney-to-body weight ratios, a reduced cystic index in MCC950 vs. vehicle-treated animals (Fig. 6 B–D). MCC950 treatment resulted in better renal function: BUN and serum creatinine were lower when compared with vehicle-treated mice (Fig. 6 E and F). Vehicle-treated Pkd1 mutant mice showed significantly higher urinary levels of IL-18 normalized for creatinine compared to mice treated with the inflammasome inhibitor (Fig. 6G and SI Appendix, Fig. S4 D and E). No urinary excretion of IL-1β was detected. The number of F4/80 positive macrophages tended to be lower in MCC950-treated Pkd1 mutant mice (Fig. 7 A and B), which was accompanied by a lower expression level of Ccl2 (Fig. 7C), a macrophage-associated chemokine that is involved in the pathogenesis of ADPKD. Both differences did not reach statistical difference. Kidney fibrosis was significantly lower in Pkd1 mutant animals treated with the inflammasome inhibitor compared to vehicle-treated animals (Fig. 7 D and E and SI Appendix, Fig. S5A). These data demonstrate that treatment of Pkd1 mutant ADPKD mice with the inflammasome inhibitor MCC950 significantly reduces cyst growth and improves renal function.

Fig. 6.

Pharmacologic inflammasome inhibition ameliorates the ADPKD phenotype. (A) Protocol timeline. (B) Kidney weight (kw)‐to‐body weight (bw) ratio in 14-wk-old Pkd1 ko and wt mice, treated with intraperitoneal injections of MCC950 or vehicle. Total weight of both kidneys was used. (C) Cystic index of 14-wk-old mice with the indicated genotypes and treatment. (D) Representative images of PAS staining of kidneys from 14-wk-old mice with the indicated genotypes and treatment. (Scale bar, 50 µm.) See also SI Appendix, Fig. S6 for low magnification views. (E and F) Serum creatinine (E) and blood urea nitrogen (BUN) (F) in 14-wk-old mice with the indicated genotypes and treatment. (G) Urine IL-18 to urine creatinine ratio in 14-wk-old mice with the indicated genotypes and treatment. B, C, and E–G. Each dot represents one individual mouse. Two-way ANOVA followed by Tukey–Kramer test: * P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Effect of inflammasome inhibition on macrophage infiltration and fibrosis. (A) Representative images of DAB immunohistochemical staining of 14-wk-old mice with the indicated genotypes and treatment. (Scale bar, 50 µm.) (B) Proportion of F4/80 positive cells according to indicated genotype and treatment. (C) mRNA expression of Ccl2 in kidneys from 14-wk-old mice with the indicated genotypes and treatment. (D) AFOG staining of 14-week-old pkd1 ko mouse kidneys, treated with intraperitoneal injections of MCC950 or vehicle. Arrows indicate collagen fibers. (Scale bar, 50 µm.) (E) Interstitial fibrosis scores from AFOG stainings of 14-wk-old mouse kidneys with the indicated genotypes and treatment. B, C, and E. Each dot represents one individual mouse. Two-way ANOVA followed by Tukey–Kramer test: **P < 0.01. ns: not statistically different.

Discussion

The data presented here suggest that inflammasomes function as drivers of ADPKD and demonstrate that pharmacological interference with a specific inflammasome inhibitor in an orthologous model of ADPKD slows both cyst formation and renal function decline. Differential genetic targeting strategies reveal different pools of inflammasomes in the kidney.

Gene set enrichment analysis of transcriptomic profiles, analyzed at a very early stage, revealed that 29 from the top 50 upregulated GO:BP terms were directly linked to inflammation. Differential gene expression analysis showed upregulation of Ccl2, a chemokine that has previously been identified as an important driver of cyst formation in ADPKD (39, 43), as well as Il-1β and Il-18, which are key effectors of activated inflammasomes. Other significantly regulated transcripts were Aim2, Pycard, Casp1, Casp4, and Casp12. These findings suggest that inflammasome activation occurs early in ADPKD.

We used a genetic approach to target Pycard/Asc in ADPKD mice, thereby specifically inactivating the inflammasome. This abolished the upregulation of IL-18 and IL1β in ADPKD tissue and decreased urinary IL18 excretion below wild type levels, indicating that no escape phenomena occurred. Crucially, we found that inflammasome inactivation reduced cyst formation and improved renal function. A previous report found that targeting of Caspase 1 in RC/RC mice ameliorated cyst formation significantly only in female but not in male mice (55). The seeming differences between our observations and the aforementioned study may be due to several factors. We used a fast progressive model of Pkd1 inactivation vs. the more slowly progressing RC/RC model, with severe cystic disease at 3 mo in our model vs. mild cystic disease at 6 mo in the RC/RC model. Importantly, in our model, the target was Pycard/Asc, while in the RC/RC model Caspase 1 was inactivated. Caspase 1 can be substituted by other caspases, e.g., caspase 8 (58) potentially causing an escape phenomenon in caspase 1 inactivated mice, thus reducing the effect of this strategy on the disease phenotype.

Inflammatory cells are the most likely source of inflammasome activity and release of IL-1β (59). In many renal diseases also IL-18 is upregulated and contributes to disease severity (60–62). IL-18 is secreted in the urine of patients with renal disease and IL-18, as well as NLRP3 have been identified in tubular cells by immunostaining, suggesting these may be the predominant source of IL-18 in the kidney (60, 63). Indeed, we find both IL-18 and IL-1$\beta$ upregulated in Pkd1 ko kidneys, but only IL-18 in the urine, supporting this concept. To date, functional evidence that urinary IL-18 is processed in renal tubular cells has been lacking. Our findings in Pkd1 ko kidneys with inflammasome inactivation restricted to tubular epithelial cells demonstrate robustly that urinary IL-18 derives from epithelial cells and provide the first functional proof that inflammasomes exist in renal tubular cells. Interestingly, while urinary IL-18 excretion in these animals is reduced below that of wild-type animals, Il-18 expression in whole kidneys is only minimally affected. This observation strongly suggests that 2 pools of inflammasome activity exist in ADPKD: immune cells and tubular epithelial cells. Both contribute to the pathology of ADPKD, although the trend toward higher IL-1β expression in Pkd1 ko kidneys with tubular inactivation of the inflammasome may suggest a communication between these 2 pools and the existence of counterregulatory pathways. It is interesting to note that despite the unaffected interstitial pool of inflammasome activity in mice with tubule targeted Asc deletion, the values of the renal function parameters creatinine and BUN were similar compared to Asc/Pkd1 ko mice.

There is a need to find new treatments for ADPKD. Here, we investigated the inflammasome inhibitor MCC950, which has been studied in animal models of a variety of nonrenal diseases where inflammasomes play a role, such as cryopyrin-associated periodic syndrome, an orphan disease caused by NLRP3 gain-of-function mutations, gout, atherosclerosis, heart failure, stroke, neurodegeneration, asthma, ulcerative colitis, and arthritis (56). We observed that application of the inflammasome inhibitor concurrent to genetic inactivation of Pkd1 resulted in much slower cyst formation compared to mice treated with vehicle. This was accompanied by a significant decrease of urinary IL-18 excretion. Since IL-18 is a main inflammasome target, these findings support the conclusion that the amelioration of ADPKD is a result of inflammasome inhibition.

Current interventions have a moderate impact on ADPKD disease progression. The only specifically approved drug for ADPKD, tolvaptan, when taken over 20 years is estimated to delay renal failure by 4 years (64), while conferring significant side effects, most importantly polyuria to an extent which is prohibitive for a large number of people. The long-time course of ADPKD, however, represents a window of opportunity for drugs with a low risk of side effects, that may be administered for years. Blockers of IL-1β and IL-18 are available, and some have been employed in large studies (65). In the largest study with >6,000 patients treated with IL-1β blockade, there was a modest increase in infections (66), however, their safety profile during long-term use is less clear. Direct inflammasome inhibitors may be more favorable. In mice, genetic ablation of Nlrp3 or Pycard/Asc is not associated with obvious adverse phenotypes. Clinical development of MCC950 has not been pursued due to concerns over liver toxicity, but novel NLRP3 inflammasome inhibitors are under development (56). Our observations provide a solid rationale to study them in patients with ADPKD if they have a favorable safety profile.

Materials and Methods

Mice.

For mouse strains and protocols see SI Appendix.

The primary outcome measure was the kidney weight-to-body weight ratio. The experimental group size was predetermined by a biostatistician (unpaired t test, two-tailed, significance level of 5%, 80% power, standardized effect size of 1.06).

In total, 111 mice were analyzed. 3 animals were excluded: One animal died on week 7 due to nonrenal causes; 2 mice were excluded due to missing data.

Sex as a Biological Variable.

For statistical reasons, the experiments were conducted on males, since female mice have a weaker phenotype (67).

Pathology.

Kidney samples were processed by the Institute of Surgical Pathology, University of Freiburg according to standardized procedures. Antibodies and scoring are described in SI Appendix.

RNA Sequencing Analysis.

Sequencing of fragmented cDNA was carried out on a HiSeq 2500 instrument (Illumina) at GATC Biotech Laboratory (Konstanz, Germany). After quality control and preprocessing, processed reads were aligned against the mouse reference genome GRCm39 from Ensembl (v. 108) (68) Gene set enrichment analysis was carried out using the R package gage (v. 2.46.1) (69). For a detailed description, see SI Appendix.

Statistics.

Data are expressed as means ± SD. Differences between the experimental groups were evaluated using two-way ANOVA, followed by the Tukey’s multiple comparisons test. A P-value less than 0.05 was considered statistically significant. The statistical analysis was performed using GraphPad Prism V10 software. Image analysis (immunohistochemistry) and mouse phenotypic analysis were performed in a blinded fashion. All other analyses were done unblinded.

Study Approval.

All animal experiments were conducted according to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals, as well as the German law for the welfare of animals, and were approved by regional authorities (Regierungspräsidium Freiburg G-19/29).

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

RNA sequencing data sets data have been deposited in National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus (GSE273186) (70).