Salsalate, but not metformin or canagliflozin, slows kidney cyst growth in an adult-onset mouse model of polycystic kidney disease

Abstract

Background: Multiple preclinical studies have highlighted AMP-activated protein kinase (AMPK) as a potential therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). Both metformin and canagliflozin indirectly activate AMPK by inhibiting mitochondrial function, while salsalate is a direct AMPK activator. Metformin, canagliflozin and salsalate (a prodrug dimer of salicylate) are approved for clinical use with excellent safety profile. Although metformin treatment had been shown to attenuate experimental cystic kidney disease, there are concerns that therapeutic AMPK activation in human kidney might require a higher oral metformin dose than can be achieved clinically.

Methods: In this study, we tested metformin-based combination therapies for their additive (metformin plus canagliflozin) and synergistic (metformin plus salsalate) effects and each drug individually in an adult-onset conditional Pkd1 knock-out mouse model (n = 20 male/group) using dosages expected to yield clinically relevant drug levels.

Findings: Compared to untreated mutant mice, treatment with salsalate or metformin plus salsalate improved kidney survival (i.e. blood urea nitrogen <20 mmol/L at the time of sacrifice) and reduced cystic kidney disease severity. However, the effects of metformin plus salsalate did not differ from salsalate alone; and neither metformin nor canagliflozin was effective. Protein expression and phosphorylation analyses indicated that salsalate treatment was associated with reduction in mTOR (mammalian target of rapamycin) activity and cellular proliferation in Pkd1 mutant mouse kidneys. Global gene expression analyses suggested that these effects were linked to restoration of mitochondrial function and suppression of inflammation and fibrosis.

Interpretation: Salsalate is a highly promising candidate for drug repurposing and clinical testing in ADPKD.

Keywords: AMPK; Canagliflozin; Metformin; Polycystic kidney disease; Salsalate.

Fig. 1

Experimental protocol. Twenty male Tam-Ksp-Pkd1^loxlox mice/group were treated with tamoxifen at days P18 and P19 to inactivate Pkd1 and drug treatment began on day P40 (Rx). Untreated mutant mice began to develop advanced kidney failure at around ~P105. BUN monitoring via tail-vein blood sampling began at P75 to identify those mice with kidney failure; all mice with a BUN >20 mmol/L were considered to have ESRD and sacrificed. When ~50% of the untreated mutant mice reached ESRD, all the mice from all study groups were sacrificed.

Fig. 2

Salsalate treatment slowed progression of polycystic kidney disease in iKsp-Pkd1^del mice. (a) Only treatment with salsalate (SAL) or metformin plus salsalate (MET+SAL) was associated with a significant improvement in kidney survival. Only treatment with salsalate or metformin plus salsalate was associated with a significant reduction in (b) the ratio of two-kidney weight to body weight (2KW/BW) and (c) cystic index. There was no difference between treatment with MET+SAL and SAL suggesting the therapeutic effect of the former treatment group was likely due to salsalate alone. (d) Representative histological kidney sections (i.e. the median) from different study groups. NS denotes not significant. Data presented as mean ± SEM; WT (n = 8), untreated (n = 26), SAL (n = 21), MET (n = 19), MET+SAL (n = 20).

Fig. 3

Salsalate reversed multiple pathogenic mediators of cystic kidney disease in iKsp-Pkd1^del mice. Western blot analyses showed that kidneys from mutant Pkd1 mice (compared to WT mice) displayed increased expression of protein markers for (a) mTORC1 activity (i.e. levels of pS6K), (b) cell proliferation (i.e. PCNA), (c) cell cycle progression (i.e. CDK2), and (d) cAMP; all these changes were significantly attenuated by salsalate treatment. Data presented as mean ± SEM. For kidney cAMP levels, WT (n = 7), KO (n = 8), SAL (n = 9).

Fig. 4

Salsalate treatment improved defective metabolism in Pkd1 mutant kidneys. (a) Gene expression profiling showing changes consistent with a generalized defective metabolism with reduced oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in mutant (compared to WT) Pkd1 kidneys; these changes were attenuated by salsalate treatment (all changes shown were identified using a FDR adjusted p-value <0∙01). Each column represents an individual kidney sample and each row represents the expression value of a specific gene; red indicates greater expression than the mean (white) value and blue, less than the mean value. (b) Western blot analysis confirming a decreased expression of PGC-1α in Pkd1 mutant kidneys which was less pronounced by salsalate treatment. (c) An inverse correlation between mtDNA/nDNA and KW/BW in Pkd1 mutant kidneys (r = −0∙8, p < 0∙0001) was noted consistent with defective mitochondrial biogenesis in ADPKD. (d) Salsalate treatment increased the ratio of mtDNA/nDNA in Pkd1 mutant kidneys. Data presented as mean ± SEM. For kidney mtDNA qPCR, WT (n = 8), KO (n = 26), SAL (n = 21). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Salsalate treatment reduced inflammation in Pkd1 mutant kidneys. (a) Gene expression profiling showing an increased expression of markers for damage-associated molecular patterns (DAMPs) and their receptors, macrophages (MΦ), inflammation and fibrosis in Pkd1 mutant kidneys. Each column represents an individual kidney sample and each row represents the expression value of a specific gene; red indicates greater expression than the mean (white) value and blue, less than the mean value. (b) Western blot analysis showing an increased expression of NFκB p65 in Pkd1 mutant kidneys. Histological analysis showing increased (c) fibrosis by Sirius Red staining and (d) macrophage infiltration by F4/80 staining in Pkd1 mutant kidneys; all these changes were less severe by salsalate treatment. TF denotes transcriptional factor. Data presented as mean ± SEM. For fibrotic index, WT (n = 8), KO (n = 10), SAL (n = 21); for F4/80 staining, WT (n = 4), KO (n = 6), SAL (n = 6). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Salsalate treatment reduced fibrosis and inflammation in Pkd1 mutant kidneys. Representative kidney sections showing (a) Sirius Red staining for collagen deposition as early marker for fibrosis and (b) F4/80 staining for macrophage infiltration. Salsalate treated Pkd1 mutant (KO_SAL) mice had less fibrosis and macrophage infiltration compared to untreated Pkd1 mutant (KO) mice.