Short salsalate administration affects cell proliferation, metabolism, and inflammation in polycystic kidney disease.

Metabolic reprogramming is a driver of autosomal dominant polycystic kidney disease (ADPKD) progression and a potential therapeutic intervention route. We showed before that the AMP-associated protein kinase (AMPK) activator salsalate attenuates cystic disease progression. Here, we aim to study the early, direct effects of short salsalate treatment in adult-onset conditional Pkd1 deletion mice. Cystic mice were treated with salsalate for two weeks, after which NMR metabolomics and RNA sequencing analyses were performed. Pkd1 deletion resulted in clear metabolomic dysregulation. Short salsalate treatment has small, but significant, effects, reverting acetylcarnitine and phosphocholine concentrations back to wildtype levels, and showing associations with altered purine metabolism. RNA sequencing revealed that short salsalate treatment, next to restoring energy metabolism toward wildtype levels, also affects cell proliferation and inflammation, in PKD. We show that salsalate positively affects major dysregulated processes in ADPKD: energy metabolism, cell proliferation, and inflammation, providing more insights into its working mechanisms.

Preclinical evaluation of tolvaptan and salsalate combination therapy in a Pkd1-mouse model.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder and an important cause of end stage renal disease (ESRD). Tolvaptan (a V2R antagonist) is the first disease modifier drug for treatment of ADPKD, but also causes severe polyuria. AMPK activators have been shown to attenuate cystic kidney disease. Methods: In this study, we tested the efficacy of the combined administration of salsalate (a direct AMPK activator) and tolvaptan using clinically relevant doses in an adult-onset conditional Pkd1 knock-out (KO) mouse model. Results: Compared to untreated Pkd1 mutant mice, the therapeutic effects of salsalate were similar to that of tolvaptan. The combined treatment tended to be more effective than individual drugs used alone, and was associated with improved kidney survival (p < 0.0001) and reduced kidney weight to body weight ratio (p < 0.0001), cystic index (p < 0.001) and blood urea levels (p < 0.001) compared to untreated animals, although the difference between combination and single treatments was not statistically significant. Gene expression profiling and protein expression and phosphorylation analyses support the mild beneficial effects of co-treatment, and showed that tolvaptan and salsalate cooperatively attenuated kidney injury, cell proliferation, cell cycle progression, inflammation and fibrosis, and improving mitochondrial health, and cellular antioxidant response. Conclusion: These data suggest that salsalate-tolvaptan combination, if confirmed in clinical testing, might represent a promising therapeutic strategy in the treatment of ADPKD.

High Dietary Fat Consumption Impairs Axonal Mitochondrial Function In Vivo.

Peripheral neuropathy (PN) is the most common complication of prediabetes and diabetes. PN causes severe morbidity for Type 2 diabetes (T2D) and prediabetes patients, including limb pain followed by numbness resulting from peripheral nerve damage. PN in T2D and prediabetes is associated with dyslipidemia and elevated circulating lipids; however, the molecular mechanisms underlying PN development in prediabetes and T2D are unknown. Peripheral nerve sensory neurons rely on axonal mitochondria to provide energy for nerve impulse conduction under homeostatic conditions. Models of dyslipidemia in vitro demonstrate mitochondrial dysfunction in sensory neurons exposed to elevated levels of exogenous fatty acids. Herein, we evaluated the effect of dyslipidemia on mitochondrial function and dynamics in sensory axons of the saphenous nerve of a male high-fat diet (HFD)-fed murine model of prediabetes to identify mitochondrial alterations that correlate with PN pathogenesis in vivo We found that the HFD decreased mitochondrial membrane potential (MMP) in axonal mitochondria and reduced the ability of sensory neurons to conduct at physiological frequencies. Unlike mitochondria in control axons, which dissipated their MMP in response to increased impulse frequency (from 1 to 50 Hz), HFD mitochondria dissipated less MMP in response to axonal energy demand, suggesting a lack of reserve capacity. The HFD also decreased sensory axonal Ca2+ levels and increased mitochondrial lengthening and expression of PGC1α, a master regulator of mitochondrial biogenesis. Together, these results suggest that mitochondrial dysfunction underlies an imbalance of axonal energy and Ca2+ levels and impairs impulse conduction within the saphenous nerve in prediabetic PN.SIGNIFICANCE STATEMENT Diabetes and prediabetes are leading causes of peripheral neuropathy (PN) worldwide. PN has no cure, but development in diabetes and prediabetes is associated with dyslipidemia, including elevated levels of saturated fatty acids. Saturated fatty acids impair mitochondrial dynamics and function in cultured neurons, indicating a role for mitochondrial dysfunction in PN progression; however, the effect of elevated circulating fatty acids on the peripheral nervous system in vivo is unknown. In this study, we identify early pathogenic events in sensory nerve axons of mice with high-fat diet-induced PN, including alterations in mitochondrial function, axonal conduction, and intra-axonal calcium, that provide important insight into potential PN mechanisms associated with prediabetes and dyslipidemia in vivo.

Renal cyst growth is attenuated by a combination treatment of tolvaptan and pioglitazone, while pioglitazone treatment alone is not effective.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic disorders, characterized by the progressive formation of fluid-filled cysts. Tolvaptan is an approved drug for ADPKD patients, but is also associated with multiple side effects. The peroxisome proliferator-activator receptor gamma (PPARγ) agonist pioglitazone slows disease progression in the PCK rat model for PKD. Here, we tested whether a combination treatment of relevant doses of tolvaptan and pioglitazone leads to improved efficacy in an adult-onset PKD mouse model. Tolvaptan indeed slowed PKD progression, but the combination treatment was not more effective than tolvaptan alone. In addition, although pioglitazone raised plasma levels of its surrogate drug marker adiponectin, the drug unexpectedly failed to slow PKD progression. The pioglitazone target PPARγ was expressed at surprisingly low levels in mouse, rat and human kidneys. Other pioglitazone targets were more abundantly expressed, but this pattern was comparable across various species. The data suggest that several potential pharmacokinetic and pharmacodynamic (PK/PD) differences between different species may underlie whether or not pioglitazone is able to slow PKD progression. The ongoing phase II clinical trial with low-dose pioglitazone treatment (NCT02697617) will show whether pioglitazone is a suitable drug candidate for ADPKD treatment.

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

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.

Salsalate activates brown adipose tissue in mice.

Salsalate improves glucose intolerance and dyslipidemia in type 2 diabetes patients, but the mechanism is still unknown. The aim of the current study was to unravel the molecular mechanisms involved in these beneficial metabolic effects of salsalate by treating mice with salsalate during and after development of high-fat diet-induced obesity. We found that salsalate attenuated and reversed high-fat diet-induced weight gain, in particular fat mass accumulation, improved glucose tolerance, and lowered plasma triglyceride levels. Mechanistically, salsalate selectively promoted the uptake of fatty acids from glycerol tri[(3)H]oleate-labeled lipoprotein-like emulsion particles by brown adipose tissue (BAT), decreased the intracellular lipid content in BAT, and increased rectal temperature, all pointing to more active BAT. The treatment of differentiated T37i brown adipocytes with salsalate increased uncoupled respiration. Moreover, salsalate upregulated Ucp1 expression and enhanced glycerol release, a dual effect that was abolished by the inhibition of cAMP-dependent protein kinase (PKA). In conclusion, salsalate activates BAT, presumably by directly activating brown adipocytes via the PKA pathway, suggesting a novel mechanism that may explain its beneficial metabolic effects in type 2 diabetes patients.