Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease

Area of Science:

• Molecular Biology
• Renal Physiology
• Aging Research

Background:

• Klotho is a renal protein crucial for phosphate homeostasis and considered an antiaging factor.
• Two Klotho gene transcripts exist, potentially encoding membrane-bound and secreted forms, but their origins are unclear.
• Soluble Klotho is found in bodily fluids, yet the mechanisms of its production (secretion vs. shedding) remain elusive.

Purpose of the Study:

• To investigate the production mechanisms of soluble Klotho.
• To elucidate the role of nonsense-mediated mRNA decay (NMD) in regulating Klotho mRNA.
• To understand how renal disease and noxious stimuli affect Klotho mRNA splicing and protein levels.

Main Methods:

• Analysis of alternative Klotho mRNA processing and its relationship with NMD.
• RNA immunoprecipitation (RIP) targeting the NMD core factor UPF1.
• Assessment of mRNA localization to P bodies and polysomes.
• In vitro experiments to detect secreted Klotho and study mRNA splicing under various conditions.
• Investigation of Klotho mRNA splicing in acute kidney injury models.

Main Results:

• Alternative Klotho mRNA, potentially encoding secreted Klotho, is subject to continuous degradation by NMD.
• Disruption of NMD led to the accumulation of alternative Klotho mRNA, indicating its normal degradation.
• Alternative Klotho mRNA was enriched in UPF1 immunoprecipitates and not associated with polysomes, suggesting no active translation.
• Alternative Klotho mRNA transcripts were found in P bodies, sites of mRNA degradation.
• Secreted Klotho was not detected in vitro, supporting the hypothesis that soluble Klotho originates from membrane shedding.
• Splicing of Klotho mRNA transcripts was dysregulated in acute kidney injury and by noxious stimuli in vitro.
• This dysregulation likely contributes to the downregulation of membrane-bound Klotho.

Conclusions:

• Soluble Klotho is primarily derived from the cleavage of membrane-bound Klotho, not direct secretion.
• Nonsense-mediated mRNA decay plays a significant role in regulating Klotho mRNA levels.
• Dysregulated mRNA splicing, particularly in response to kidney injury and stress, represents a novel mechanism for the reduction of membrane-bound Klotho.