Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1 F121A ) with increased autophagic flux, and αKlothohypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/ BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1 F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately 3130 | SHI et al.
Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho’s known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 ( Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 ( Becn1+/−), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 ( Bcl2AAA). Klotho was increased by transgenic overexpression ( Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/− and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/− ;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/− or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.
Cellular senescence is an irreversible cell growth arrest and is associated with aging and age-related diseases. High plasma phosphate (Pi) and deficiency of Klotho contribute to aging and kidney fibrosis, a pathological feature in the aging kidney and chronic kidney disease. This study examined the interactive role of Pi and Klotho in kidney senescence and fibrosis. Homozygous Klotho hypomorphic mice had high plasma Pi, undetectable Klotho in plasma and kidney, high senescence with massive collagen accumulation in kidney tubules, and fibrin deposits in peritubular capillaries. To examine the Pi effect on kidney senescence, a high (2%) Pi diet was given to wild-type mice. One week of high dietary Pi mildly increased plasma Pi, and upregulated kidney p16/p21 expression, but did not significantly decrease Klotho. Two weeks of high Pi intake led to increase in plasminogen activator inhibitor (PAI)-1, and decrease in kidney Klotho, but still without detectable increase in kidney fibrosis. More prolonged dietary Pi for 12 weeks exacerbated kidney senescence and fibrosis; more so in heterozygous Klotho hypomorphic mice compared to wild-type mice, and in mice with chronic kidney disease (CKD) on high Pi diet compared to CKD mice fed a normal Pi diet. In cultured kidney tubular cells, high Pi directly induced cellular senescence, injury and epithelialmesenchymal transition, and enhanced H 2 O 2-induced cellular senescence and injury, which were abrogated by Klotho. Fucoidan, a bioactive molecule with multiple biologic functions including senescence inhibition, blunted Pi-induced cellular senescence, oxidation, injury, epithelial-mesenchymal transition, and senescence-associated secretary phenotype. In conclusion, high Pi activates senescence through distinct but interconnected mechanisms: upregulating p16/p21 (early), and elevating plasminogen activator inhibitor-1 and downregulating Klotho (late). Klotho may be a promising agent to attenuate senescence and ameliorate age-associated, and Pi-induced kidney degeneration such as kidney fibrosis.
Cisplatin-induced acute kidney injury (CIAKI) is a common complication in patients receiving cisplatin-based chemotherapy. But the effective therapies for CIAKI are not available. Retinoic acid (RA), the main derivative of vitamin A, has the potential to reduce inflammation and fibrosis in renal injury. However, the effect and mechanism of RA on CIAKI are still unclear. The aim of this study is to investigate whether RA can alleviate CIAKI through activation of autophagy. In this study, we evaluated the effect of RA, RA's effect on autophagy and apoptosis after cisplatin-induced injury on renal tubular epithelial cells (RTECs) by LDH assay, immunoblotting and TUNEL staining. Then we established Atg5 flox/flox :Cagg-Cre mice in which Cagg-Cre is tamoxifen inducible, and Atg5 is conditional deleted after tamoxifen injection. The effect of RA and RA's effect on autophagy on CIAKI model were evaluated by biochemical assessment, hematoxylin and eosin (HE) staining, and immunoblotting in the control and autophagy deficient mice. In vitro, RA protected RTECs against cisplatin-induced injury, activated autophagy, and inhibited cisplatin-induced apoptosis. In vivo, RA attenuated cisplatin-induced tubular damage, shown by improved renal function, decreased renal cast formation, decreased NGAL expression, and activated autophagy in the control mice. Furthermore, the nephrotoxicity of cisplatin was aggravated, and the protective effect of RA was attenuated in autophagy deficient mice, indicating that RA works in an autophagydependent manner on CIAKI. RA activates autophagy and alleviates CIAKI in vivo and in vitro.Thus RA may be a renoprotective adjuvant for cisplatin-based chemotherapy.
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