#1316 Sirtuin 3 promotes renal gluconeogenesis in fibrotic kidneys
Authors: Lin Pinglan , Wu Ming , Hu Jing , Ye Chaoyang 
Published: May 2024

Abstract

Background and Aims
Decreased renal gluconeogenesis is currently identified as a hallmark of chronic kidney disease (CKD), which may contribute to renal fibrosis. Sirt3 is a crucial metabolic sensor that regulates ATP generation, mitochondrial adaptive response to stress, and glucose metabolism. It has been shown that Sirt3 is renal protective in fibrotic kidneys, however its underlying mechanisms is not completely known. In this study, we aimed to investigated whether Sirt3 inhibits CKD associated renal fibrosis through restoring renal gluconeogenesis.

Method
1. Animal model of renal fibrosis (in vivo)
For unilateral ureter obstructed (UUO) mouse model, a. Twenty-eight C57 mice were divided into four groups: sham + Vehicle, UUO + Vehicle, UUO + 3-TYP and UUO + HKL group. Mice were treated with saline or 20 mg/kg 3-TYP, an inhibitor of SIRT3 or 1 mg/kg HKL, an activator of SIRT3 daily (i.p.). b. Fourteen C57 mice were divided into two groups: Ad-Sirt 3 or Ad-LacZ (0.5 × 109 pfu/100 μL) was injected into the left renal artery before the UUO operation. Systemic glucose metabolism was evaluated by insulin and glucose tolerance tests. 7 or 14 days after operation, kidney tissues were collected and performed histopathological and molecular examination.
2. Primary murine renal tubules fibrosis (ex vivo)
Primary renal tubules were isolated from C57 mouse of 4 weeks of age. 5 ng/mL TGF-β was added to culture medium for inducing fibrosis. After this Sirt3 was inhibited by 3-TYP (25 μm) or activated by HKL (2 μm) or overexpressed by adenovirus for different time periods (1, 3, 24, 48 hours). Protein or RNA samples were taken to evaluate renal fibrosis and gluconeogenesis.
3. Human renal epithelial (HK2) cells culture and treatment (in vitro)
TGF-β stimulated HK2 cells were used as an in vitro model. Sirt3 was inhibited by 3-TYP or activated by HKL or overexpressed by adenovirus. Protein and RNA samples were taken to evaluate renal fibrosis and gluconeogenesis. Glucose and lactate were measured in supernatant of culture medium.

Results
In vivo, Masson staining showed that 3-TYP treatment increased the tubulointerstitial collagen deposition of UUO kidneys (Fig. 1A). WB results indicated that 3-TYP enhanced the increased expression of fibrotic marks (Fibronectin, Collagen-I, Vimentin) (Fig. 1B), and further reduced the expression of gluconeogenesis limitation enzyme (PCK1, FBP1) and transcription factors (PGC-1α and FOXO1) 14 days after UUO operation (Fig. 1C). Conversely, HKL treatment or overexpression of Sirt3 in UUO kidney reduced expression of fibrotic marks, increased the expression of gluconeogenesis markers (Fig. 1D-E). 3-TYP increased the lactate concentration in UUO kidneys (Fig. 1F). Insulin sensitivity and glucose clearance tests showed that treatment with 3-TYP further damaged the glucose homeostasis in UUO mice (Fig. 1G-J).
Ex vivo, q-PCR results showed that the mRNA of Sirt3 was significantly decreased by time after TGF-β stimulation while the mRNA of FN, Snail was up-regulated and PCK1 and FBP1 were down-regulated (Fig. 2A-D). WB showed that 3-TYP treatment inhibited the expression of PCK1, FBP1 and PGC-1α, FOXO1 (Fig. 2E), however, HKL treatment or overexpressed of Sirt3 by adenovirus increased the expression of gluconeogenesis markers respectively in primary renal tubules (Fig. 2F).
In vitro, WB showed that transfection of Sirt3 increased the expression of gluconeogenesis markers and decreased the expression of fibrotic marks of HK2 cells in a fibrotic condition (Fig. 3A-B). Moreover, 3-TYP treatment further decreased the mRNA expression of PCK1 and FBP1 in TGF-β stimulated HK2 cells (Fig. 3C-D). The increased lactate concentration and decreased glucose concentration in the culture medium of TGF-β stimulated HK2 cells were further escalated after 3-TYP treatment (Fig. 3E-F) while reversed after overexpression of Sirt3 (Fig. 3G-H).

Conclusion
Sirt 3 promotes renal gluconeogenesis in fibrotic kidneys through up-regulation of renal gluconeogenic enzyme PCK1 and FBP1 expression, which is associated with activation of renal metabolic transcription factors PGC-1α and FOXO1.