Re: '스퍼미딘' 신장 섬유화 방지.. 2023 네이처!!

[문형철]

1. nature  
2. communications biology  
3. articles  
4. article

Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

Download PDF

• Article
• Open access
• Published: 28 June 2023

Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

• Seishi Aihara, 
• Kumiko Torisu, 
• Yushi Uchida, 
• Noriyuki Imazu, 
• Toshiaki Nakano & 
• Takanari Kitazono 

Communications Biology volume 6, Article number: 676 (2023) Cite this article

• 7081 Accesses

• 14 Citations

• 4 Altmetric

• Metricsdetails

Abstract

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.

요약 

만성 신장 질환에서 

신장 대사 과정은 크게 변화될 수 있습니다. 

본 연구에서는 대사체 분석을 통해 

단측 요관 폐쇄(UUO)로 인한 신장 섬유화에서 

아르기닌 대사 과정이 가장 크게 변화되었음을 보고합니다. 

아르기닌의 대사산물 중 

스퍼미딘이 가장 크게 증가했습니다. 

인간 신장염에서 면역염색으로 측정된 스퍼미딘의 양은 

섬유화 정도와 연관되어 있습니다. 

인간 근위세관 세포에서 스퍼미딘은 

핵인자 에리트로이드 2 관련 인자 2(Nrf2)를 유도합니다. 

이후 스퍼미딘은 

변형 성장 인자 β1 분비, 콜라겐 1 mRNA, 산화 스트레스(미토콘드리아 막 전위 감소로 나타남)와 같은 

섬유화 신호를 억제합니다. 

Arg2 결손 마우스의 UUO 신장은 

야생형 마우스에 비해 스페르미딘 농도가 낮고 

섬유화가 현저히 악화되었습니다. 

Arg2 결손 UUO 신장에서

Nrf2 활성화가 감소했습니다.

스페르미딘 투여는 

Arg2 결손 마우스에서 섬유화 진행을 유의미하게 억제했습니다. 

신장 섬유화에서 스페르미딘 농도가 증가하지만, 

추가적인 스페르미딘 증가가 섬유화를 감소시킬 수 있습니다.

Similar content being viewed by others

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) drives chronic kidney disease progression in male mice

Article Open access11 March 2023

Asymmetric dimethylarginine (ADMA) accelerates renal cell fibrosis under high glucose condition through NOX4/ROS/ERK signaling pathway

Article Open access29 September 2020

NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury

Article 13 March 2023

Introduction

Kidney fibrosis is the final common pathway for most types of progressive kidney diseases and eventually leads to end-stage kidney disease1. Chronic kidney disease (CKD) with kidney fibrosis is associated with complications, such as cardiovascular disease, anemia, inflammation, and malnutrition2. Therefore, not only systemic metabolism, but also kidney metabolism, may be greatly altered in patients with CKD. Recent advances in omics analysis have reported that fatty acid3 or lysine4 metabolism is altered in renal injury. The human kidney plays an important role in maintaining homeostasis of amino acid pools throughout the body through synthesis, degradation, filtration, reabsorption, and urinary excretion of amino acids and peptides. The kidney is the major excretion site for glutamine and proline, as well as the production site for serine, cysteine, and arginine5. The balance between amino acid excretion and metabolism is considered important for renal protection6.

Arginine is not only a material for protein synthesis, but also a precursor for urea, nitric oxide (NO)7, polyamines, proline, glutamic acid, creatine, and agmatine8. Therefore, arginine is involved in a variety of biological processes. l-arginine is the substrate for nitric oxide synthase (NOS) and also the substrate for arginase7. Kidney mass reduction by uninephrectomy causes changes in arginine metabolism and increases blood pressure9. Arginase hydrolyzes l-arginine into urea and l-ornithine, and ornithine-derived polyamines are essential for the growth and function of cells10. Spermidine (Spd) is an ornithine-derived polyamine. One of five conserved metabolites released from apoptotic cells is Spd. Apoptotic metabolites are selectively released as “good-bye” signals to suppress inflammation in surrounding cells, leading to wound healing11. Arginine metabolism in the kidney may be important because arginase 2 (ARG2) is predominantly expressed in renal tubular epithelial cells (RTECs)12,13. Our previous study showed that ischemia–reperfusion injury in the kidney was attenuated in Arg2 knockout (KO) mice or arginase inhibitor-treated mice through regulating nitrosative stress12. We have previously investigated the importance of arginine metabolism in acute kidney injury, but not in chronic kidney injury. Therefore, in this study, we investigated the role of arginine metabolism involving ARG2 and its metabolites in kidney fibrosis using comprehensive metabolic analysis. We used mice with unilateral ureteral obstruction (UUO) as a model of CKD. The fibrotic inhibitory effect of Spd was investigated.

소개

신장 섬유화는

대부분의 진행성 신장 질환의 최종 공통 경로이며

결국 말기 신장 질환으로 진행됩니다1.

신장 섬유화를 동반한 만성 신장 질환(CKD)은

심혈관 질환, 빈혈, 염증, 영양 장애 등 다양한 합병증과 연관되어 있습니다2.

따라서

CKD 환자는 전신 대사뿐만 아니라

신장 대사도 크게 변화될 수 있습니다.

최근 오믹스 분석의 발전은

신장 손상 시 지방산3 또는 라이신4 대사가 변화된다는 사실을 보고했습니다.

인간 신장은

아미노산 풀의 항상성을 유지하는 데 중요한 역할을 하며,

아미노산과 펩타이드의 합성, 분해, 필터링, 재흡수, 요로 배설을 통해 이를 조절합니다.

신장은 글루타민과 프로린의 주요 배설 부위이며,

세린, 시스테인, 아르기닌의 생산 부위이기도 합니다5.

아미노산 배설과 대사 사이의 균형은

신장 보호에 중요하다고 여겨집니다6.

아르기닌은

단백질 합성의 재료일 뿐만 아니라

요소, 일산화질소(NO)7, 폴리아민, 프로린, 글루탐산, 크레아틴, 아그마틴8의 전구체입니다.

따라서

아르기닌은 다양한 생물학적 과정에 관여합니다.

L-아르기닌은

일산화질소 합성효소(NOS)의 기질이며

아르기나제의 기질이기도 합니다7.

단측 신장 절제술로 인한 신장 질량 감소는

아르기닌 대사 변화를 유발하고 혈압을 증가시킵니다9.

아르기나제는

L-아르기닌을 요산과 L-오르니틴으로 가수분해하며,

오르니틴 유래 폴리아민은 세포의 성장과 기능에 필수적입니다10.

스퍼미딘(Spd)은

오르니틴 유래 폴리아민입니다.

세포 사멸 시 방출되는 5가지 보존된 대사산물 중 하나가

Spd입니다.

세포 사멸 대사산물은

주변 세포의 염증을 억제하여 상처 치유를 유도하는

'작별 신호'로 선택적으로 방출됩니다11.

신장에서의 아르기닌 대사는

아르기나제 2(ARG2)가 신장 관상 상피 세포(RTECs)에서 주로 발현되기 때문에

중요할 수 있습니다12,13.

우리의 이전 연구에서는

Arg2 결손 마우스 또는 아르기나제 억제제 처리 마우스에서

신장 허혈-재관류 손상이 질소화 스트레스 조절을 통해 완화되었음을 보여주었습니다12.

우리는

급성 신장 손상에서의 아르기닌 대사 중요성을 조사했지만

만성 신장 손상에서는 조사하지 않았습니다.

따라서

본 연구에서는 ARG2 및 그 대사물을 포함한 아르기닌 대사가

신장 섬유화에 미치는 역할을 포괄적 대사 분석을 통해 조사했습니다.

우리는 CKD 모델로 단측 요관 폐쇄(UUO) 마우스를 사용했습니다.

Spd의 섬유화 억제 효과를 조사했습니다.

Results

Arginine metabolism is upregulated in the UUO kidney in mice

To examine metabolic changes in UUO, we compared metabolites from UUO kidneys and sham-operated kidneys. “Arginine and proline metabolism” and “arginine biosynthesis” were most altered when pathway analysis was performed on significantly increased metabolites in the UUO kidney among 110 detected amino acids (Fig. 1a and Table 1). When we focused on “arginine and proline metabolism”, we found that ornithine and proline concentrations were significantly higher (>2 times, both P < 0.01) in the UUO kidney than in the sham-operated kidney (Fig. 1b, c). With regard to ornithine-derived polyamines, putrescine and Spd concentrations were approximately twice as high in the UUO kidney as those in the sham-operated kidney, while spermine (Spm) concentrations showed no difference (Fig. 1b). Although arginine metabolism was activated, the substrate l-arginine was not decreased in UUO kidneys, but increased instead (Fig. 1b). A schematic diagram of “arginine biosynthesis” and “arginine and proline metabolism” is shown in Fig. 1c.

결과

UUO 신장에서 아르기닌 대사 활성이 증가합니다

UUO에서의 대사 변화를 조사하기 위해 UUO 신장과 가짜 수술을 받은 신장의 대사물을 비교했습니다. 110개의 검출된 아미노산 중 UUO 신장에서 유의미하게 증가한 대사물을 대상으로 경로 분석을 수행한 결과, “아르기닌 및 프로린 대사”와 “아르기닌 생합성”이 가장 크게 변화했습니다(그림 1a 및 표 1).

“아르기닌과 프로린 대사”에 초점을 맞추었을 때,

UUO 신장에서 가짜 수술을 받은 신장보다

오르니틴과 프로린 농도가 유의미하게 높았으며(>2배, 모두 P < 0.01)(그림 1b, c).

오르니틴 유래 폴리아민 중 푸트레신과 Spd 농도는

UUO 신장에서 가짜 수술 신장보다 약 2배 높았으며,

스퍼민(Spm) 농도는 차이가 없었습니다(그림 1b).

아르기닌 대사가 활성화되었지만,

UUO 신장에서 기질인 l-아르기닌은 감소하지 않고 오히려 증가했습니다(그림 1b).

“아르기닌 생합성”과 “아르기닌 및 프로린 대사”의 개념도는

그림 1c에 표시되어 있습니다.

Fig. 1: Arginine metabolism is upregulated in the UUO kidney of mice.

a Results of pathway analysis using MetaboAnalyst. Of the 110 metabolites, pathway analysis was performed only for metabolites with a fold change of ≥1.5 in the UUO kidney relative to shams (n = 4 in each group). Red squares indicate “arginine and proline metabolism” and “arginine biosynthesis”. b Absolute quantitative values of metabolites related to arginine metabolites in the UUO kidney compared with those in shams (n = 4 in each group). Data are indicated as means ± SD. c Schematic diagram of “arginine biosynthesis” and “arginine and proline metabolism”. *P < 0.05, **P < 0.01. NS not significant, UUO unilateral ureteral obstruction, ARG2 arginase 2, SMOX spermine oxidase.

Full size image

Table 1 Network enrichment analysis results from metabolites in unilateral ureteral obstruction.

Spermidine increases in response to kidney fibrosis

We focused on Spd, which was the most increased arginine metabolite. Spd was fifth among the 110 measured metabolites that was specifically increased in the UUO kidney relative to controls (Table 2). Immunostaining showed that Spd levels were remarkably increased in the UUO kidney, especially in the tubules (Fig. 2a, b). Western blot analysis of mouse UUO kidneys showed that ARG2 protein levels were approximately two times higher in the UUO kidney relative to the sham-operated kidney (Fig. 2c, d). Immunostaining of ARG2 was significantly greater in the UUO kidney than in the sham-operated kidney and was localized mostly in RTECs (Fig. 2e, f, P < 0.05. We hypothesized that Spd levels are correlated with the severity of interstitial fibrosis in the human kidney. Human kidney specimens from donor kidneys at the time of living donor kidney transplantation as a control or kidneys of IgA nephropathy were eval‎uated. The characteristics of patients at diagnosis by renal biopsy are shown in Table 3. The tubular atrophy/interstitial fibrosis score (T score) was significantly associated with higher urinary protein and kidney function. Spd levels were remarkably enhanced in the kidney with fibrosis, especially in the tubules (Fig. 2g, h). A toxic compound produced from Spm, acrolein, was also increased in the UUO kidney (Supplementary Fig. S1a, b). The expression‎ of spermine oxidase (Smox) mRNA, which generates Spd from Spm, was also significantly higher in the UUO kidney than in the sham-operated kidney (Supplementary Fig. S1c, P < 0.01). This finding is consistent with the finding that only Spm among the polyamines was not increased in the UUO kidney in metabolomic analysis (Fig. 1b). These findings indicate that arginine metabolism is altered by fibrotic stimuli in mice.

스페르미딘은 신장 섬유화에 반응하여 증가합니다

우리는

가장 크게 증가한 아르기닌 대사산물인

Spd에 초점을 맞췄습니다.

Spd는

UUO 신장에서 대조군에 비해 특이적으로 증가한 110개의 측정된 대사산물 중 5위를 차지했습니다(표 2).

면역염색 결과, Spd 수준은

UUO 신장에서 특히 세뇨관 부위에서 현저히 증가했습니다(그림 2a, b).

마우스 UUO 신장의 서양 블롯 분석 결과, ARG2 단백질 수준은 UUO 신장에서 가짜 수술을 받은 신장에 비해 약 2배 높았습니다(그림 2c, d). ARG2 면역염색은 UUO 신장에서 가짜 수술 신장보다 유의미하게 높았으며 주로 RTECs에 국한되었습니다(그림 2e, f, P < 0.05. 우리는 Spd 수준이 인간 신장의 간질 섬유화 심각도와 관련이 있을 것으로 가설을 세웠습니다. 생체 신장 이식 시 기증자 신장 또는 IgA 신증 환자의 신장 표본을 대조군으로 평가했습니다. 신장 생검 시 진단 시 환자의 특성은 표 3에 표시되어 있습니다. 관 위축/간질 섬유화 점수(T 점수)는 요단백 수치와 신장 기능과 유의미하게 연관되었습니다. Spd 수준은 섬유화가 있는 신장에서, 특히 관에서 현저히 증가했습니다(그림 2g, h). Spm에서 생성되는 독성 화합물인 아크롤레인은 UUO 신장에서 증가했습니다(보충 그림 S1a, b). Spm에서 Spd를 생성하는 스퍼민 산화효소(Smox) mRNA의 발현은 UUO 신장에서 가짜 수술을 받은 신장보다 유의미하게 높았습니다(보충 그림 S1c, P < 0.01).

이 결과는

대사체 분석에서 UUO 신장에서

폴리아민 중 Spm만 증가하지 않았다는 결과와 일치합니다(그림 1b).

이러한 결과는

섬유화 자극이 쥐에서 아르기닌 대사 과정을 변화시킨다는 것을 시사합니다.

Table 2 Top ten metabolites that were significantly increased in unilateral ureteral obstruction model compared with those in shams.

Full size table

Fig. 2: UUO increases spermidine and ARG2 protein levels in the mouse kidney.

a Confocal immunofluorescence microscopic images of Spd in control and UUO kidneys. Green, anti-Spd antibody; blue, DAPI. Scale bars, 50 µm. b Quantification of the Spd-positive area in control and UUO kidneys (n = 6 in each group). Data are indicated as means ± SD. c Western blot analysis of ARG2 protein levels in the whole kidney of sham and UUO mice. d Relative levels of ARG2 protein normalized to GAPDH in sham and UUO kidneys are shown (n = 6 in each group). Data are indicated as means ± SD. e Immunohistochemistry of ARG2 in control and UUO kidneys. Green, anti-ARG2 antibody; blue, DAPI. Scale bars, 50 µm. White arrowheads indicate ARG2-positive tubules. f Quantification of the ARG2-positive area in sham and UUO kidneys (n = 6 in each group). Data are indicated as means ± SD. *P < 0.05, **P < 0.01. g Immunostaining of Spd in the human kidney. Control, donor kidneys; T0–T2, interstitial fibrosis/tubular atrophy scores from the Oxford classification of IgA nephropathy. The percentages of lesions in the cortical area were as follows: T0 = 0%–25%, T1 = 26%–50%, and T2 = > 50%. Scale bars, 50 µm. h Quantification of the Spd-positive area in human kidneys (n = 6 in each group). Data are indicated as means ± SD. **P < 0.01. Spd spermidine, UUO unilateral ureteral obstruction, ARG2 arginase 2, DAPI 4′,6-diamidino-2-phenylindole, GAPDH glyceraldehyde-3-phosphate dehydrogenase.

Full size image

Table 3 Patients’ characteristics at the time of a renal biopsy in relation to the degree of fibrosis.

Full size table

Oxidative stress upregulates ARG2 protein levels and polyamine including spermidine in RTECs

We focused on ARG2 as an important enzyme that regulates arginine biosynthesis. During the fibrotic process, tubules are expected to be exposed to a large amount of reactive oxygen species stress. Therefore, HK-2 cells were exposed to 500 µM hydrogen peroxide (H2O2) for 24 h. Oxidative stress increased ARG2 protein levels in HK-2 cells (Fig. 3a, b). This result is consistent with the enhanced ARG2 protein levels in the UUO kidney, especially in RTECs. We then eval‎uated changes in the production of polyamine or Spd during oxidative stress. Polyamine red staining showed that polyamine production was induced by H2O2 (Fig. 3c). ARG2 levels were reduced to ~10% of control levels in HK-2 cells transfected with Arg2-siRNA (Supplementary Fig. S2a). Polyamine was reduced by Arg2 knockdown in HK-2 cells (Fig. 3c, d). This suppression of polyamine production in Arg2 knockdown cells was observed even in the absence of oxidative stress. In an immunofluorescence study, Spd formed puncta, which were diffusely distributed in the cytoplasm (Fig. 3e). Overexpression‎ of ARG2 (Supplementary Fig. S2b) increased the amount of Spd, while knockdown of Arg2 decreased the amount of Spd (Fig. 3e, f). These findings suggest that the amount of Spd is largely dependent on ARG2 expression‎ levels.

Fig. 3: Oxidative stress upregulates ARG2 protein levels in renal tubular epithelial cells.

a Western blot analysis of ARG2 protein levels in HK-2 cells stimulated with H2O2. The arrowhead shows a nonspecific signal. b Relative levels of ARG2 protein normalized to GAPDH are shown (n = 6 in each group). Data are indicated as means ± SD. c Polyamine red staining in control or Arg2 siRNA-transfected HK-2 cells stimulated with H2O2. Red, polyamine; blue, DAPI. Scale bars, 20 µm. d Quantification of polyamine red fluorescence intensity/cell shown in Fig. 3c (n = 5 in each group). Data are indicated as means ± SD. e Immunofluorescence images of Spd in HK-2 cells with Arg2 overexpression‎ or knockdown. Green, anti-Spd antibody; blue, DAPI. Scale bars, 20 µm. f Quantification of the Spd-positive area corrected for nuclei (n = 10 in each group). Data are indicated as means ± SD. *P < 0.05, **P < 0.01. Spd spermidine, ARG2 arginase 2, DAPI 4′,6-diamidino-2-phenylindole, GAPDH glyceraldehyde-3-phosphate dehydrogenase.

Full size image

Spd activates the transcription factor nuclear factor erythroid 2-related factor 2 in RTECs

We hypothesized that Spd plays an important role in the development of kidney fibrosis. Initially, cell survival during 24-h exposure of Spd to tubular cells was investigated (Supplementary Fig. S3a). Below a concentration of 20 µM, cell numbers did not change, but above 100 µM, cell death occurred. Therefore, HK-2 cells were exposed to 20 µM Spd for 24 h to increase Spd levels in cells. HK-2 cells incubated with Spd showed high Spd levels in the cytoplasm (Supplementary Fig. S3b). Surprisingly, Spd caused activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which was increased by ~15 times that of controls (Fig. 4a, b). There was no significant change in protein levels of kelch-like ECH-associated protein 1 (Keap1), which is an adapter of the ubiquitin ligase complex that targets Nrf2 in HK-2 cells (Fig. 4a, c). Although the activation of Nrf2 was expected to be associated with the activation of inflammatory signaling, there was no significant change in the phosphorylation of nuclear factor-κB (NF-κB) by Spd in HK-2 cells (Fig. 4d, e). Immunocytochemistry showed the nuclear translocation of Nrf2 by Spd in HK-2 cells (Fig. 4f, g). Consistent with the nuclear translocation of Nrf2, the expression‎ of Nrf2-targeted genes, HO-1, NQO1, and GCLM mRNA, was induced in HK-2 cells by Spd (Fig. 4h–j). These data suggest that Spd activates the transcription factor Nrf2 in RTECs.

Fig. 4: Spd activates the transcription factor Nrf2 in renal tubular epithelial cells.

a Western blot analysis of Nrf2 and Keap1 protein levels in HK-2 cells incubated with Spd. The arrow shows two Nrf2 bands. b Relative levels of Nrf2 protein normalized to β-actin are shown (n = 6 in each group). Data are indicated as means ± SD. c Relative levels of Keap1 protein normalized to β-actin are shown (n = 6 in each group). Data are indicated as means ± SD. d Western blot analysis of phospho- and total NF-κB in HK-2 cells incubated with Spd. e Relative levels of phospho-NF-κB protein normalized to total NF-κB are shown (n = 6 in each group). Data are indicated as means ± SD. f Immunofluorescence images of Nrf2 in HK-2 cells incubated with Spd. Red, anti-Nrf2 antibody; blue, DAPI. Scale bars, 20 µm. g Quantification of the nuclear Nrf2-positive area corrected for nuclei. Data are indicated as means ± SD. h HO-1, i NQO1, and j GCLM mRNA expression‎ determined by real-time PCR in HK-2 cells incubated with Spd (n = 4 in each group). Data are indicated as means ± SD. *P < 0.05, **P < 0.01. Spd spermidine, Nrf2 nuclear factor erythroid 2-related factor 2, Keap1 kelch-like ECH-associated protein 1, NF-κB nuclear factor-κB, DAPI 4′,6-diamidino-2-phenylindole.

Full size image

Spd activates autophagy, promoting dissociation of Keap1 from Nrf2

We then investigated the detailed mechanism by which Spd activates Nrf2 in HK-2 cells. Polyamines, including Spd, are potent inducers of autophagy14. While Spd increased conversion of microtubule-associated protein 1 A/1B-light chain 3 (LC3)-I to LC3-II, the amount of p62, which is a typical substrate for autophagy, did not decrease, but increased instead (Fig. 5a–c). Although there was no significant difference in Keap1 protein levels between the control and Spd-treated HK-2 cells (Fig. 4c), co-localization of Keap1 and p62 was significantly increased by Spd (Fig. 5d, e, P < 0.05). Consistent with the increased co-localization of Keap1 and p62, the co-localization of Keap1 with LC3-positive autophagosomes increased in Spd-treated cells (Supplementary Fig. S4a). These results suggest that Keap1 translocates to autophagosomes by binding to p62. To assess autophagic flux, LC3-II levels in cells were examined in the presence of 100 µM hydroxychloroquine (HCQ). LC3-II levels were remarkably increased (Fig. 5f, g) in the presence of HCQ. These results suggest that Spd enhances autophagic flux and simultaneously activates degradation in lysosomes. Furthermore, p62, which was increased by Spd, was not further increased by the addition of HCQ (Fig. 5f, h). The activation of Nrf2 (Fig. 5i) and the expression‎ of target genes, such as HO-1 and GCLM (Fig. 5j, k), were partially reduced by HCQ. Significant Nrf2 activation by Spd was suppressed to two-thirds of normal levels in Atg5 knockdown cells (Supplementary Fig. S4b, c; P < 0.01, which changed to not significant after knockdown), which was accompanied by a trend towards reduced HO-1 mRNA expression‎ (Supplementary Fig. S4d). A similar trend was observed in Atg5 KO mouse embryonic fibroblasts, although the activation of Nrf2 by Spd was not as strong as that in tubular cells (Supplementary Fig. S4e–g). Nrf2 activation by Spd is expected to be mediated in part through autophagy.

Spd는 자가포식을 활성화하여

Keap1과 Nrf2의 분리를 촉진합니다.

Spd activates autophagy,

promoting dissociation of Keap1 from Nrf2

배경: 유비퀴틴-프로테아좀 시스템의 기능 장애는 기능하지 않고 잠재적으로 독성이 있는 단백질 응집체를 축적시킵니다. 결과: 단백질 응집체는 Nrf2를 활성화한 다음 생체 내에서 자가포식에 의해 제거됩니다. 결론: Nrf2와 자가포식은 모두 프로테아좀 장애에 대한 생체 내 세포의 적응 기능으로 작용합니다. 의의: 세포에는 프로테오스타시스 장애에 대항하는 세포 방어 메커니즘의 네트워크가 존재합니다.

유비퀴틴-프로테아좀 시스템과 자가포식은 세포의 단백질 항상성 유지에 매우 중요합니다. 이 두 경로는 상호 의존적이며, 어느 한 경로의 기능 장애는 인간 병리학의 특징인 유비퀴틴 양성 응집체의 축적을 유발합니다. 프로테아좀 기능 장애에 대한 생체 내 보상 작용을 밝히기 위해, 우리는 간에서 프로테아좀 활성이 감소된 마우스를 개발했습니다. 이 돌연변이 마우스는 심각한 간 손상을 보였으며, 유비퀴틴과 선택적 자가포식 및 항산화 Keap1-Nrf2 경로의 어댑터 단백질인 p62/Sqstm1에 양성인 응집체가 형성되었습니다. 이러한 응집체는 자가포식 소체에 선택적으로 포획되었으며, 자가포식이 동시에 억제되면 프로테아좀 활동이 손상된 간의 병리학적 특징이 악화되었습니다. 반면, p62/Sqstm1의 동시 결손은 간 병리학에 명백한 영향을 미치지 않았지만, p62/Sqstm1은 집합체 형성에 필수적이었습니다. 또한, 프로테아좀 기능 결손은 Nrf2의 전사 활성화를 유발했으며, 이는 생리적 적응 메커니즘으로 작용했습니다. 우리 in vivo 데이터는 세포가 결손된 프로테오스타시스(proteostasis)에 대항하는 세포 방어 메커니즘의 네트워크를 포함하고 있음을 시사합니다.

NRF2는 산화 스트레스에 반응하여 세포를 보호하는 역할을 하는 단백질이에요. KAP1은 NRF2를 억제하는 단백질이죠. 자가포식은 세포 내 불필요한 물질을 제거하는 과정이고요. NRF2 활성화는 자가포식을 유도하고, KAP1은 이를 억제하는 역할을 합니다. 이 세 가지 요소는 서로 밀접하게 연결되어 세포 항상성을 유지하는 데 중요한 역할을 합니다.

그런 다음 HK-2 세포에서

Spd가 Nrf2를 활성화하는 자세한 메커니즘을

조사했습니다.

Spd를 포함한 폴리아민은

강력한 자가포식 유도제입니다14.

Spd는

미세소관 관련 단백질 1A/1B-경쇄 3 (LC3)-I의 LC3-II로의 전환을 증가시켰지만,

자가포식의 전형적인 기질인 p62의 양은 감소하지 않고

오히려 증가했습니다 (그림 5a-c).

Keap1 단백질 수준은 대조군과 Spd 처리 HK-2 세포 간 유의미한 차이를 보이지 않았습니다(그림 4c). 그러나 Spd는 Keap1과 p62의 공위치를 유의미하게 증가시켰습니다(그림 5d, e, P < 0.05). Keap1과 p62의 공위화 증가와 일치하게, Spd 처리된 세포에서 Keap1과 LC3 양성 자식소체 간의 공위화가 증가했습니다(보조 그림 S4a). 이 결과는 Keap1이 p62와 결합하여 자식소체로 이동함을 시사합니다. 자식작용 유동성을 평가하기 위해 100μM 하이드록시클로로퀸(HCQ) 존재 하에서 세포 내 LC3-II 수준을 측정했습니다. HCQ 존재 시 LC3-II 수준은 현저히 증가했습니다(그림 5f, g).

이 결과는

Spd가 자가포식 유동성 autophagic flux, 을 증강시키고

동시에 리소좀 내 분해를 활성화함을 시사합니다.

또한 Spd에 의해 증가된 p62는 HCQ 추가 시 추가로 증가하지 않았습니다(그림 5f, h). Nrf2 활성화(그림 5i) 및 HO-1과 GCLM과 같은 표적 유전자 발현(그림 5j, k)은 HCQ에 의해 부분적으로 감소되었습니다. Spd에 의한 Nrf2 활성화는 Atg5 노크다운 세포에서 정상 수준의 2/3로 억제되었으며(보조 그림 S4b, c; P < 0.01, 노크다운 후 유의미하지 않음으로 변경), 이는 HO-1 mRNA 발현 감소 경향과 동반되었습니다(보조 그림 S4d). Atg5 KO 마우스 배아 섬유모세포에서도 유사한 경향이 관찰되었지만, Spd에 의한 Nrf2의 활성화는 관형 세포에서만큼 강하지는 않았습니다 (보충 그림 S4e–g). Spd에 의한 Nrf2의 활성화는 부분적으로 자가포식을 통해 매개되는 것으로 예상됩니다.

Fig. 5: Spd activates autophagy, resulting in acceleration of Keap1 degradation in renal tubular epithelial cells.

a Western blot analysis of LC3-I and II and p62 in HK-2 cells incubated with Spd. b Relative levels of LC3-II protein normalized to β-actin are shown (n = 6 in each group). Data are indicated as means ± SD. c Relative levels of p62 protein normalized to β-actin are shown (n = 6 in each group). Data are indicated as means ± SD. d Confocal immunofluorescence microscopic images of Keap1 and p62 in HK-2 cells incubated with Spd. Red, anti-Keap1 antibody; green, anti-p62 antibody; blue, DAPI. White arrowheads indicate co-localized areas. Scale bars, 5 µm. e Quantification of co-localization areas of Keap1 and p62 in each cell. Data are indicated as means ± SD. f Western blot analysis of LC3-I and II, p62, and Nrf2 in HK-2 cells incubated with Spd in the presence of HCQ. g LC3-II, h p62, and i Nrf2 relative protein levels normalized to β-actin are shown (n = 4 in each group). Data are indicated as means ± SD. j HO-1 and k GCLM mRNA expression‎ determined by real-time PCR (n = 4 in each group). Data are indicated as means ± SD. *P < 0.05, **P < 0.01. Spd spermidine, LC3 microtubule-associated protein 1A/1B-light chain

3, Keap1 kelch-like ECH-associated protein 1, HCQ hydroxychloroquine.

Full size image

Spd has antifibrotic effects, but does not inhibit the endothelin pathway like bardoxolone methyl

To further investigate whether Spd has a protective effect on fibrotic signaling in tubular cells, transforming growth factor β1 (TGFβ1), which is a fibrotic signal, secreted from HK-2 cells was measured with or without Spd. A certain amount of TGFβ1 was released from HK-2 cells, but the addition of Spd significantly suppressed this release (Fig. 6a, P < 0.01). Collagen 1 mRNA expression‎ in tubular cell fibrosis induced by TGFβ1 was significantly suppressed by Spd (Fig. 6b, P < 0.01). TGFβ1 stimuli alone did not increase HO-1 mRNA expression‎. Spd induced HO-1 gene expression‎ even in the presence of TGFβ1 (Fig. 6c). We investigated whether Spd inhibits oxidative stress because oxidative stress accelerates the progression of fibrosis in the kidney. Oxidative stress, which was represented by a decrease in mitochondrial membrane potential caused by H2O2, was significantly suppressed by the addition of Spd (Fig. 6d, e). These results suggest that Spd plays a protective role in fibrotic signaling in RTECs. The effect of Spd on the endothelin pathway was then examined. Bardoxolone methyl (CDDO-me), which is a representative Nrf2 inducer, increases the incidence of heart failure via inhibition of the endothelin pathway15. Real-time polymerase chain reaction (PCR) showed that CDDO-me 40 nM and Spd 20 µM were equivalent in their induction of HO-1 mRNA expression‎ (Fig. 6f). CDDO-me concentrations in the medium were determined with reference to the patient’s blood concentrations16. Endothelin-1 (ET-1) mRNA expression‎ and ET-1 protein concentrations were suppressed with CDDO-me, but not with Spd (Fig. 6g, h).

Fig. 6: Spd suppresses fibrotic signaling, but does not inhibit the endothelin pathway like bardoxolone methyl.

a Secretion of TGFβ1 from HK-2 cells treated with Spd measured by ELISA (n = 6 in each group). Data are indicated as means ± SD. b Collagen 1 and c HO-1 mRNA expression‎ determined by real-time PCR in HK-2 cells incubated with TGFβ1 in the presence of Spd (n = 3 in each group). Data are indicated as means ± SD. d MitoTracker Red CMXRos staining in HK-2 cells when exposed to H2O2 and Spd. Scale bar, 100 µm. e Quantification of Mitotracker Red CMXRos intensity measured by a multimode plate reader (n = 8 in each group). Data are indicated as means ± SD. f HO-1 and g ET-1 mRNA expression‎ determined by real-time PCR in HK-2 cells incubated with Spd and CDDO-me (n = 3 in each group). Data are indicated as means ± SD. h Secretion of ET-1 from HK-2 cells treated with Spd and CDDO-me measured by ELISA (n = 3 in each group). Data are indicated as means ± SD. **P < 0.01. NS not significant, Spd spermidine, TGFβ1 transforming growth factor β1, CDDO-me bardoxolone methyl.

Full size image

UUO-induced kidney fibrosis is aggravated and Nrf2 is decreased in the Arg2 KO kidney

To determine whether ARG2 is involved in kidney fibrosis, we used the UUO model with Arg2 KO mice. There was no significant difference in body weight or kidney function between wild-type (WT) and Arg2 KO mice with UUO. Systolic blood pressure in Arg2 KO mice with UUO tended to be higher than that in WT mice, but this was not significant (Supplementary Table S1). Spd protein levels were remarkably reduced in the Arg2 KO UUO kidney (Fig. 7a, b). Interstitial fibrosis of the kidney determined by Sirius red or Masson trichrome staining was significantly aggravated in Arg2 KO mice compared with that in WT mice (Fig. 7c–f, P < 0.05). Collagen 1 protein levels were significantly higher in Arg2 KO mice than in WT mice (Fig. 7g, P < 0.01). In Arg2 KO mice, α smooth muscle actin (αSMA) protein levels in the Arg2 KO kidney tended to be increased (Fig. 7h). Mature TGFβ protein levels were also increased in Arg2 KO mice (Fig. 7i). There was no difference in L-arginine concentrations between WT and Arg2 KO mice (Supplementary Fig. S5). Protein levels of ARG1, which is another arginase isoform, were not increased in the Arg2 KO UUO kidney (Supplementary Fig. S6a, b). In addition to a decrease in Spd, Smox mRNA expression‎ and SMOX protein levels were significantly decreased in Arg2 KO mice (Supplementary Fig. S7a–c, both P < 0.05). Acrolein protein levels were not obviously increased in Arg2 KO mice. (Supplementary Fig. S7d, e). In Arg2 KO mice, the expression‎ of enzymes related to arginine metabolism other than ARG2 was also altered. Protein levels of endothelial NOS (eNOS) and the endothelial marker CD31 were significantly increased in the Arg2 KO UUO kidney (Supplementary Fig. S8a–c, both P < 0.01), which suggested that angiogenesis was associated with fibrosis. On the basis of the above-mentioned results, inflammation and oxidative stress are expected to be enhanced in the Arg2 KO UUO kidney. However, Nrf2 protein levels in the Arg2 KO UUO kidney were not increased, but decreased by two-thirds those of the WT UUO kidney (Fig. 7j, k). Accordingly, HO-1 mRNA expression‎ was significantly reduced in the Arg2 KO UUO kidney (Fig. 7l, P < 0.01). Consistent with the results of the cell experiments, the lack of Spd in Arg2 KO mice may inhibit the activation of Nrf2. On the basis that Arg2 KO mice had reduced Spd levels, we hypothesized that fibrosis is exacerbated by inadequate Nrf2 activation in Arg2 KO mice. We investigated whether Spd supplementation suppresses fibrosis. Kidney fibrosis, which was significantly higher in Arg2 KO mice than in WT mice, was suppressed by treatment with Spd and the significant difference between WT and Arg2 KO mice disappeared (Fig. 7m, n). In the Arg2 KO kidney, HO-1 mRNA expression‎ of the Nrf2 target gene was significantly reduced, but tended to increase with Spd treatment (Supplementary Fig. S9).

Fig. 7: UUO-induced kidney fibrosis is aggravated in the Arg2 knockout mouse kidney.

a Confocal immunofluorescence images of Spd in the UUO kidney of WT and Arg2 KO mice. Green, anti-Spd antibody; blue, DAPI. Scale bars, 20 µm. b Quantification of the Spd-positive area (n = 6 in each group). Data are indicated as means ± SD. c Representative images of Sirius red staining in the UUO kidney of WT and Arg2 KO mice. Scale bars, 100 µm. d Quantification of the Sirius red staining-positive area (n = 6 in each group). Data are indicated as means ± SD. e Representative images of Masson trichrome staining in the UUO kidney of WT and Arg2 KO mice. Scale bars, 100 µm. f Quantification of the Masson trichrome staining-positive area (n = 6 in each group). Data are indicated as means ± SD. g Western blot analysis of collagen type 1 protein in the UUO kidney of WT and Arg2 KO mice. Quantification of relative levels of collagen type 1 protein normalized to αβ tubulin are shown on the right (n = 6 in each group). Data are indicated as means ± SD. h Western blot analysis of αSMA protein in the UUO kidney of WT and Arg2 KO mice. Quantification of relative levels of αSMA protein normalized to αβ tubulin are shown on the right (n = 6 in each group). Data are indicated as means ± SD. i Western blot analysis of mature TGFβ protein in the UUO kidney of WT and Arg2 KO mice. Quantification of relative levels of mature TGFβ protein normalized to αβ tubulin are shown on the right (n = 6 in each group). Data are indicated as means ± SD. j Western blot analysis of Nrf2 protein in the UUO kidney of WT and Arg2 KO mice. k Quantification of relative levels of Nrf2 protein normalized to αβ tubulin (n = 6 in each group). Data are indicated as means ± SD. l HO-1 mRNA expression‎ in the UUO kidney of WT and Arg2 KO mice determined by real-time PCR (n = 6 in each group). Data are indicated as means ± SD. m Representative images of Sirius red staining in the UUO kidney of WT and Arg2 KO mice treated with Spd. Scale, 100 µm. n Quantification of the Sirius red staining-positive area (n = 5 in each group). Data are indicated as means ± SD. *P < 0.05, **P < 0.01. NS not significant, UUO unilateral ureteral obstruction, Col1 collagen 1, Spd spermidine, DAPI 4′,6-diamidino-2-phenylindole, αSMA α smooth muscle actin, TGFβ transforming growth factor β1, Nrf2 nuclear factor erythroid 2-related factor 2.

Full size image

Discussion

In recent years, kidney injury and energy metabolism have been popular areas of focus in drug discovery. Amino acid metabolism is activated in the mouse diabetic kidney model17 or in hypertensive kidney damage4, but few studies have investigated whether metabolites are involved in kidney disorders. To the best of our knowledge, this study shows that the metabolite Spd is involved in antifibrosis.

Although arginine metabolism was expected to be impaired in Arg2 KO mice, there was no difference in L-arginine concentrations between WT and Arg2 KO UUO kidneys (Supplementary Fig. S5a). Parallel to the exacerbation of fibrosis, eNOS and CD31 protein levels were increased in the Arg2 KO UUO kidney (Supplementary Fig. S8a–c). L-arginine, which accumulates without undergoing degradation in Arg2 KO mice, may be degraded by angiogenesis-induced eNOS. In UUO kidneys in our study, ornithine (metabolite of ARG2) was increased 1.9 fold, whereas citrulline (metabolite of NOS) was increased 4.2-fold (Fig. 1b). NO production modifies renal hemodynamics in the early phase of UUO18 and plays a protective role against fibrosis in the chronic phase of UUO19,20. In a previous study, acute ischemia–reperfusion injury in Arg2 KO mice was reduced compared with that in WT mice in contrast to the present study12. There was no increase in eNOS, inducible NOS, or neuronal NOS expression‎ in the Arg2 KO kidney with acute injury compared with that in the WT12. The differences in NOS expression‎ changes may have led to phenotypic differences in acute and chronic renal injury in our study because eNOS is increased in the UUO kidney.

In recent years, there have been many important reports on the benefits of polyamines in pathological mouse models. Increased colonic luminal polyamines promote longevity in mice21, and bacterial-derived polyamines ameliorate symptoms in colitis model mice22. Oral supplementation of Spd extends the lifespan of mice and exerts cardioprotective effects in old mice23. In our UUO model, Spd administration also reduced fibrosis. SMOX is an enzyme that generates Spd and the highly toxic compound acrolein from Spm. A previous study showed that Spm concentrations were decreased with high SMOX activity, and plasma acrolein concentrations were increased in patients with CKD24. Acrolein was found to increase with renal ischemia-reperfusion injury and induce tubular cell death25. Cisplatin-induced acute kidney injury is alleviated in Smox KO mice in which Spd is reduced26. Chronic kidney injury may also be promoted because it is exacerbated in Smox KO mice in the chronic inflammatory model of dextran sulfate sodium-induced enteritis27. Not only a lack of ARG2, but also a decrease in SMOX, may contribute to the low Spd expression‎ in Arg2 KO mice.

In a lipopolysaccharide-induced acute kidney injury model, Spd inhibited inflammasome activation and promoted mitochondrial respiration in macrophages through the activation of eukaryotic translation initiation factor 5A-228. In renal ischemia–reperfusion injury model mice, Spd reduced poly (ADP-ribose) polymerase 1 activation and DNA nitrative stress29. Although Spd was administered in both of these studies28,29, Spd may be released from the injured tubules in the context of acute kidney injury. The biological defense response of increased spermidine levels in the impaired kidney may be more effective if it is supplemented by the administration of spermidine. The administration of Spd to 5/6 nephrectomized rats activated SIRT1 in vascular smooth muscle cells and reduced vascular calcification30. The protective effect of Spd on the kidneys has been observed in various cells of the kidney and may be more diverse than only transcriptional activation of Nrf2 via the activation of autophagy. We also found that acrolein protein levels were not reduced in Arg2 KO mice (Supplementary Fig. S7d, e). These findings suggest that acrolein is produced from other pathways, including heat-induced dehydration of glycerol, retro-aldol cleavage of dehydrated carbohydrates, lipid peroxidation of polyunsaturated fatty acids, and degradation of methionine and threonine31, rather than from polyamines, in Arg2 KO mice.

Nrf2 is a redox-sensitive transcription factor that regulates antioxidant proteins, cell cycle-related regulators, and detoxification enzymes32. Nrf2 has a renal protective role in various CKD models33,34,35,36,37. Nrf2 deficiency promotes the progression from acute tubular damage to chronic kidney fibrosis following UUO38. The Nrf2 pathway suppresses TGFβ1-SMAD-mediated fibrosis signaling in HK-2 cells39. Intriguingly, previous studies have shown that L-arginine induces an antioxidant response in the liver40 or ameliorates cardiac oxidative stress via the Nrf2 pathway41. Although arginine supplementation activates Nrf2, whether arginine itself or arginine metabolites activate Nrf2 remains unclear. A higher incidence of cardiovascular events with CDDO-me than with placebo prompted termination of a trial on advanced CKD42. Suppression of the endothelin pathway, which affects sodium and water homeostasis in the renal tubules, by CDDO-me is a mechanism contributing to adverse cardiovascular events15. The use of Spd in the treatment of CKD progression may avoid the issue of heart failure by activating Nrf2.

Spd is a natural polyamine involved in inducing autophagy14. The induction of autophagy in RTECs protects against apoptosis and promotes TGFβ degradation, thereby reducing development of renal interstitial fibrosis43. Spd also protects against liver fibrosis by enhancing Nrf2 signaling through activating autophagy44. In our study, co-localization of Keap1 and p62 was significantly increased by Spd in the cytoplasm of HK-2 cells (Fig. 5d, e). The binding of Keap1 to p62 was thought to increase free Nrf2. In a previous study, phosphorylation of p62 markedly increased p62’s binding affinity for Keap1, and consequently, p62 phosphorylation induced expression‎ of cytoprotective Nrf2 targets45. Another study showed that Nrf2 positively regulated p62 gene expression‎, which suggested a positive feedback loop46. These reports explain the phenomenon that p62 is increased by Spd, but its amount is not changed by autophagy inhibition in our experiments.

A limitation of this study is that Spd affects diverse pathways and cells, and effects on pathways other than the autophagy and Nrf2 pathways in tubular cells have not been investigated. Spd treatment of Arg2 KO mice did not suppress fibrosis to WT levels. Therefore, we consider that the phenotype of Arg2 KO mice may not be solely due to the amount of Spd.

In summary, arginine metabolism is activated and Spd is increased by fibrotic stimuli, resulting in acceleration of the Nrf2 pathway, which protects against interstitial inflammation and fibrosis in RTECs. Moreover, Spd activates the Nrf2 pathway in RTECs partly through the induction of autophagy. Increasing Spd levels may be effective in preventing chronic renal interstitial fibrosis.