Effects of HIF-1α on renal fibrosis in cisplatin-induced chronic kidney disease.

Cisplatin (Cis) can cause chronic kidney disease (CKD) and promote renal fibrosis, but the underlying mechanism is not fully understood. Hypoxia inducible factor-1α (HIF-1α) can promote renal fibrosis in some kidney diseases, but its role in Cis-induced CKD is still unknown. Notch-1 is a recognized molecule that promotes renal fibrosis under pathological circumstances, and evidence shows that HIF-1α and Notch-1 are closely related to each other. In the present study, mice with HIF-1α gene knockout in proximal tubular cells (PTCs) (PT-HIF-1α-KO) were generated and treated with Cis to induce CKD. A human proximal tubular cell line (HK-2) and primary mouse PTCs were used for in vitro studies. The results showed that HIF-1α was increased in the kidneys of Cis-treated wild-type mice, accompanied by elevated Notch-1, Notch-1 intracellular domain (N1ICD), Hes-1 and renal fibrosis. However, these alterations were partially reversed in PT-HIF-1α-KO mice. Similar results were observed in HK-2 cells and primary mouse PTCs. In addition, treating the cells with Cis induced a marked interaction of HIF-1α and N1ICD. Further inhibiting Notch-1 significantly reduced cellular fibrogenesis but did not affect HIF-1α expression. The data suggested that HIF-1α could promote renal fibrosis in Cis-induced CKD by activating Notch-1 both transcriptionally and posttranscriptionally and that HIF-1α may serve as a potential therapeutic target for Cis-induced CKD.

Renal hypoxia is a common pathophysiological condition in kidney disease [9, 10].

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Hypoxia inducible factor 1α (HIF-1α) is a key regulatory molecule in mammals under 13 hypoxic conditions [11,12]. HIF-1α is involved in CKD progression caused by various 14 stimuli, such as ischemia-reperfusion, aristolochic acid and unilateral ureteral occlusion In the present study, we found that HIF-1α was obviously elevated in the kidney 2 cortex of Cis-treated wild-type (WT) mice, accompanied by elevated Notch-1, N1ICD, 3 Hes-1 and renal fibrosis compared to the WT control group, but these alterations were 4 partially reversed in mice with PTC-specific HIF-1α knockout (PT-HIF-1α-KO). Similar 5 results were observed in HK-2 cells cultured with Cis and were further confirmed in 6 mouse primary PTCs. In addition, a marked interaction of HIF-1α and N1ICD was 7 observed in Cis-treated HK-2 cells and mouse primary PTCs. Furthermore, inhibiting 8 Notch-1 decreased cellular fibrogenesis but had no obvious effects on HIF-1α expression. 9 These data indicated that HIF-1α could promote renal fibrosis by activating Notch-1 in 10 Cis-induced CKD.  13 PT-HIF-1α-KO mice were generated and identified as previously described [14]. The  Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20210061 Primary mouse PTCs were isolated and cultured as previously described [14]. HK-2 1 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) containing 10% 2 FBS. All cells were incubated in a cell incubator with 5% CO 2 and 95% air at 37°C. For  The body weight and kidney weight were measured by electronic balance before and 11 after the mice were sacrificed, respectively. To measure the serum creatinine (Scr) and   19 Hematoxylin and eosin (HE) staining and Masson trichrome staining were performed 20 to analyze kidney morphological changes. As previously described [ to the percentage of damaged tubules in HE-stained sections and the stained 4 tubulointerstitial area in Masson trichrome-stained sections, respectively, as previously     Cell IF staining was performed as previously described with some modifications [14]. Briefly, HK-2 cells and primary mouse PTCs were fixed with 4% paraformaldehyde for 5 1 minutes, permeabilized with methanol at -20°C for approximately 10 minutes, blocked 2 with blocking buffer (phosphate-buffered saline+1% bovine serum albumin+0.3% Triton 3 X-100) for 1 h at 37°C, and then incubated with primary antibodies against HIF-1α (1:50, 4 Abcam, ab179483), Notch-1 (1:100, Abcam, ab27526), and N1ICD (1:100, Abcam, 5 ab8925) overnight at room temperature. After washing with PBS, the cells were incubated 6 with Alexa Fluor 488-conjugated goat anti-rabbit antibodies for 1 h at 37°C, followed by 7 Hoechst staining to delineate the nuclei. Images were obtained by a confocal laser 8 scanning microscope (Zeiss LSM 780). 9 2.8 qPCR analysis 10 qPCR was performed as previously described [24]. Briefly, total RNA was extracted 11 from the kidney superficial cortex or cultured cells, and then the RNA was reverse 12 transcribed into cDNA using a cDNA Synthesis Kit (TaKaRa, Japan) according to the 13 instructions. qPCR was performed using SYBR premix EXTaq TM reagents (TaKaRa,       Cis treatment ( Figure 1G). Further semiquantitative analysis of HE and Masson trichrome 1 staining showed similar tubular damage and tubulointerstitial fibrosis (TIF) scores 2 between the WT mice and PT-HIF-1α-KO mice without Cis treatment, whereas the scores 3 were markedly higher in WT mice than in PT-HIF-1α-KO mice after Cis intervention 4 ( Figure 1H). The degree of apoptosis in the renal tubular epithelial cells of the WT mice 5 was significantly higher than that in the renal tubular epithelial cells of the 6 PT-HIF-1α-KO mice after two months of Cis intervention, as indicated by TUNEL 7 staining ( Figure 1I and 1J).

HIF-1α gene deficiency inhibited Notch-1 signaling in mice with Cis-induced
17 CKD. 18 The qPCR results showed the HIF-1α mRNA expression was slightly higher in the  Notch-1 and N1ICD expression showed a trend consistent with qPCR ( Figure 4B and 4C), 5 as well as cell IF staining ( Figure 4D). Co-IP experiments followed by WB analyses were 6 performed in HK-2 cells using antibodies against HIF-1α and N1ICD. The results showed 7 that HIF-1α antibody pulled down N1ICD and that N1ICD antibody also pulled down  N1ICD expression was much higher in the primary PTCs of WT mice than in those from 1 PT-HIF-1α-KO mice after treatment with Cis ( Figure 5B and 5C). The IF analysis of 2 HIF-1α, Notch-1 and N1ICD expression in cells showed results consistent with the WB 3 results ( Figure 5D). Co-IP experiments followed by WB analysis showed that N1ICD was 4 specifically pulled down by HIF-1α antibody and that HIF-1α could also be pulled down 5 by N1ICD antibody in Cis-treated primary PTCs from WT mice ( Figure 5E and 5F).  with Cis but not HIF-1α expression.

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The qPCR results showed that the HIF-1α, Notch-1 and Hes-1 mRNA levels were 14 notably increased in HK-2 cells treated with Cis compared to the control group and were 15 further increased by transfection with HIF-1α overexpression plasmid. When HK-2 cells 16 were cotreated with Notch-1 siRNA, the Notch-1 and Hes-1 mRNA levels were 17 significantly decreased, but there was no obvious effect on HIF-1α mRNA expression 18 ( Figure 6A). The western blot analysis of HIF-1α, Notch-1 and N1ICD showed consistent 19 results with qPCR ( Figure 6B and 6C). qPCR analysis showed that the mRNA levels of with HIF-1α plasmid but notably reduced by Notch-1 siRNA ( Figure 6D). The WB 1 analysis of FN and α-SMA expression showed consistent results with qPCR ( Figure 6E 2 and 6F).

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Primary PTCs were isolated from WT mice to further verify these results. As shown 4 in Figure 6G to Figure   our previous study [14]. However, the role of HIF-1α in Cis-induced CKD is still unclear. In the present study, we found that compared to PT-HIF-1α-KO mice, WT mice 1 experienced much more severe renal dysfunction and renal fibrosis two months after Cis 2 injection (Figure 1 and 2). These data indicated for the first time that HIF-1α might play  Here, we found that the expression of HIF-1α was increased by Cis in vivo and in vitro.

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Perhaps the differences in the experimental protocols, such as the doses of Cis, the time of 10 Cis intervention and the animal strains, may contribute to these discrepancies.

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The next question is why HIF-1α expression was increased by Cis treatment without 12 hypoxic stimulation in our study. HIF-1α is conventionally regulated by oxygen content,  This evidence may explain why the HIF-1α level was increased in this study, but the 21 precise mechanism still needs to be further studied. in the mouse kidney cortex increased the most among the four types after two months of 5 Cis treatment, and the change in Notch-1 was tightly associated with HIF-1α (Figure 3).

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Therefore, we further studied the precise relationship between HIF-1α and Notch-1 in 7 Cis-induced CKD. It is known that there is an intricate relationship between HIF-1α and Notch-1 signaling appear to be cell type-and disease-dependent. In the present study, we 18 observed that HIF-1α could induce the mRNA and protein expression of Notch-1 in 19 Cis-treated HK-2 cells and primary mouse PTCs. We also observed an obvious interaction Cis-induced CKD will be the focus of our future study.

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In conclusion, the current study demonstrated for the first time that HIF-1α plays a 17 key role in renal fibrosis in Cis-induced CKD by activating Notch-1 signaling both 18 transcriptionally and posttranscriptionally (Figure 7). HIF-1α may serve as a potential 19 therapeutic target to prevent renal fibrosis in Cis-induced CKD. The data that support the findings of the present study are available from the 10 corresponding author, Lin Sun, upon reasonable request.

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The authors declare they have no competing interests and have nothing to disclose.       The increased Notch-1 in PTCs combines with its ligands and releases the intracellular 5 domain (N1ICD). HIF-1α interacts with and stabilizes the released N1ICD, facilitating 6 N1ICD translocation into the nucleus to promote its target gene expression and aggravate