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Biomedicines
Special Issues
Kidney Injury/Failure: Molecular Mechanisms and Clues for Intervention
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Kidney Injury/Failure: Molecular Mechanisms and Clues for Intervention

A special issue of Biomedicines (ISSN 2227-9059). This special issue belongs to the section "Cell Biology and Pathology".

Deadline for manuscript submissions: 30 June 2025 | Viewed by 5052

Special Issue Editor

Dr. Chang Chu

E-Mail Website
Guest Editor
1. Fifth Department of Medicine (Nephrology/Endocrinology/Rheumatology), University Medical Centre Mannheim, University of Heidelberg, 68167 Mannheim, Germany
2. Department of Nephrology, Charite University Berlin, 13353 Berlin, Germany
Interests: acute kidney injury; chronic kidney disease; dialysis; sodium–glucose cotransporter 2 inhibitors; angiogenesis biomarkers

Special Issue Information

Dear Colleagues,

Kidney injury/failure is a significant health concern worldwide and has been classified into two syndromes—acute kidney injury (AKI) and chronic kidney disease (CKD). However, a close interconnection between these two syndromes has recently been found. These two syndromes are independent risk factors for each other, and they are both risk factors for cardiovascular disease, resulting in greater mortality in patients with kidney failure. Currently, therapeutic approaches for patients with AKI and/or early-stage CKD include the prevention of nephrotoxic substances, lifestyle modifications, supportive care, Renin–angiotensin–aldosterone system (RAAS) inhibitors, and comorbidities management, but interventions for pathophysiological changes in the kidney itself remain limited. New therapeutic approaches are therefore needed to slow the deterioration of kidney function and even halt kidney impairment through early intervention. Therefore, this Special Issue aims to explore the latest advancements in understanding the complicated molecular and cellular pathways, and novel treatments of kidney injury. It provides a platform for researchers to share their insights and contribute to improving patient outcomes in kidney injury/failure with topics including but not limited to:

  1. Cellular and molecular pathways involved in kidney injury.
  2. Oxidative stress, inflammation, and immune responses in acute kidney injury or chronic kidney failure.
  3. Clinical trials/observational studies investigating novel treatments and interventions for kidney injury.
  4. Basic experiment of new therapeutic strategies for kidney injury.

Dr. Chang Chu
Guest Editor

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Keywords

  • acute kidney injury
  • chronic kidney diseases
  • molecular mechanisms
  • cellular mechanisms
  • new interventions
  • therapeutic approaches

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Published Papers (3 papers)

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Research

14 pages, 1561 KiB  
Article
Chrysin Attenuates Gentamicin-Induced Renal Injury in Rats Through Modulation of Oxidative Damage and Inflammation via Regulation of Nrf2/AKT and NF-kB/KIM-1 Pathways
by Talat A. Albukhari, Rehab M. Bagadood, Bayan T. Bokhari, Waheed A. Filimban, Hatem Sembawa, Nani Nasreldin, Hossam E. Gadalla and Mohamed E. El-Boshy
Biomedicines 2025, 13(2), 271; https://doi.org/10.3390/biomedicines13020271 - 23 Jan 2025
Viewed by 802
Abstract
Background: Gentamicin (GM) is extensively used as an antibiotic for the treatment of infections caused by Gram-negative bacteria. Oxidative stress and proinflammatory cytokines are implicated in GM-induced renal damage. Chrysin (CH), also known as 5,7-dihydroxyflavone, has been used in traditional medicine to treat [...] Read more.
Background: Gentamicin (GM) is extensively used as an antibiotic for the treatment of infections caused by Gram-negative bacteria. Oxidative stress and proinflammatory cytokines are implicated in GM-induced renal damage. Chrysin (CH), also known as 5,7-dihydroxyflavone, has been used in traditional medicine to treat various kidney disorders. The aim of this study was to investigate the antioxidant, anti-apoptotic, and anti-inflammatory effects of CH against nephrotoxicity induced by GM. Methods: Male rats were separated into four equal groups: a negative control group (NC), a CH-treated group (100 mg/kg/day per os), a group treated with GM (100 mg/kg/day IM), and a group treated with both GM and CH (100 mg/kg/day), for 10 days. Blood and urine renal markers were investigated. Results: GM caused increases in the serum creatinine and urea levels and decreases in creatinine clearance, urine flow, and urine volume in the GM-treated rats. Moreover, there were increases in the levels of IL-1β, TNF-α, IL-18, and MDA in the renal tissues, with an augmented expression of NF-κB/KIM-1, as well as decreases in antioxidant marker (GSH, GPx, CAT, and SOD) activities and decreased expressions of the anti-inflammatory transcription factors Nrf2 and AKT. The simultaneous treatment with CH in the GM-treated group protected renal tissues against the nephrotoxicity induced by GM, as demonstrated by the normalization of renal markers and improvement in histopathological damage. Conclusions: This study reveals that CH may attenuate GM-induced renal toxicity in rats. Full article
(This article belongs to the Special Issue Kidney Injury/Failure: Molecular Mechanisms and Clues for Intervention)
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Figure 1

Figure 1
<p>Interleaved scatter plots presenting the mean ± SD of the relative expression of NF-kB (<b>A</b>), KIM-1 (<b>B</b>), AKT (<b>C</b>), and Nrf2 (<b>D</b>) in the renal tissues obtained from the GM and GM + CH groups compared with their relative expression in the NC and CH groups. Dots, triangles, stars, and asterisks indicate the values of NF-kB, KIM-1, AKT, and Nrf2 for the rats in each group (<span class="html-italic">n</span> = 8). Red error bars represent the ± standard deviation of the mean, and the blue lines indicate the mean values of the relative expression of these genes in each group. Statistical significance is indicated as follows: * = <span class="html-italic">p</span> &lt; 0.05 compared with the NC group; <sup>#</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the CH group; <sup><span>$</span></sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the positive GM group; and <sup>¥</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the GM + CH group.</p> Full article ">Figure 2
<p>Interleaved scatter plots presenting the mean ± SD of the relative expression of Caspase-3 (<b>A</b>) and BCL-2 (<b>B</b>) in the renal tissues of the GM and GM + CH groups compared with their relative expression in the NC and CH groups. Dots, triangles, stars, and asterisks indicate the values of Caspase-3 and BCL-2 for the rats in each group (<span class="html-italic">n</span> = 8). Red error bars represent the ± standard deviation of the mean, and the blue lines indicate the mean values of the relative expression of these genes in each group. Statistical significance is indicated as follows: * = <span class="html-italic">p</span> &lt; 0.05 compared with the NC group; <sup>#</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the CH group; <sup><span>$</span></sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the positive GM group; and <sup>¥</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the GM + CH group.</p> Full article ">Figure 3
<p>Renal histological features in all study groups, visualized via H&amp;E staining (scale bar = 10 µm). (<b>A</b>) In the CH group, the kidney tissue shows normal renal glomeruli and renal tubules with a normal lining epithelium (as indicated by the arrow) (H&amp;E, 400×). (<b>B</b>) In the GM-treated group, the kidney tissue shows the dissolution of renal glomeruli (as indicated by the arrow) (H&amp;E, 400×). (<b>C</b>) In the GM-treated group, the kidney tissue also shows hemorrhaging in the interstitial tissue (as indicated by the arrow) that had replaced renal tubules and degenerative changes in the renal glomeruli (H&amp;E, 400×). (<b>D</b>) In the GM + CH-treated group, the kidney tissue shows normal renal glomeruli (as indicated by the arrow) and cloudy swelling in the renal tubular epithelium (as indicated by the arrowhead). (<b>E</b>) In the GM + CH-treated group, the kidney tissue also shows scattered necrosis of the renal tubular epithelium (as indicated by the arrowhead) and normal renal glomeruli (as indicated by the arrow) (H&amp;E, 400×).</p> Full article ">Figure 4
<p>The histopathological damage scores in the renal tissues of all groups, displayed as bars in the graph (data are shown as mean ± SD). Triangles, stars, and asterisks represent the values of histological scores for each rat per group (<span class="html-italic">n</span> = 8), red error bars represent the ± standard deviation of the mean, and the blue line indicates the mean values of histological scores in each group. Statistical significance is indicated as follows: <sup>#</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the CH group; and <sup>¥</sup> = <span class="html-italic">p</span> &lt; 0.05 compared with the GM + CH group.</p> Full article ">
21 pages, 4439 KiB  
Article
Potential Nephroprotective Effect of uPA against Ischemia/Reperfusion-Induced Acute Kidney Injury in αMUPA Mice and HEK-293 Cells
by Heba Abd Alkhaleq, Israel Hacker, Tony Karram, Shadi Hamoud, Aviva Kabala and Zaid Abassi
Biomedicines 2024, 12(10), 2323; https://doi.org/10.3390/biomedicines12102323 - 12 Oct 2024
Viewed by 1106
Abstract
Background/Objectives: The incidence of acute kidney injury (AKI) has been steadily increasing. Despite its high prevalence, there is no pathogenetically rational therapy for AKI. This deficiency stems from the poor understanding of the pathogenesis of AKI. Renal ischemia/hypoxia is one of the leading [...] Read more.
Background/Objectives: The incidence of acute kidney injury (AKI) has been steadily increasing. Despite its high prevalence, there is no pathogenetically rational therapy for AKI. This deficiency stems from the poor understanding of the pathogenesis of AKI. Renal ischemia/hypoxia is one of the leading causes of clinical AKI. This study investigates whether αMUPA mice, overexpressing the urokinase plasminogen activator (uPA) gene are protected against ischemic AKI, thus unraveling a potential renal damage treatment target. Methods: We utilized an in vivo model of I/R-induced AKI in αMUPA mice and in vitro experiments of uPA-treated HEK-293 cells. We evaluated renal injury markers, histological changes, mRNA expression of inflammatory, apoptotic, and autophagy markers, as compared with wild-type animals. Results: the αMUPA mice exhibited less renal injury post-AKI, as was evident by lower SCr, BUN, and renal NGAL and KIM-1 along attenuated adverse histological alterations. Notably, the αMUPA mice exhibited decreased levels pro-inflammatory, fibrotic, apoptotic, and autophagy markers like TGF-β, IL-6, STAT3, IKB, MAPK, Caspase-3, and LC3. By contrast, ACE-2, p-eNOS, and PGC1α were higher in the kidneys of the αMUPA mice. In vitro results of the uPA-treated HEK-293 cells mirrored the in vivo findings. Conclusions: These results indicate that uPA modulates key pathways involved in AKI, offering potential therapeutic targets for mitigating renal damage. Full article
(This article belongs to the Special Issue Kidney Injury/Failure: Molecular Mechanisms and Clues for Intervention)
►▼ Show Figures

Figure 1

Figure 1
<p>In vivo and in vitro levels/expression of kidney function/renal injury markers in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Serum creatinine level in mice; (<b>B</b>) Blood urea nitrogen (BUN) level in mice; (<b>C</b>) q-PCR of renal NGAL in mice; (<b>D</b>) q-PCR of renal KIM-1 in mice; (**, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—WT vs. αMUPA which underwent similar procedure); (<b>E</b>) q-PCR of NGAL in cells; (<b>F</b>) q-PCR of KIM-1 in cells; (*, <span class="html-italic">p</span> &lt; 0.05—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 2
<p>Effects of AKI on renal histology in WT and αMUPA mice: Representative histological images of kidney sections from WT and αMUPA mice, including both sham-operated and AKI models, were taken. The images show cortical, outer medullary, and inner medullary regions stained with hematoxylin and eosin. The first column displays a kidney section from a sham-operated WT mouse, followed by an AKI WT mouse, sham-operated αMUPA mouse, and an AKI αMUPA mouse in the subsequent columns. Long arrows point to areas of tubular collapse, brush border loss, and detachment of cells from the tubular basement membrane, while short arrows indicate areas of congestion. The images were captured at 20× magnification with a scale bar of 100 µm.</p> Full article ">Figure 3
<p>In vivo and in vitro expression/abundance of uPA and uPAR in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Renal Urokinase Plasminogen activator (uPA) expression in mice; (<b>B</b>) Renal Urokinase Plasminogen receptor (PlauR/uPAR) expression in mice; (<b>C</b>) Renal Plasminogen activator inhibitor 1 (PAI-1) expression in mice; (<b>D</b>) Immunoreactive levels of urokinase plasminogen activator (uPA) in mice; (<b>E</b>) Immunoreactive levels of urokinase plasminogen receptor (uPAR) in mice; (**, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—WT vs. αMUPA which underwent similar procedure); (<b>F</b>) uPA expression in cells with/out uPA treatment; (<b>G</b>) uPAR expression in cells; (<b>H</b>) PAI-1 expression in cells; (<b>I</b>) uPA abundance in cells; (<b>J</b>) uPAR abundance in cells (*, <span class="html-italic">p</span> &lt; 0.05—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 4
<p>In vivo and in vitro expression of renal leptin, insulin receptor and PGC1-α in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Insulin receptor (InsR) in mice; (<b>B</b>) PGC1α in mice; (<b>C</b>) Leptin in mice (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05—WT vs. αMUPA which underwent similar procedure); (<b>D</b>) InsR in cells; (<b>E</b>) PGC1α in cells (*, <span class="html-italic">p</span> &lt; 0.05—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 5
<p>In vivo and in vitro expression/abundance of renal inflammatory and fibrotic markers in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Expression of IL-6 in mice; (<b>B</b>) Expression of Toll like receptor 4 (TLR4) in mice; (<b>C</b>) Expression of TGF-β in mice; (<b>D</b>) Immunoreactive levels of STAT-3 in mice; (<b>E</b>) Immunoreactive levels of p-STAT3 in mice; (<b>F</b>) Immunoreactive levels of Cathepsin L in mice; (<b>G</b>) Immunoreactive levels of IKB in mice; (<b>H</b>) Immunoreactive levels of MAPK in mice; (<b>I</b>) Immunoreactive levels of TGF-β in mice (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—WT vs. αMUPA which underwent similar procedure); (<b>J</b>) Expression of IL-6 in cells; (<b>K</b>) Expression of TGF-β in cells; (<b>L</b>) Immunoreactive levels of STAT-3 in cells; (<b>M</b>) Immunoreactive levels of p-STAT-3 in cells; (<b>N</b>) Immunoreactive levels of IL-6 in cells; (<b>O</b>) Immunoreactive levels of IKB in cells; (<b>P</b>) Immunoreactive levels of MAPK in cells; (<b>Q</b>) Immunoreactive levels of TGF-β in cells (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 5 Cont.
<p>In vivo and in vitro expression/abundance of renal inflammatory and fibrotic markers in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Expression of IL-6 in mice; (<b>B</b>) Expression of Toll like receptor 4 (TLR4) in mice; (<b>C</b>) Expression of TGF-β in mice; (<b>D</b>) Immunoreactive levels of STAT-3 in mice; (<b>E</b>) Immunoreactive levels of p-STAT3 in mice; (<b>F</b>) Immunoreactive levels of Cathepsin L in mice; (<b>G</b>) Immunoreactive levels of IKB in mice; (<b>H</b>) Immunoreactive levels of MAPK in mice; (<b>I</b>) Immunoreactive levels of TGF-β in mice (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—WT vs. αMUPA which underwent similar procedure); (<b>J</b>) Expression of IL-6 in cells; (<b>K</b>) Expression of TGF-β in cells; (<b>L</b>) Immunoreactive levels of STAT-3 in cells; (<b>M</b>) Immunoreactive levels of p-STAT-3 in cells; (<b>N</b>) Immunoreactive levels of IL-6 in cells; (<b>O</b>) Immunoreactive levels of IKB in cells; (<b>P</b>) Immunoreactive levels of MAPK in cells; (<b>Q</b>) Immunoreactive levels of TGF-β in cells (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 6
<p>In vivo and in vitro expression/abundance of renal of apoptotic and autophagy markers in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Expression of Caspase-3 in mice; (<b>B</b>) Expression of Caspase-7 in mice; (<b>C</b>) Expression of LC3 in mice; (<b>D</b>) Expression of P62 in mice; (<b>E</b>) Immunoreactive levels of LC3 in mice; (<b>F</b>) Expression of Galectin-8 in mice (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01—WT vs. αMUPA which underwent similar procedure); (<b>G</b>) Expression of Caspase-3 in cells; (<b>H</b>) Expression of Caspase-7 in cells; (<b>I</b>) Expression of LC3 in cells; (<b>J</b>) Expression of P62 in cells; (<b>K</b>) Immunoreactive levels of LC3 in cells; (<b>L</b>) Expression of Galectin-8 in cells (<span>$</span>, <span class="html-italic">p</span> &lt; 0.05—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 7
<p>In vivo and in vitro expression/abundance of renal ACE-2, MasR and Renin in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) Expression of ACE-2 in mice; (<b>B</b>) Expression of Mas receptor in mice; (<b>C</b>) Expression of Renin in mice; (<b>D</b>) ACE-2 immunoreactive levels amount in mice (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01—Sham vs. AKI in the same mice strain, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01—WT vs. αMUPA which underwent similar procedure); (<b>E</b>) Expression of ACE-2 in cells; (<b>F</b>) Expression of Mas receptor in cells; (<b>G</b>) ACE-2 immunoreactive levels amount in cells (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01—with vs. without uPA treatment in the same cultured cells group, <span>$</span><span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.001—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 8
<p>In vivo and in vitro expression/abundance of renal of eNOS and p-eNOS in WT and αMUPA mice following sham/AKI (in vivo) and in cultured HEK-293 and ACE-2 overexpressing cells with/out uPA treatment (in vitro): (<b>A</b>) eNOS Immunoreactivity in mice; (<b>B</b>) p-eNOS Immunoreactivity in mice; (<b>C</b>) Expression of eNOS in mice (*, <span class="html-italic">p</span> &lt; 0.05—Sham vs. AKI in the same mice strain, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01—WT vs. αMUPA which underwent similar procedure); (<b>D</b>) eNOS Immunoreactivity in cells; (<b>E</b>) p-eNOS Immunoreactivity in cells; (<b>F</b>) Expression of eNOS in cells (*, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001—with vs. without uPA treatment in the same cultured cells group, <span>$</span>, <span class="html-italic">p</span> &lt; 0.05, <span>$</span><span>$</span>, <span class="html-italic">p</span> &lt; 0.01—HEK-293 vs. ACE-2 overexpressing cells which underwent similar treatment).</p> Full article ">Figure 9
<p>Schematic description of potential mechanisms underlying the nephroprotective actions of uPA against I/R-induced AKI. Figure created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">
17 pages, 9734 KiB  
Article
YAP/ACSL4 Pathway-Mediated Ferroptosis Promotes Renal Fibrosis in the Presence of Kidney Stones
by Lei Li, Zehua Ye, Yuqi Xia, Bojun Li, Lijia Chen, Xinzhou Yan, Tianhui Yuan, Baofeng Song, Weimin Yu, Ting Rao, Fangyou Lin, Xiangjun Zhou and Fan Cheng
Biomedicines 2023, 11(10), 2692; https://doi.org/10.3390/biomedicines11102692 - 1 Oct 2023
Cited by 10 | Viewed by 2374
Abstract
The potential association between calcium oxalate stones and renal fibrosis has been extensively investigated; however, the underlying mechanisms remain unclear. Ferroptosis is a novel form of cell death characterized by iron-dependent lipid peroxidation and regulated by acyl coenzyme A synthase long-chain family member [...] Read more.
The potential association between calcium oxalate stones and renal fibrosis has been extensively investigated; however, the underlying mechanisms remain unclear. Ferroptosis is a novel form of cell death characterized by iron-dependent lipid peroxidation and regulated by acyl coenzyme A synthase long-chain family member 4 (ACSL4). Yes-associated protein (YAP), a transcriptional co-activator in the Hippo pathway, promotes ferroptosis by modulating ACSL4 expression. Nevertheless, the involvement of YAP–ACSL4 axis-mediated ferroptosis in calcium oxalate crystal deposition-induced renal fibrosis and its molecular mechanisms have not been elucidated. In this study, we investigated ACSL4 expression and ferroptosis activation in the kidney tissues of patients with calcium oxalate stones and in mice using single-cell sequencing, transcriptome RNA sequencing, immunohistochemical analysis, and Western blot analysis. In vivo and in vitro experiments demonstrated that inhibiting ferroptosis or ACSL4 mitigated calcium oxalate crystal-induced renal fibrosis. Furthermore, YAP expression was elevated in the kidney tissues of patients with calcium oxalate stones and in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines. Mechanistically, in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines, activated YAP translocated to the nucleus and enhanced ACSL4 expression, consequently inducing cellular ferroptosis. Moreover, YAP silencing suppressed ferroptosis by downregulating ACSL4 expression, thereby attenuating calcium oxalate crystal-induced renal fibrosis. Conclusively, our findings suggest that YAP–ACSL4-mediated ferroptosis represents an important mechanism underlying the induction of renal fibrosis by calcium oxalate crystal deposition. Targeting the YAP–ACSL4 axis and ferroptosis may therefore hold promise as a potential therapeutic approach for preventing renal fibrosis in patients with kidney stones. Full article
(This article belongs to the Special Issue Kidney Injury/Failure: Molecular Mechanisms and Clues for Intervention)
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<p>Ferroptosis is activated in mice with CaOx kidney stones. (<b>A</b>) HE, Von Kossa and Masson staining results to evaluate tubular damage, CaOx crystals and collagen fibrillation deposition (<span class="html-italic">n</span> = 5). The scale bar represents 50 µm. (<b>B</b>) GO enrichment analysis. (<b>C</b>) TEM showed shrunken mitochondria with outer membrane ruptured (indicated by red arrows) in mice with CaOx kidney stones. The scale bar represents 1 µm. (<b>D</b>,<b>E</b>) BUN and Scr levels of kidney tissue (<span class="html-italic">n</span> = 5). (<b>F</b>) Western blot analysis showed the expressions of SLC7A11, GPX4, fibronectin, and α-SMA in kidney tissues and quantification by densitometry. Data were presented as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 2
<p>Inhibition of ferroptosis alleviates renal fibrosis. (<b>A</b>–<b>E</b>) Measurement of the levels of BUN, Scr, GSH, CAT, and MDA in the serum of mice (<span class="html-italic">n</span> = 5). (<b>F</b>) HE, Von Kossa and Masson staining results to evaluate tubular damage, CaOx crystals and collagen fibrillation deposition (<span class="html-italic">n</span> = 5). The scale bar represents 50 µm. (<b>G</b>) Immunofluorescence staining analysis of SLC7A11, GPX4 and α-SMA expressions in mouse kidney tissues and semi-quantitative analysis (<span class="html-italic">n</span> = 5). The scale bar represents 50 µm. (<b>H</b>) Western blot analysis showed the expressions of SLC7A11, GPX4, fibronectin and α-SMA in kidney tissues and quantification by densitometry. Data were presented as mean ± SD; ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. control group; # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01 vs. Gly group.</p> Full article ">Figure 3
<p>ACSL4 expression is upregulated in CaOx kidney stones. (<b>A</b>) Single-cell sequencing database of ACSL4 expression in normal subjects and patients with CKD. (<b>B</b>) RNA sequencing analysis of the transcriptomes of control and Gly group mice, red represents highly expressed genes and blue represents low expressed genes (<span class="html-italic">n</span> = 3). (<b>C</b>) Western blot analysis showed the expressions of ACSL4 expression in kidney tissues and quantification by densitometry (<span class="html-italic">n</span> = 5). (<b>D</b>,<b>E</b>) qPCR and immunofluorescence analysis of ACSL4 expression in the kidney of normal and kidney stone patients and semi-quantitative analysis (<span class="html-italic">n</span> = 6). The scale bar represents 50 µm. Data were presented as mean ± SD; ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 4
<p>ACSL4-induced ferroptosis promotes renal fibrosis in mice with CaOx kidney stones. (<b>A</b>) Overall docking plot of abemaciclib and ACSL4. (<b>B</b>) Body weight changes in mice (<span class="html-italic">n</span> = 5). (<b>C</b>–<b>G</b>) Measurement of the levels of BUN, Scr, GSH, CAT, and MDA in the serum of mice (<span class="html-italic">n</span> = 5). (<b>H</b>) HE, Von Kossa, Masson, and Perls staining to assess tubular damage, CaOx crystals, collagen fibrillation and iron deposition (<span class="html-italic">n</span> = 5). The scale bar represents 50 µm. (<b>I</b>) Western blot analysis showed the expressions of ACSL4, P53, SLC7A11, GPX4, fibronectin, and α-SMA in mouse kidney tissues and quantification by densitometry. (<b>J</b>) Immunofluorescence staining analysis of ACSL4, SLC7A11, GPX4, and α-SMA expressions in mouse kidney tissues and semi-quantitative analysis (<span class="html-italic">n</span> = 5). The scale bar represents 50 µm. (<b>K</b>) TEM revealed mitochondrial damage (indicated by red arrows) in mice with CaOx kidney stones. The scale bar represents 1 µm. Data were presented as mean ± SD; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. control group; # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01 vs. Gly group.</p> Full article ">Figure 5
<p>ACSL4-induced ferroptosis promotes COM-induced fibrosis in HK-2 cells. (<b>A</b>) Western blot analysis showed the expressions of ACSL4, GPX4, fibronectin and α-SMA after stimulation of HK-2 cells with different concentrations of COM and quantification by densitometry. (<b>B</b>) Cell viability of HK-2 under different concentrations of COM stimulation. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. control group. (<b>C</b>) Western blots validated ACSL4-siRNA knockdown efficiency. ** <span class="html-italic">p</span> &lt; 0.01 vs. COM group. (<b>D</b>) Western blot analysis showed the expressions of P53, SLC7A11, GPX4, fibronectin and Collagen I after knockdown of ACSL4 and quantification by densitometry. (<b>E</b>) Immunofluorescence analysis of SLC7A11, GPX4 and α-SMA expressions in HK-2 cells after knockdown of ACSL4 and semi-quantitative analysis. The scale bar represents 50 µm. (<b>F</b>) Measurement of lipid peroxidation in HK-2 cells by the C11 BODIPY581/591 fluorescent probe. The scale bar represents 50 µm. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. Con-siRNA group; # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001 vs. Con-siRNA + COM group. Data were presented as mean ± SD.</p> Full article ">Figure 6
<p>YAP is involved in regulating the expression of ACSL4 transcript levels, and inhibition of YAP inhibits ACSL4-mediated ferroptosis and reverses renal fibrosis. (<b>A</b>) Western blots validated YAP-siRNA knockdown efficiency. * <span class="html-italic">p</span> &lt; 0.05 vs. COM group.(<b>B</b>) Western blot analysis showed the expressions of ACSL4, SLC7A11, GPX4, fibronectin, and Collagen I after knockdown of YAP and quantification by densitometry. (<b>C</b>) Measurement of changes in intracellular ROS in HK-2 cells after knockdown of YAP by flow cytometry. (<b>D</b>) Immunofluorescence analysis of ACSL4, SLC7A11, and GPX4 expressions in HK-2 cells following knockdown of YAP and semi-quantitative analysis. The scale bar represents 50 µm. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. Con-siRNA group; # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01 vs. Con-siRNA + COM group. (<b>E</b>) Western blot analysis showed the expressions of SLC7A11, GPX4, fibronectin, and Collagen I expressions after overexpression of YAP, with or without ACSL4-siRNA and quantification by densitometry. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. COM group; # <span class="html-italic">p</span> &lt; 0.05 vs. ACSL4-siRNA + COM group; ns, no statistical significance. Data were presented as mean ± SD.</p> Full article ">Figure 7
<p>Inhibition of YAP mediates the downregulation of ACSL4 expression which, in turn, inhibits CaOx crystal-induced ferroptosis and renal fibrosis.</p> Full article ">
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