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22 pages, 1297 KiB

Open AccessReview

Progress in Lactate Metabolism and Its Regulation via Small Molecule Drugs

by Jin Liu, Feng Zhou, Yang Tang, Linghui Li and Ling Li

Molecules 2024, 29(23), 5656; https://doi.org/10.3390/molecules29235656 - 29 Nov 2024

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Lactate, once viewed as a byproduct of glycolysis and a metabolic “waste”, is now recognized as an energy-providing substrate and a signaling molecule that modulates cellular functions under pathological conditions. The discovery of histone lactylation in 2019 marked a paradigm shift, with subsequent [...] Read more.

Lactate, once viewed as a byproduct of glycolysis and a metabolic “waste”, is now recognized as an energy-providing substrate and a signaling molecule that modulates cellular functions under pathological conditions. The discovery of histone lactylation in 2019 marked a paradigm shift, with subsequent studies revealing that lactate can undergo lactylation with both histone and non-histone proteins, implicating it in the pathogenesis of various diseases, including cancer, liver fibrosis, sepsis, ischemic stroke, and acute kidney injury. Aberrant lactate metabolism is associated with disease onset, and its levels can predict disease outcomes. Targeting lactate production, transport, and lactylation may offer therapeutic potential for multiple diseases, yet a systematic summary of the small molecules modulating lactate and its metabolism in various diseases is lacking. This review outlines the sources and clearance of lactate, as well as its roles in cancer, liver fibrosis, sepsis, ischemic stroke, myocardial infarction, and acute kidney injury, and summarizes the effects of small molecules on lactate regulation. It aims to provide a reference and direction for future research. Full article

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23 pages, 1016 KiB

Open AccessReview

Exploring Endogenous Tryptamines: Overlooked Agents Against Fibrosis in Chronic Disease? A Narrative Review

by Hunter W. Korsmo

Livers 2024, 4(4), 615-637; https://doi.org/10.3390/livers4040043 - 28 Nov 2024

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Abstract

Long regarded as illicit substances with no clinical value, N-dimethylated tryptamines—such as N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and bufotenine—have been found to produce naturally in a wide variety of species, including humans. Known for their psychoactive effects through [...] Read more.

Long regarded as illicit substances with no clinical value, N-dimethylated tryptamines—such as N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and bufotenine—have been found to produce naturally in a wide variety of species, including humans. Known for their psychoactive effects through serotonin receptors (5-HTRs), N-dimethylated tryptamines are currently being reinvestigated clinically for their long-term benefits in mental disorders. Endogenous tryptamine is methylated by indolethylamine-N-methyltransferase (INMT), which can then serve as an agonist to pro-survival pathways, such as sigma non-opioid intracellular receptor 1 (SIGMAR1) signaling. Fibrogenic diseases, like metabolic-associated fatty liver disease (MAFLD), steatohepatitis (MASH), and chronic kidney disease (CKD) have shown changes in INMT and SIGMAR1 activity in the progression of disease pathogenesis. At the cellular level, endothelial cells and fibroblasts have been found to express INMT in various tissues; however, little is known about tryptamines in endothelial injury and fibrosis. In this review, I will give an overview of the biochemistry, molecular biology, and current evidence of INMT’s role in hepatic fibrogenesis. I will also discuss current pre-clinical and clinical findings of N-methylated tryptamines and highlight new and upcoming therapeutic strategies that may be adapted for mitigating fibrogenic diseases. Finally, I will mention recent findings for mutualistic gut bacteria influencing endogenous tryptamine signaling and metabolism. Full article

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(A) Tryptamine Structure. Tryptamine is a heterocyclic indole derivative with an ethylamine at the C3 position. (A,B) Tryptamine Metabolism. Tryptophan (i) is decarboxylated by AADC to form (ii), the precursor to DMT. Serotonin (iii) may be methylated by INMT as well as 5-MeO-tryptamine. R = H-, HO- or MeO- groups. (C) Trimethylselenonium production. Abbreviations: aromatic L-amine decarboxylase (AADC); aldehyde dehydrogenase, (ALDH); N-acetylserotonin O-methyltransferase, (ASMT); indoleamine 2,3-dioxygenase, (IDO); indolethylamine-N-methyltransferase, (INMT); monoamine oxidase A, (MAO-A); S-adenosylhomocysteine, (SAH); S-adenosylmethionine, (SAM); tryptophan 2,3-dioxygenase, (TDO); tryptophan hydroxylase, (TPH); (i) = tryptophan; (ii) = tryptamine; (iii) = serotonin; (iv) = 5-methoxy-tryptamine; (v′) = N-methyltryptamine or derivative; (vi′) = N,N-methyltryptamine or derivative; (vii′) N,N,N-trimethyltryptamine or derivative. (gray) = possible metabolite. Figure were generated using ChemDraw v22.2.0. Full article ">Figure 2
INMT’s potential in mediating fibrogenic diseases through N-methylated tryptamines and selenium metabolism. Red arrows denote insults that promote fibrosis. Green arrows denote resolving of fibrosis through SIGMAR1. PDB: 2A14. Abbreviations: endothelin-1, (ET-1); hypoxia-inducible factor 1-alpha, (HIF1α); indolethylamine-N-methyltransferase, (INMT); reactive oxygen species, (ROS); sigma non-opioid intracellular receptor 1, (SIGMAR1); transforming growth factor beta, (TGFβ). Figure were generated using BioRender. Full article ">

16 pages, 9442 KiB

Open AccessArticle

Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

by Ishita Kathuria, Aditi Prasad, Bal Krishan Sharma, Ravi Varma Aithabathula, Malvin Ofosu-Boateng, Maxwell A. Gyamfi, Jianxiong Jiang, Frank Park, Udai P. Singh and Bhupesh Singla

Int. J. Mol. Sci. 2024, 25(23), 12782; https://doi.org/10.3390/ijms252312782 - 28 Nov 2024

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Abstract

Clinical and genetic studies strongly support a significant connection between nonalcoholic fatty liver disease (NAFLD) and atherosclerotic cardiovascular disease (ASCVD) and identify ASCVD as the primary cause of death in NAFLD patients. Understanding the molecular factors and mechanisms regulating these diseases is critical [...] Read more.

Clinical and genetic studies strongly support a significant connection between nonalcoholic fatty liver disease (NAFLD) and atherosclerotic cardiovascular disease (ASCVD) and identify ASCVD as the primary cause of death in NAFLD patients. Understanding the molecular factors and mechanisms regulating these diseases is critical for developing novel therapies that target them simultaneously. Our preliminary immunoblotting experiments demonstrated elevated expression of nidogen 2 (NID2), a basement membrane glycoprotein, in human atherosclerotic vascular tissues and murine steatotic livers. Therefore, we investigated the role of NID2 in regulating hepatosteatosis and atherosclerosis utilizing Western diet-fed Apoe^−/− mice with/without NID2 overexpression. Quantitative real-time PCR confirmed increased NID2 mRNA expression in multiple organs (liver, heart, kidney, and adipose) of NID2-overexpressing mice. Male mice with NID2 overexpression exhibited higher liver and epididymal white adipose tissue mass, increased hepatic lipid accumulation, and fibrosis. Additionally, these mice developed larger atherosclerotic lesions in the whole aortas and aortic roots, with increased necrotic core formation. Mechanistic studies showed reduced AMPK activation in the livers of NID2-overexpressing mice compared with controls, without any effects on hepatic inflammation. In conclusion, these findings suggest that NID2 plays a deleterious role in both hepatosteatosis and atherosclerosis, making it a potential therapeutic target for these conditions. Full article

(This article belongs to the Special Issue Atherosclerosis 2.0: From Molecular Mechanisms, Pathophysiology to Novel Therapeutic Approaches)

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Expression of NID2 protein is elevated in human atherosclerotic arteries and murine steatotic livers. (A) Representative western blot images for NID2 and β-tubulin protein expression in human atherosclerotic inner curvature (IC) and non-atherosclerotic descending aorta (DA) vascular tissue. The bar diagram shows mean protein levels expressed as a ratio of NID2 to β-tubulin. (B) Representative Western blot images for NID2 (red arrowhead points to the correct band) and GAPDH in the livers of control diet (CD)- and calorie-matched high-fat diet (HFD, 12 weeks)-fed C57BL/6J mice. The bar diagram represents the mean NID2 protein expression (n = 4). Statistical analyses were performed using a two-tailed unpaired t-test (A,B). Data represent mean ± SEM. * p < 0.05, and **** p < 0.0001. Full article ">Figure 2
NID2 overexpression enhances liver and epididymal white adipose tissue mass in male mice. (A) The schematic diagram illustrates the experimental plan. Apoe−/− mice were injected with control (Ctrl) and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. (B–E) Male control and NID2-AAV-injected Apoe−/− mice were utilized to measure NID2 mRNA levels in various organs by qRT-PCR at least in duplicate. Bar diagrams represent mRNA expression in the liver (B, n = 10), kidney (C, n = 6), epididymal white adipose tissue (EpiWAT, D, n = 7–10), and heart (E, n = 10). Bar diagrams show body weight gain (F), plasma total cholesterol (G), fasting blood glucose (H), whole-body fat/lean mass (I), liver weight (J), adipose tissue weight (K), and spleen weight (L) (n = 5–6). A two-tailed unpaired t-test (C,G–K), two-tailed unpaired Mann–Whitney test (B,D,E,L), and two-way ANOVA followed by Sidak post hoc test for multiple comparisons (F) were utilized for statistical analyses. Data represent mean ± SEM. ns: non-significant. * p < 0.05, *** p < 0.001 and **** p < 0.0001. Full article ">Figure 3
NID2 overexpression in mice promotes hepatic lipid accumulation and fibrosis. Male Apoe−/− mice were injected with control and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. (A) Representative Western blot images for NID2 and GAPDH protein expression in the livers of control and NID2-overexpressing mice (n = 3). (B) Representative images of liver sections stained with H & E (lipid droplets), ORO (neutral lipid accumulation), and Sirius red (fibrosis); scale bar 100 μm. (C–H) Bar diagrams represent lipid accumulation (C, n = 6), fibrosis area (D, n = 5), hepatic triglyceride (E, n = 3–4), NEFA levels (F, n = 3–4), plasma triglyceride (G, n = 5) and NEFA levels (H, n = 5), in control and NID2-AAV-injected mice. Statistical analyses were performed using a two-tailed unpaired t-test (C–H). Data represent mean ± SEM. ns: non-significant. * p < 0.05, and ** p < 0.01. Full article ">Figure 4
NID2 overexpression augments atherosclerosis in male hypercholesterolemic mice. Male Apoe−/− mice were injected with control (Ctrl) and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks and analyzed. (A) Representative in situ images of the aortic arch (red arrowheads point to atherosclerotic lesions). (B) Representative ORO staining of whole aortas; scale bar 5 mm. The bar diagram represents ORO-positive areas in whole aortas (n = 6). (C) Representative images of aortic root cross-sections stained with H & E (lesion area and necrotic core), ORO (lipid accumulation), and Masson’s trichrome (collagen content); scale bar 200 μm. (D–G) Bar diagrams show lesion area (D), lipid deposition (E), collagen content (F), and necrotic core area (G) (n = 5–6). Statistical analyses were performed using a two-tailed unpaired t-test (B,D–G). Data represent mean ± SEM. * p < 0.05, and ** p < 0.01. Full article ">Figure 5
NID2 overexpression inhibits the activation of the lipid metabolism-related protein AMPK. (A) Representative Western blot images for lipid metabolism and pro-inflammatory proteins utilizing liver lysates from control and NID2-AAV-injected mice. Bar diagrams represent mean protein expression (B,C) as the ratios of phospho-total proteins ACC (B) and AMPK (C), and protein levels of IL-6 (D) and TNFα (E) (n = 5). Statistical analyses were performed using a two-tailed unpaired t-test. Data represent mean ± SEM. ns: non-significant. * p < 0.05. Full article ">

47 pages, 3709 KiB

Open AccessReview

Oxidative Stress in Kidney Injury and Hypertension

by Willaim J. Arendshorst, Aleksandr E. Vendrov, Nitin Kumar, Santhi K. Ganesh and Nageswara R. Madamanchi

Antioxidants 2024, 13(12), 1454; https://doi.org/10.3390/antiox13121454 - 27 Nov 2024

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Abstract

Hypertension (HTN) is a major contributor to kidney damage, leading to conditions such as nephrosclerosis and hypertensive nephropathy, significant causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD). HTN is also a risk factor for stroke and coronary heart disease. Oxidative [...] Read more.

Hypertension (HTN) is a major contributor to kidney damage, leading to conditions such as nephrosclerosis and hypertensive nephropathy, significant causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD). HTN is also a risk factor for stroke and coronary heart disease. Oxidative stress, inflammation, and activation of the renin–angiotensin–aldosterone system (RAAS) play critical roles in causing kidney injury in HTN. Genetic and environmental factors influence the susceptibility to hypertensive renal damage, with African American populations having a higher tendency due to genetic variants. Managing blood pressure (BP) effectively with treatments targeting RAAS activation, oxidative stress, and inflammation is crucial in preventing renal damage and the progression of HTN-related CKD and ESRD. Interactions between genetic and environmental factors impacting kidney function abnormalities are central to HTN development. Animal studies indicate that genetic factors significantly influence BP regulation. Anti-natriuretic mechanisms can reset the pressure–natriuresis relationship, requiring a higher BP to excrete sodium matched to intake. Activation of intrarenal angiotensin II receptors contributes to sodium retention and high BP. In HTN, the gut microbiome can affect BP by influencing energy metabolism and inflammatory pathways. Animal models, such as the spontaneously hypertensive rat and the chronic angiotensin II infusion model, mirror human essential hypertension and highlight the significance of the kidney in HTN pathogenesis. Overproduction of reactive oxygen species (ROS) plays a crucial role in the development and progression of HTN, impacting renal function and BP regulation. Targeting specific NADPH oxidase (NOX) isoforms to inhibit ROS production and enhance antioxidant mechanisms may improve renal structure and function while lowering blood pressure. Therapies like SGLT2 inhibitors and mineralocorticoid receptor antagonists have shown promise in reducing oxidative stress, inflammation, and RAAS activity, offering renal and antihypertensive protection in managing HTN and CKD. This review emphasizes the critical role of NOX in the development and progression of HTN, focusing on its impact on renal function and BP regulation. Effective BP management and targeting oxidative stress, inflammation, and RAAS activation, is crucial in preventing renal damage and the progression of HTN-related CKD and ESRD. Full article

(This article belongs to the Special Issue NADPH Oxidases in Health and Aging—2nd Edition)

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An intricate network of pathways leads to renal nephrosclerosis in hypertension. Hypertension and RAAS (renin–angiotensin–aldosterone system) activation initiate a cascade of events, including NOX activation and ROS generation, which induce endothelial dysfunction, inflammation, and epithelial-to-mesenchymal transition (EMT). The resulting podocyte injury and apoptosis contribute to the denudation of the glomerular basement membrane. Concurrently, endothelial-to-mesenchymal transition (EndoMT), vascular smooth muscle cell (VSMC) proliferation, and myofibroblast activation occur, leading to hyalinosis and narrowing of the afferent arteriole, the collapse of capillary loops, and retraction of the glomerular tuft. Epithelial cell damage and EMT lead to tubular dilation, atrophy, inflammation, and fibrosis, ultimately resulting in tubulointerstitial fibrosis and the filling of Bowman’s space with collagen. Full article ">Figure 2
Oxidative stress is central to the pathophysiological pathways leading to hypertension. Unhealthy dietary habits and antibiotic use disrupt the gut microbiome, leading to an imbalance that promotes the release of pro-inflammatory cytokines and metabolites and the activation of immune cells. The presence of inflammatory mediators in conjunction with other comorbidities results in reduced nitric oxide (NO) bioavailability and impaired activities of SOD and catalase, inducing mitochondrial dysfunction and increased ROS generation. This, in turn, triggers endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Critical signaling pathways like TGFβ/SMAD2/3, NFκB, and RAAS) are activated, further worsening oxidative stress by upregulating NOX enzyme levels. Oxidative stress is a pivotal downstream event in the pathophysiological cascade, causing cellular dysfunction, damage, and apoptosis. In the kidney, oxidative stress induces renal inflammation, fibrosis, endothelial dysfunction, and VSMC proliferation. These processes result in renal vasoconstriction, vascular hypertrophy, remodeling, and increased sodium and water retention, leading to elevated systemic vascular resistance and blood pressure, ultimately causing hypertension. Full article ">Figure 3
Mechanisms affecting renal function and pressure–natriuresis relation in the development of hypertension. The pressure–natriuresis relationship describes the kidney’s ability to excrete Na+ in response to changes in BP. Increased SNS and RAAS activity, renal oxidative stress, and inflammation at normotensive BP result in impaired pressure–natriuresis, characterized by reduced Na+ excretion. That leads to elevated BP and a “reset” of the pressure–natriuresis curve, normalizing Na+ excretion to match sodium intake levels. This normalization is characterized by improved Na+ filtration capacity and reduced Na+ retention. Up arrows indicate an increased effect, while down arrows signify a decreased effect. Full article ">Figure 4
NADPH oxidases expression in the renal cells. NADPH oxidases (NOX) and mitochondria are the primary sources of ROS in renal and vascular cells, including VSMC, podocytes, mesangial cells, and tubular epithelial cells. Full article ">Figure 5
Animal models of hypertension. In the Dahl salt-sensitive rat model, a high salt diet leads to renal inflammation, increased ROS levels, activation of the SNS and RAAS, decreased GFR, and changes in Na+ and water excretion. The chronic infusion of Ang II leads to similar pathophysiological changes, including increased ROS levels, RAAS activation, impaired Na+ excretion, reduced GFR, endothelial dysfunction, and heightened renal vascular reactivity and resistance. In the spontaneously hypertensive rat model, HTN develops through genetic predisposition when the animals are fed a normal salt diet. Pathophysiological changes involve renal inflammation, oxidative stress, RAAS activation, Na+ retention, and impaired pressure–natriuresis. Preventive measures across all models include RAAS inhibition (ACEi/ARB), antioxidants, and dietary modifications such as a low or normal salt diet. Full article ">Figure 6
Renal and systemic effects of proximal tubular SGLT2 (sodium–glucose cotransporter-2) inhibition. SGLT2 inhibitors lead to glucosuria and promote renal natriuresis by enhancing mitochondrial function and reducing oxidative stress. This improvement helps to enhance glomerular function and decrease tubulointerstitial fibrosis. On a systemic level, SGLT2 inhibition results in lower plasma glucose levels, reduced renal and systemic inflammation, decreased vasoconstriction and vascular injury, Na+ retention, and BP. Full article ">

15 pages, 9352 KiB

Open AccessArticle

Therapeutic Potential of Oligo-Fucoidan in Mitigating Peritoneal Dialysis-Associated Fibrosis

by Yu-Wei Chen, Mei-Yi Wu, Nai-Jen Huang, Mai-Szu Wu, Yung-Ho Hsu, Chia-Te Liao and Cheng-Hsien Chen

Mar. Drugs 2024, 22(12), 529; https://doi.org/10.3390/md22120529 - 25 Nov 2024

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Abstract

Peritoneal dialysis (PD) serves as a home-based kidney replacement therapy with increasing utilization across the globe. However, long-term use of high-glucose-based PD solution incites repeated peritoneal injury and inevitable peritoneal fibrosis, thus compromising treatment efficacy and resulting in ultrafiltration failure eventually. In the [...] Read more.

Peritoneal dialysis (PD) serves as a home-based kidney replacement therapy with increasing utilization across the globe. However, long-term use of high-glucose-based PD solution incites repeated peritoneal injury and inevitable peritoneal fibrosis, thus compromising treatment efficacy and resulting in ultrafiltration failure eventually. In the present study, we utilized human mesothelial MeT-5A cells for the in vitro experiments and a PD mouse model for in vivo validation to study the pathophysiological mechanisms underneath PD-associated peritoneal fibrosis. High-glucose PD solution (Dianeal 4.25%, Baxter) increased protein expression of mesothelial–mesenchymal transition (MMT) markers, such as N-cadherin and α-SMA in MeT-5A cells, whereas it decreased catalase expression and stimulated the production of reactive oxygen species (ROS). Furthermore, macrophage influx and increased serum pro-inflammatory cytokines, such as IL-1β, MCP-1, and TNF-α, were observed in the PD mouse model. Interestingly, we discovered that oligo-fucoidan, an oligosaccharide extract from brown seaweed, successfully prevented PD-associated peritoneal thickening and fibrosis through antioxidant effect, downregulation of MMT markers, and attenuation of peritoneal and systemic inflammation. Hence, oligo-fucoidan has the potential to be developed into a novel preventive strategy for PD-associated peritoneal fibrosis. Full article

(This article belongs to the Special Issue Marine Polysaccharides and Oligosaccharides: Extraction and Biological Activities)

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Graphical abstract

Graphical abstract
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Influence of oligo-fucoidan (Fc) on the signal transduction of epithelial–mesenchymal transition and inflammation in MeT-5A cells. The cells were pretreated with Fc for 30 min and then cultured in high glucose (HG, containing 60 mM glucose) or normal medium. Protein expression was analyzed by Western blotting. α-SMA, E-cadherin, and N-cadherin are the markers of epithelial–mesenchymal transition. Phospho-JNK and TNF-α are the markers of inflammation. The relative increase in protein bands is also presented as a chart. Results are expressed as mean ± SD (n = 3). Full article ">Figure 2
The protective effect of oligo-fucoidan (Fc) on high-glucose-induced apoptosis in MeT-5A cells. The cells were pretreated with Fc for 30 min and then cultured in high glucose (HG, containing 60 mM glucose) or normal medium (control). (A) The Western blots of cleaved caspases-3 and Bcl-2. Cleaved caspases-3 and Bcl-2 are the markers of apoptosis. The relative increase in protein bands is also presented as a chart. Results are expressed as mean ± SD (n = 3). (B) The representative flow cytometric plots of cell apoptosis. In each plot, the lower left quadrant represents viable cells, the upper left quadrant necrotic cells, the lower right quadrant early apoptotic cells, and the upper right quadrant necrotic or late apoptotic cells. The apoptotic rate is also presented as a chart. Results are expressed as mean ± SD (n = 3). High-glucose-induced apoptosis in MeT-5A cells, which was inhibited by 0. 5 mg/mL Fc. AnnV: Annexin V-FITC, PI: propidium iodide. Full article ">Figure 3
Effects of oligo-fucoidan (Fc) on high-glucose-induced reactive oxygen species (ROS) generation in MeT-5A cells. MeT-5A cells were pretreated with Fc for 30 min and then cultured in high glucose (HG, containing 60 mM glucose) or normal medium. (A) The Western blots of catalase and SOD. Catalase and SOD are the markers of antireactive oxygen species. The relative increase in protein bands is also presented as a chart. Results are expressed as mean ± SD (n = 3). (B) Representative images by fluorescent staining with 2′,7′-dichlorofluorescin (DCF). The image represents a combination of visible light microscopy and fluorescence microscopy images of MeT-5A cells. Green signifies the fluorescence of DCF when stained with ROS. (C) The intensity of DCF fluorescence in the cells measured by a fluorescence microplate reader. Fluorescence intensities of cells are shown as the relative intensity of experimental groups compared with untreated control cells. High-glucose-induced ROS in MeT-5A cells, which was inhibited by 0.1 and 0.5 mg/mL Fc. Data are shown in mean ± S.D. (n = 5). Full article ">Figure 4
Masson trichrome staining of the parietal peritoneum of PD mice. PD mice received 4.25% dextrose dialysate or without (control) and were then treated with oligo-fucoidan (Fc) orally (100 mg/kg/d). Average parietal peritoneum thickness in PD mice was presented as a chart. Dialysate increased parietal peritoneum thickness in PD mice, which was inhibited by Fc. Data are shown in mean ± S.D. (n = 5). Full article ">Figure 5
Morphology of the parietal peritoneum of PD mice with or without the oral oligo-fucoidan (Fc) treatment. Sections of paraffin-embedded mouse parietal peritoneum were stained by IHC using antibodies for α-SMA, fibronectin, TNF-α, F4/80, and cleaved caspase-3 (A). Red arrows highlight F4/80-positive macrophages and cleaved caspase-3-positive cells in the parietal peritoneum. Charts also display the positively stained areas in the peritoneum for α-SMA (B), fibronectin (C), and TNF-α (D), as well as the positively stained cells for F4/80 (E) and cleaved caspase-3 (F). Data are presented as the mean ± SD from 3 mice in each group. Full article ">Figure 6
The reduction effect of oligo-fucoidan (Fc) on serum TNF-α, MCP-1, and IL-1β in PD mice. PD mice received 4.25% glucose dialysate or without (control) and were then treated with Fc orally (100 mg/kg/d). Blood was collected from each mouse to measure serum TNF-α (A), MCP-1 (B), and IL-1β (C). Dialysate induced serum TNF-α, MCP-1, and IL-1β in PD mice, which was inhibited by Fc. The results are expressed as the mean ± SD (n = 3). Full article ">

24 pages, 8027 KiB

Open AccessArticle

Renal Epithelial Complement C3 Expression Affects Kidney Fibrosis Progression

by Ganna Stepanova, Anna Manzéger, Miklós M. Mózes and Gábor Kökény

Int. J. Mol. Sci. 2024, 25(23), 12551; https://doi.org/10.3390/ijms252312551 - 22 Nov 2024

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Abstract

Kidney fibrosis is a hallmark of chronic kidney diseases. Evidence shows that genetic variability and complement component 3 (C3) might influence tubulointerstitial fibrosis. Still, the role of renal C3 production in the epithelial-to-mesenchymal transition (EMT) and genetically determined fibrosis progression remains undiscovered. The [...] Read more.

Kidney fibrosis is a hallmark of chronic kidney diseases. Evidence shows that genetic variability and complement component 3 (C3) might influence tubulointerstitial fibrosis. Still, the role of renal C3 production in the epithelial-to-mesenchymal transition (EMT) and genetically determined fibrosis progression remains undiscovered. The kidneys of fibrosis-resistant C57Bl/6J (B6) and fibrosis-prone CBA/J (CBA) and BALB/cJ (BalbC) mice (n = 4–8/group) were subjected to unilateral ureteral obstruction (UUO) and analyzed after 1, 3, and 7 days, along with human focal glomerular sclerotic (FSGS) and healthy kidneys. Mouse primary tubular epithelial cells (PTECs) were investigated after 24 h of treatment with transforming growth factor β (TGFβ) or complement anaphylatoxin 3a (C3a) agonist (n = 4/group). UUO resulted in delayed kidney injury in fibrosis-resistant B6 mice, but very early renal C3 messenger RNA (mRNA) induction in fibrosis-prone CBA and BalbC mice, along with collagen I (Col1a1) and collagen III (Col3a1). CBA depicted the fastest fibrosis progression with the highest C3, lipocalin-2 (Lcn2), Tgfb1, and chemokine (C-C motif) ligand 2 (Ccl2) expression. Human FSGS kidneys depicted C3 mRNA over-expression and strong tubular C3 immunostaining. In PTECs, C3a agonist treatment induced pro-fibrotic early growth response protein 1 (EGR1) expression and the EMT, independent of TGFβ signaling. We conclude that de novo renal tubular C3 synthesis is associated with the genetically determined kidney fibrosis progression rate in mice and the pathogenesis of FSGS in humans. This tubular C3 overproduction can, through local pro-fibrotic effects, influence the progression of chronic kidney disease. Full article

(This article belongs to the Special Issue Kidney Diseases: Molecular Pathogenesis and Therapeutic Strategies—2nd Edition)

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29 pages, 2260 KiB

Open AccessReview

Hedgehog Signaling Pathway in Fibrosis and Targeted Therapies

by Yuchen Hu, Linrui Peng, Xinyu Zhuo, Chan Yang and Yuwei Zhang

Biomolecules 2024, 14(12), 1485; https://doi.org/10.3390/biom14121485 - 22 Nov 2024

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Abstract

Hedgehog (Hh) signaling is a well-established developmental pathway; it is crucial for early embryogenesis, cell differentiation, and damage-driven regeneration. It is being increasingly recognized that dysregulated Hh signaling is also involved in fibrotic diseases, which are characterized by excessive extracellular matrix deposition that [...] Read more.

Hedgehog (Hh) signaling is a well-established developmental pathway; it is crucial for early embryogenesis, cell differentiation, and damage-driven regeneration. It is being increasingly recognized that dysregulated Hh signaling is also involved in fibrotic diseases, which are characterized by excessive extracellular matrix deposition that compromises tissue architecture and function. As in-depth insights into the mechanisms of Hh signaling are obtained, its complex involvement in fibrosis is gradually being illuminated. Notably, some Hh-targeted inhibitors are currently under exploration in preclinical and clinical trials as a means to prevent fibrosis progression. In this review, we provide a concise overview of the biological mechanisms involved in Hh signaling. We summarize the latest advances in our understanding of the roles of Hh signaling in fibrogenesis across the liver, kidneys, airways, and lungs, as well as other tissues and organs, with an emphasis on both the shared features and, more critically, the distinct functional variations observed across these tissues and organs. We thus highlight the context dependence of Hh signaling, as well as discuss the current status and the challenges of Hh-targeted therapies for fibrosis. Full article

(This article belongs to the Section Molecular Medicine)

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Biological processes of the canonical Hedgehog (Hh) signaling pathway. The major steps in the canonical Hh signaling pathway in mammals have been elucidated: Hh biogenesis, release, transmission, reception, and intracellular transduction. (A). In the endoplasmic reticulum (ER), the Hh ligand undergoes two sequential lipid modifications, including cholesterol added by an autoprocessing reaction and palmitate attached by Hh acetyltransferase (Hhat). Dispatched homologue 1 (DISP1) then transfers the Hh ligand to its carrier, the cubulin (CUB) domain of epidermal growth factor (EGF)-like protein 2 (SCUBE2). This SCUBE2-mediated soluble complex enables the long-range transport of the hydrophobic Hh ligand in the intercellular matrix to the Hh-receiving cell. (B). In the absence of the Hh ligand (pathway-off), patched (PTCH), located on the primary cilium (PC), inhibits Smoothened (SMO) as a sterol transporter, which means sterols are barely accessible to SMO. As a result, SMO remains inactive in the cytoplasm. Simultaneously, glioma-associated oncogene (GLI) interacts with the suppressor of fused (SUFU) to form a complex, which undergoes phosphorylation by protein kinase A (PKA), glycogen synthase kinase-3 (GSK3β), and casein kinase 1 (CK1). This is followed by ubiquitin-mediated proteolysis via β-transducin repeat-containing protein (βTRCP), producing a truncated repressor form of GLI (GLIR), which inhibits the transcription of target genes. (C). In the presence of the Hh ligand (pathway-on), the binding of the Hh ligand to PTCH is facilitated via co-receptors such as those cell adhesion molecule-related/downregulated by oncogenes (CDO), brother of CDO (BOC), and growth arrest-specific protein 1 (GAS1). The Hh-PTCH compound then leaves the ciliary membrane and is degraded, which can generate the environment where SMO can be accessible to sterols and be activated. Kinesin protein 3 (Kif3) and β-arrestin mediate the movement of SMO from the cytoplasm into the PC. CK1 and G protein-coupled receptor kinase 2 (GRK2) phosphorylate SMO to be fully activated, allowing the SUFU/ GLI complex to enter the PC with the assistance of Kif7. Once at the tip of the PC, GLI dissociates from SUFU and remains in its full-length active form (GLIA). GLIA then translocates to the nucleus, where it drives the transcription of target genes. Full article ">Figure 2
Hh signaling pathway mediated-fibrogenesis. Prolonged and repetitive stimuli lead to consistent injury of parenchymal cells and the recruitment of various inflammatory cells, including neutrophils, lymphocytes, macrophages, T cells, B cells, and dendritic cells. These cells secrete the Hh ligand in either an autocrine or paracrine manner. Several precursor cell types, such as resident fibroblasts, quiescent stellate cells, pericytes, bone marrow-derived fibrocytes/mesenchymal stem cells (MSCs), endothelial cells undergoing endothelial–mesenchymal transition (EndMT), epithelial cells undergoing epithelial–mesenchymal transition (EMT), and GLI1+ MSCs, which are responsive to the Hh ligand, contribute to the myofibroblasts’ activation, proliferation, differentiation, and sustained extracellular matrix (ECM) production. This process is accompanied by the activation of the Hh signaling pathway (Hh/PTCH/SMO/GLIA), ultimately resulting in increased tissue stiffness, hypoxia, and tissue remodeling, which together foster a vicious cycle. Full article ">

21 pages, 6832 KiB

Open AccessArticle

Identification of Spatial Specific Lipid Metabolic Signatures in Long-Standing Diabetic Kidney Disease

by Yiran Zhang, Hai-Long Piao and Di Chen

Metabolites 2024, 14(11), 641; https://doi.org/10.3390/metabo14110641 - 20 Nov 2024

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Abstract

Background: Diabetic kidney disease (DKD) is a major complication of diabetes leading to kidney failure. Methods: This study investigates lipid metabolism profiles of long-standing DKD (LDKD, diabetes duration > 10 years) by integrative analysis of available single-cell RNA sequencing and spatial multi-omics data [...] Read more.

Background: Diabetic kidney disease (DKD) is a major complication of diabetes leading to kidney failure. Methods: This study investigates lipid metabolism profiles of long-standing DKD (LDKD, diabetes duration > 10 years) by integrative analysis of available single-cell RNA sequencing and spatial multi-omics data (focusing on spatial continuity samples) from the Kidney Precision Medicine Project. Results: Two injured cell types, an injured thick ascending limb (iTAL) and an injured proximal tubule (iPT), were identified and significantly elevated in LDKD samples. Both iTAL and iPT exhibit increased lipid metabolic and biosynthetic activities and decreased lipid and fatty acid oxidative processes compared to TAL/PT cells. Notably, compared to PT, iPT shows significant upregulation of specific injury and fibrosis-related genes, including FSHR and BMP7. Meanwhile, comparing iTAL to TAL, inflammatory-related genes such as ANXA3 and IGFBP2 are significantly upregulated. Furthermore, spatial metabolomics analysis reveals regionally distributed clusters in the kidney and notably differentially expressed lipid metabolites, such as triglycerides, glycerophospholipids, and sphingolipids, particularly pronounced in the inner medullary regions. Conclusions: These findings provide an integrative description of the lipid metabolism landscape in LDKD, highlighting injury-associated cellular processes and potential molecular mechanisms. Full article

(This article belongs to the Special Issue New Analytical Techniques and Applications of Metabolomics and Lipidomics)

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Figure 1
Single-cell RNA sequencing (scRNA-seq) analysis reveals kidney cell types in LDKD and healthy donors. (a) Clinical profiles of LDKD patients in the study. RAAS, renin–angiotensin–aldosterone system. (b) UMAP diagram of the identified cell types. Different colors correspond to distinct cell types. (c) Dot plot of the markers corresponding to the cell types. (d) UMAP diagram of the expression of canonical markers for the cell types. The color scales across multiple plots were adjusted by gene scaling. (e) Bar plot of the composition of different cell types in each sample. Alongside are the proportions of iTAL and iPT cell types in LDKD and healthy samples. Wilcoxon test. * p ≤ 0.05, **** p ≤ 0.0001. Full article ">Figure 2
Metabolic pathway profiles of different kidney cell types based on the scRNA-seq data. (a) Dot plot showing lipid-associated metabolism process enrichment (from GO BP) across cell types. (b,c) UMAP plots of sphingolipid (b) and cholesterol (c) metabolism pathway activities in LDKD vs. healthy samples. Box plots compare pathway activities between LDKD and healthy samples by cell type (Wilcoxon test: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ns: not significant). The black points are outliers in each boxplot. (d) Violin plots comparing sphingolipid and cholesterol metabolism pathway activities between iTAL/TAL and iPT/PT in LDKD (left) vs. healthy (right). Wilcoxon test. (e) Chord diagram of cholesterol metabolism pathway in cell–cell interaction networks for healthy and LDKD samples. (f) Differentially expressed genes in iPT (up/downregulated) and iTAL (upregulated) pathways. Full article ">Figure 3
Spatial transcriptomics analysis for LDKD and healthy samples. (a) Spatial distribution of cell types in LDKD and healthy samples. Scale bar: 1 mm. (b) Dot plot showing characteristic markers for distinct cell types. (c) Loess smoothed curves of cell type proportion changes with iTAL (left) and iPT (right). The x-axis ranks cells by iTAL/iPT proportion; the lower half of the y-axis shows changes in other cell types’ proportions. (d) Scatter plot of differentially expressed genes in iTAL vs. TAL and iPT vs. PT comparisons in LDKD samples. Colors indicate avg_logFC direction, with a 0.5 threshold for separation. Wilcoxon test. (e) Pathway activities comparison between iTAL vs. TAL and iPT vs. PT in LDKD and healthy samples. Dark pink/blue indicates positive/negative activity with intensity reflecting adjusted p-value. (f) Differentially expressed genes in iPT up/downregulated pathways. Genes show significant differences (p < 0.05) comparing injured cells to other types and counterparts. The black points are outliers in each boxplot. Different colors represent different cell types. (g) Chord plot of cholesterol metabolism pathway in cell–cell interactions for healthy and LDKD samples, with cell types exceeding 50% included. Full article ">Figure 4
Spatial metabolomics analysis for LDKD and healthy samples. (a) UMAP plot of spatial metabolic clusters (MCs). Different colors correspond to distinct clusters. (b) Dot plot of characteristic metabolites in different MCs. (c) Bar plot of the composition of MCs in each sample. (d) Pyramid chart of MC proportion changes between LDKD and healthy samples, ordered by absolute LDKD proportion change, with significantly changed clusters in red. (e) Spatial distribution of MCs in LDKD samples, with colors consistent with A. Scale bar: 0.5 mm. (f) Differentially expressed metabolites between LDKD and healthy samples identified via Wilcoxon test, with Bonferroni-adjusted p-values. (g) Pie chart showing proportions of different metabolite/lipid/glycerophospholipid classes among differentially expressed metabolites. Each category is colored distinctly, with the name followed by the number of matches and percentage. Full article ">Figure 5
Spatial distribution similarities between lipids and characteristic metabolites of different MCs in LDKD. (a–c) Spatial distribution similarity of TG (a), PE (b), and SM (c) with metabolic clusters in LDKD. The heatmap colors represent the correlation between each metabolite and different metabolic clusters, defined by their spatial distribution similarity. Kidney regions and metabolic clusters are distinguished by distinct colors and annotations. TG, triglycerides; PE, phosphatidylethanolamine; SM, sphingomyelin. Metabolites labelled in red are shown in figS6. For each MC, 2 or 3 characteristic metabolites were selected to characterize the spatial distribution. (d) Spatial distribution of the metabolite C2H7NO4P, which annotated PEA. Scale bar, 1 mm. PEA, phosphoethanolamine. (e) Spatial distribution similarity of metabolite C2H7NO4P with MC7 characteristic metabolites C10H14N5O7PCl and C2H8NO4PCl. The red line in each plot is the regression line for all points. Full article ">

38 pages, 531 KiB

Open AccessReview

Epigenetics of Hypertensive Nephropathy

by Yize Zhang, Hamidreza Arzaghi, Zhehan Ma, Yasmin Roye and Samira Musah

Biomedicines 2024, 12(11), 2622; https://doi.org/10.3390/biomedicines12112622 - 16 Nov 2024

Viewed by 392

Abstract

Hypertensive nephropathy (HN) is a leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD), contributing to significant morbidity, mortality, and rising healthcare costs. In this review article, we explore the role of epigenetic mechanisms in HN progression and their potential [...] Read more.

Hypertensive nephropathy (HN) is a leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD), contributing to significant morbidity, mortality, and rising healthcare costs. In this review article, we explore the role of epigenetic mechanisms in HN progression and their potential therapeutic implications. We begin by examining key epigenetic modifications—DNA methylation, histone modifications, and non-coding RNAs—observed in kidney disease. Next, we discuss the underlying pathophysiology of HN and highlight current in vitro and in vivo models used to study the condition. Finally, we compare various types of HN-induced renal injury and their associated epigenetic mechanisms with those observed in other kidney injury models, drawing inferences on potential epigenetic therapies for HN. The information gathered in this work indicate that epigenetic mechanisms can drive the progression of HN by regulating key molecular signaling pathways involved in renal damage and fibrosis. The limitations of Renin–Angiotensin–Aldosterone System (RAAS) inhibitors underscore the need for alternative treatments targeting epigenetic pathways. This review emphasizes the importance of further research into the epigenetic regulation of HN to develop more effective therapies and preventive strategies. Identifying novel epigenetic markers could provide new therapeutic opportunities for managing CKD and reducing the burden of ESRD. Full article

(This article belongs to the Special Issue Genetic and Epigenetics of Kidney Development: Pathologies of Renal Anomalies and Kidney Diseases)

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16 pages, 3007 KiB

Open AccessArticle

Modulators of Alpha-2 Macroglobulin Upregulation by High Glucose in Glomerular Mesangial Cells

by Jackie Trink, Renzhong Li, Bo Gao, Chao Lu and Joan C. Krepinsky

Biomolecules 2024, 14(11), 1444; https://doi.org/10.3390/biom14111444 - 13 Nov 2024

Viewed by 515

Abstract

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin [...] Read more.

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Kidney Diseases)

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Figure 1
A2M promoter activity is regulated by HG in MC. (A) Activity of the full-length A2M promoter was increased by HG, but not by the osmotic control mannitol (24 h, n = 18, * p < 0.05, **** p < 0.001). (B) Schematic of A2M promoter deletion constructs. “Luc” indicates the transcription start site (+1). (C) HG (24 h)-induced promoter activity was observed in −1500 bp, −925 bp, −450 bp, and −405 bp deletion constructs, but not in −375 bp or −300 bp constructs (n = 12, * p < 0.05, ** p < 0.01, *** p < 0.005, n.s.: no significant difference). Data for −2000 bp are the same as in (A). Full article ">Figure 2
NFAT5 regulates A2M promoter activity in HG. (A) Schematic of the identified NFAT5 transcription factor binding site and sequence in the A2M promoter. (B) HG (24 h) increased NFAT5 nuclear expression in MC (n = 4, * p < 0.05). (C) Increased localization of NFAT5 to the nucleus in response to HG (24 h) was confirmed using immunofluorescence (n = 4, * p < 0.05). (D) Protein–DNA interaction of NFAT5 with the −405 bp promoter region of A2M was assessed by ChIP. Interaction in response to HG was identified after immunoprecipitation using an NFAT5, but not an isotype IgG control antibody (24 h, n = 8, ** p < 0.01, **** p < 0.001). (E) Schematic of the mutated NFAT5 binding site in the −405 bp deletion construct. (F) HG (24 h)-induced activation of the −405 bp promoter was lost after site-directed mutagenesis of the NFAT5 binding site (n = 6, * p < 0.05, *** p < 0.005). Scale bar represents 10 µm. Western blot original images are in the <a href="#app1-biomolecules-14-01444" class="html-app">Supplementary Materials</a>. Full article ">Figure 3
NFAT5 inhibition prevents HG-induced A2M production. HG (24 h)-induced A2M promoter activity of the (A) −405 bp promoter and (B) full-length promoter were attenuated with siRNA inhibition of NFAT5 (n = 9, * p < 0.05, ** p < 0.01, **** p < 0.001). (C) HG (48 h)-induced A2M protein expression was also significantly reduced with NFAT5 knockdown (n = 3–7, * p < 0.05, *** p < 0.005). Inhibition of NFAT5 by KRN2 (100 nM) prevented HG (24 h)-induced A2M upregulation in both the (D) −405 bp promoter (n = 8) and (E) full-length promoter (n = 12) (* p < 0.05, *** p < 0.005). A2M (F) transcript upregulation (n = 4) and (G) protein expression (n = 6) by HG (24 h and 48 h, respectively) were inhibited by KRN2 (100 nM) (* p < 0.05, ** p < 0.01). Western blot original images are in the <a href="#app1-biomolecules-14-01444" class="html-app">Supplementary Materials</a>. Full article ">Figure 4
FOXP1 cooperates with NFAT5 to regulate A2M promoter activity. (A) FOXP1 expression was increased in the nuclear fraction after HG (24 h) (n = 6, ** p < 0.01). (B) Immunofluorescent staining of FOXP1 confirms HG (24 h)-induced nuclear localization of FOXP1 (n = 4, ** p < 0.01). (C) Immunoprecipitation of NFAT5 shows increased interaction with FOXP1 after HG (24 h) treatment (n = 5, ** p < 0.01). Knockdown of FOXP1 using siRNA prevented HG (24 h)-induced promoter activity of A2M for both the (D) −405 bp promoter (n = 9) and the (E) full-length promoter (n = 6) (* p < 0.05, ** p < 0.01). (F) HG (48 h)-induced A2M protein expression was also attenuated by FOXP1 knockdown (n = 5, ** p < 0.01). (G) The HXR9 peptide (100 nM), which prevents HOX/PBX interaction, prevented HG (48 h)-induced FOXP1 expression by MC (n = 8–9, * p < 0.05, *** p < 0.005, **** p < 0.001). FOXP1 inhibition by HXR9 (100 nM) also prevented HG (24 h)-induced (H) A2M −405 bp promoter activation (n = 6–9) and (I) A2M protein expression (n = 8–9) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001). Scale bar represents 10 µm. Western blot original images are in the <a href="#app1-biomolecules-14-01444" class="html-app">Supplementary Materials</a>. Full article ">Figure 5
Smad3 regulates A2M promoter activity upstream of −405. (A) Schematic of Smad3 and Fast1-Smad binding sites within the −1500 bp A2M promoter construct. (B) Compared to Smad3 WT cells, Smad3 KO cells had below baseline promoter activity regardless of HG treatment (24 h) with the full-length promoter as well as the −1500 bp, −925 bp, and −450 bp deletion constructs. The −405 bp construct was not affected by the absence of Smad3 and showed a response to HG (24 h) treatment in both WT and KO MC (n = 6, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001). (C) HG (48 h)-induced A2M protein upregulation was absent in Smad3 KO cells (n = 4–5, * p < 0.05). Using the Smad3 inhibitor SIS3 (5 µM), HG-induced (D) A2M promoter activity in the full-length construct (HG 24 h, n = 6–10) and (E) A2M protein expression (HG 48 h, n = 8) were reduced below basal activity or expression (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001). Western blot original images are in the <a href="#app1-biomolecules-14-01444" class="html-app">Supplementary Materials</a>. Full article ">Figure 6
HG-induced A2M expression and its effects on fibrosis are regulated by NFAT5, FOXP1, and Smad3 in isolated glomeruli. Isolated glomeruli from CD1 male mice treated ex vivo with HG (24 h) showed significantly increased (A) NFAT5 and (B) FOXP1 nuclear staining (n = 39–41, ** p < 0.01, **** p < 0.001). Glomeruli treated ex vivo with HG (48 h) showed increased expression of (C) A2M and (D) extracellular matrix proteins fibronectin (FN) and collagen Iα (Col Iα). This expression was attenuated by inhibition of either NFAT5 or FOXP1 (100 nM for either inhibitor, n= 3–4, * p < 0.05, ** p < 0.01). (E) Smad3 inhibition by SIS3 (5 µM) prevented HG (48 h)-induced A2M expression in isolated glomeruli (n= 4, * p < 0.05). (F) Knockdown of A2M using siRNA prevented HG (48 h)-induced upregulation of FN and Col Iα in isolated glomeruli (100 nM siRNA, n= 4, * p < 0.05, ** p < 0.01). Scale bar represents 5 µm. Western blot original images are in the <a href="#app1-biomolecules-14-01444" class="html-app">Supplementary Materials</a>. Full article ">

23 pages, 4613 KiB

Open AccessArticle

Renoprotective Effects of Brown-Strain Flammulina velutipes Singer in Chronic Kidney Disease-Induced Mice Through Modulation of Oxidative Stress and Inflammation and Regulation of Renal Transporters

by Min-Min Lee, Yun-Xuan Chou, Sheng-Hsiung Huang, Hsu-Tang Cheng, Chung-Hsiang Liu and Guan-Jhong Huang

Int. J. Mol. Sci. 2024, 25(22), 12096; https://doi.org/10.3390/ijms252212096 - 11 Nov 2024

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Abstract

Cisplatin, widely used in chemotherapy, acts through mechanisms such as oxidative stress to damage the DNA and cause the apoptosis of cancer cells. Although effective, cisplatin treatment is associated with considerable side effects including chronic kidney disease (CKD). Studies on brown-strain Flammulina velutipes [...] Read more.

Cisplatin, widely used in chemotherapy, acts through mechanisms such as oxidative stress to damage the DNA and cause the apoptosis of cancer cells. Although effective, cisplatin treatment is associated with considerable side effects including chronic kidney disease (CKD). Studies on brown-strain Flammulina velutipes Singer (FVB) have shown its significant antioxidant and immunomodulatory effects. High-performance liquid chromatography (HPLC) confirmed that the FVB extract contained gallic acid and quercetin. This study investigated whether FVB extract can improve and protect against cisplatin-induced CKD in mice. C57BL/6 mice were used as an animal model, and CKD was induced through intraperitoneal cisplatin injection. FVB was orally administered to the mice for 14 consecutive days. N-acetylcysteine (NAC) was administered in the positive control group. Organ pathology and serum biochemical analyses were conducted after the mice were sacrificed. Significant dose-dependent differences were discovered in body mass, kidney mass, histopathology, renal function, inflammatory factors, and antioxidant functions among the different groups. FVB extract reduced the severity of cisplatin-induced CKD in pathways related to inflammation, autophagy, apoptosis, fibrosis, oxidative stress, and organic ion transport proteins; FVB extract, thus, displays protective physiological activity in kidney cells. Additionally, orally administered high doses of the FVB extract resulted in significantly superior renal function, inflammatory factors, antioxidative activity, and fibrotic pathways. This study establishes a strategy for future clinical adjunctive therapy using edible-mushroom-derived FVB extract to protect kidney function. Full article

(This article belongs to the Special Issue New Trends and Challenges in Chronic Diseases)

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(A) Kidney sizes and (B) average kidney masses, (C) blood BUN nitrogen, and (D) CRE levels in mice with cisplatin-induced CKD. Values are presented as the means ± SD (n = 6). ### p < 0.001, significant difference between the Cis and N groups; *** p < 0.001, and ** p < 0.01, significant differences between the Cis group and Cis + NAC, Cis + GL, and Cis + GH groups. Full article ">Figure 2
(A) Renal H&E-stained sections (400×), (B) tubular damage scores, (C) renal Masson’s trichrome-stained sections showing fibrosis (400×), and (D) tubular damage scores in mice with cisplatin-induced CKD. Values are presented as the means ± SD (n = 6). ### p < 0.001, significant difference between the Cis and N groups; ** p < 0.01 and *** p < 0.001, significant differences between the Cis group and Cis + NAC, Cis + GL, and Cis + GH groups. The arrows indicate the nephron glomerulus. The bars represent 100 μm. Full article ">Figure 3
Levels of (A) glutathione, (B) nitrite, and (C) thiobarbituric acid reactive substance (TBARS) as well as (D) IL-1β, (E) IL-6, and (F) TNF-α inflammatory cytokines in mice with cisplatin-induced CKD. Values are presented as the means ± SD (n = 6). ### p < 0.001, significant difference between the Cis and N groups; * p < 0.05, ** p < 0.01, and *** p < 0.001, significant differences between the Cis group and Cis + NAC, Cis + GL, and Cis + GH groups. Full article ">Figure 4
(A) Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2); (B) TLR4, IκBα, and NF-κB; and (C) MAPKs levels in mice with cisplatin-induced CKD. The statistical analysis of these levels involved triplicate measurements. Values are expressed as the means ± SD. The bands were quantified using the densitometric program ImageJ (version 1.8.0); the protein levels were then divided by β-actin and normalized against the control protein. Full article ">Figure 5
(A) Beclin-1, LC3-I/II, and p62, (B) WNT2, β-catenin, and GSK3β, and (C) PI3K and AKT levels in the N, Cis, Cis + NAC, and Cis + GH groups of mice with cisplatin-induced chronic kidney disease. The statistical analysis of these levels involved triplicate measurements. Values are expressed as the means ± SD. The bands were quantified using the densitometric program ImageJ; the protein levels were then divided by β-actin and normalized against the control protein. Full article ">Figure 6
(A) p53, Bax, Bcl-2, and caspase-3, (B) TGF-β and SMAD3, and (C) matrix metalloproteinase 2 (MMP2) and MMP9 levels in mice with cisplatin-induced CKD. The statistical analysis of these levels involved triplicate measurements. Values are expressed as the means ± SD. The bands were quantified using the densitometric program ImageJ; the protein levels were then divided by β-actin and normalized against the control protein. Full article ">Figure 7
(A) Fibronectin, collagen, and α-smooth muscle actin (SMA), (B) nuclear factor erythroid–related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), and (C) catalase, glutathione peroxidase 3 (GPx3), and superoxide dismutase type 1 (SOD-1) levels in mice with cisplatin-induced CKD. The statistical analysis of these levels involved triplicate measurements. Values are expressed as the means ± SD. The bands were quantified using the densitometric program ImageJ; the protein levels were then divided by β-actin and normalized against the control protein. Full article ">Figure 8
Levels of organic cation transport proteins (OCT2 and OCT3) and organic anion transport proteins (OAT1 and OAT3) in mice with cisplatin-induced CKD. The statistical analysis of these levels involved triplicate measurements. Values are expressed as the means ± SD. The bands were quantified using the densitometric program ImageJ; the protein levels were then divided by β-actin and normalized against the control protein. Full article ">Figure 9
HPLC results for the FVB extract: (A) detection of gallic acid at a retention time of 7.060 min and quercetin at 39.840 min in the analysis of the FVB extract measured at 20,000 μg/g; (B) detection of gallic acid at a retention time of 7.193 min and quercetin at 39.640 min in the analysis of 100 μg/g standard samples. Full article ">Figure 10
A scheme displaying the protective effect of FVB against cisplatin-induced CDK. The green arrows denote increases and the red arrows denote decreases. Forbidden symbol denote suppression. Full article ">

16 pages, 1358 KiB

Open AccessFeature PaperReview

Metabolic Chaos in Kidney Disease: Unraveling Energy Dysregulation

by Priya Gupta, Saiya Zhu, Yuan Gui and Dong Zhou

J. Clin. Med. 2024, 13(22), 6772; https://doi.org/10.3390/jcm13226772 - 11 Nov 2024

Viewed by 712

Abstract

Background: Acute kidney injury (AKI) and chronic kidney disease (CKD) share a fundamental disruption: metabolic dysfunction. Methods: A literature review was performed to determine the metabolic changes that occur in AKI and CKD as well as potential therapeutic targets related to these changes. [...] Read more.

Background: Acute kidney injury (AKI) and chronic kidney disease (CKD) share a fundamental disruption: metabolic dysfunction. Methods: A literature review was performed to determine the metabolic changes that occur in AKI and CKD as well as potential therapeutic targets related to these changes. Results: In AKI, increased energy demand in proximal tubular epithelial cells drives a shift from fatty acid oxidation (FAO) to glycolysis. Although this shift offers short-term support, it also heightens cellular vulnerability to further injury. As AKI progresses to CKD, metabolic disruption intensifies, with both FAO and glycolysis becoming downregulated, exacerbating cellular damage and fibrosis. These metabolic alterations are governed by shifts in gene expression and protein signaling pathways, which can now be precisely analyzed through advanced omics and histological methods. Conclusions: This review examines these metabolic disturbances and their roles in disease progression, highlighting therapeutic interventions that may restore metabolic balance and enhance kidney function. Many metabolic changes that occur in AKI and CKD can be utilized as therapeutic targets, indicating a need for future studies related to the clinical utility of these therapeutics. Full article

(This article belongs to the Section Nephrology & Urology)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Metabolic shifts in AKI and their impact on cellular energy. Following AKI, key metabolic changes occur as shown in this figure, with an up arrow indicating increased levels after AKI and a down arrow indicating decreased levels after AKI. These changes include decreased FAO, impaired BCAA metabolism, and increased glycolysis. Reduced FAO leads to lipid accumulation, promoting lipotoxicity and mitochondrial dysfunction, while disrupted BCAA metabolism diminishes energy production and elevates toxic byproducts. The shift toward glycolysis compensates for the energy deficit but yields less ATP and leads to lactate buildup, exacerbating acidosis. These changes result in lower cellular energy availability, impairing cell repair and promoting kidney injury progression. AKI, acute kidney injury; FAO, fatty acid oxidation; BCAA, branched-chain amino acid. Full article ">Figure 2
Metabolic alterations driving CKD progression and fibrosis. In CKD, prolonged metabolic changes contribute to energy deficits and promote renal fibrosis. Notable disruptions include lipid accumulation in kidney cells due to impaired FAO and altered lipid metabolism, leading to lipotoxicity and mitochondrial stress. Additionally, changes in lipid profiles disrupt cellular signaling and membrane function. Reduced cellular energy availability causes cell cycle arrest and further hinders the cells’ ability to repair and regenerate. These metabolic changes create a pro-fibrotic environment, driving sustained tissue remodeling, inflammation, and scarring that ultimately progress CKD. CKD, chronic kidney disease; FAO, fatty acid oxidation. Full article ">Figure 3
FAO regulation in proximal tubule epithelial cells under normal and AKI–CKD conditions. In a healthy kidney, FAO in proximal tubule epithelial cells is efficiently regulated by essential proteins such as CPT1α, PGC-1α, and AMPK, which support energy homeostasis. Following kidney injury, changes occur as represented by the red arrows in this figure. Up arrows indicate upregulation while down arrows indicate downregulation. Thus, after kidney injury, the upregulation of CD36, a lipid transporter, and downregulation of CPT1α, PGC-1α, and AMPK disrupt FAO, leading to lipid accumulation and mitochondrial dysfunction. This metabolic imbalance contributes to cellular stress, inflammation, and fibrosis, driving AKI progression to CKD. FAO, fatty acid oxidation; CPT1α, carnitine palmitoyltransferase 1α; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1α; AMPK, AMP-activated protein kinase. Full article ">

25 pages, 21810 KiB

Open AccessArticle

Morphofunctional Features of Glomeruli and Nephrons After Exposure to Electrons at Different Doses: Oxidative Stress, Inflammation, Apoptosis

by Grigory Demyashkin, Sergey Koryakin, Mikhail Parshenkov, Polina Skovorodko, Matvey Vadyukhin, Zhanna Uruskhanova, Yulia Stepanova, Vladimir Shchekin, Artem Mirontsev, Vera Rostovskaya, Sergey Ivanov, Petr Shegay and Andrei Kaprin

Curr. Issues Mol. Biol. 2024, 46(11), 12608-12632; https://doi.org/10.3390/cimb46110748 - 6 Nov 2024

Viewed by 593

Abstract

Kidney disease has emerged as a significant global health issue, projected to become the fifth-leading cause of years of life lost by 2040. The kidneys, being highly radiosensitive, are vulnerable to damage from various forms of radiation, including gamma (γ) and X-rays. However, [...] Read more.

Kidney disease has emerged as a significant global health issue, projected to become the fifth-leading cause of years of life lost by 2040. The kidneys, being highly radiosensitive, are vulnerable to damage from various forms of radiation, including gamma (γ) and X-rays. However, the effects of electron radiation on renal tissues remain poorly understood. Given the localized energy deposition of electron beams, this study seeks to investigate the dose-dependent morphological and molecular changes in the kidneys following electron irradiation, aiming to address the gap in knowledge regarding its impact on renal structures. The primary aim of this study is to conduct a detailed morphological and molecular analysis of the kidneys following localized electron irradiation at different doses, to better understand the dose-dependent effects on renal tissue structure and function in an experimental model. Male Wistar rats (n = 75) were divided into five groups, including a control group and four experimental groups receiving 2, 4, 6, or 8 Gray (Gy) of localized electron irradiation to the kidneys. Biochemical markers of inflammation (interleukin-1 beta [IL-1β], interleukin-6 [IL-6], interleukin-10 [IL-10], tumor necrosis factor-alpha [TNF-α]) and oxidative stress (malondialdehyde [MDA], superoxide dismutase [SOD], glutathione [GSH]) were measured, and morphological changes were assessed using histological and immunohistochemical techniques (TUNEL assay, caspase-3). The study revealed a significant dose-dependent increase in oxidative stress, inflammation, and renal tissue damage. Higher doses of irradiation resulted in increased apoptosis, early stages of fibrosis (at high doses), and morphological changes in renal tissue. This study highlights the dose-dependent effects of electrons on renal structures, emphasizing the need for careful consideration of the dosage in clinical use to minimize adverse effects on renal function. Full article

(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)

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20 pages, 4472 KiB

Open AccessArticle

Hypoxia-Induced Differences in the Expression of Pyruvate Dehydrogenase Kinase 1-Related Factors in the Renal Tissues and Renal Interstitial Fibroblast-like Cells of Yak (Bos Grunniens)

by Manlin Zhou, Jun Wang, Ruirui Cao, Fan Zhang, Xuehui Luo, Yiyuan Liao, Weiji Chen, Haie Ding, Xiao Tan, Zilin Qiao and Kun Yang

Animals 2024, 14(21), 3110; https://doi.org/10.3390/ani14213110 - 29 Oct 2024

Viewed by 608

Abstract

Hypoxia is one of the factors severely affect renal function, and, in severe cases, it can lead to renal fibrosis. Although much progress has been made in identifying the molecular mediators of fibrosis, the mechanisms that govern renal fibrosis remain unclear, and there [...] Read more.

Hypoxia is one of the factors severely affect renal function, and, in severe cases, it can lead to renal fibrosis. Although much progress has been made in identifying the molecular mediators of fibrosis, the mechanisms that govern renal fibrosis remain unclear, and there have been no effective therapeutic anti-fibrotic strategies to date. Mammals exposed to low oxygen in the plateau environment for a long time are prone to high-altitude disease, while yaks have been living in the plateau for generations do not develop kidney fibrosis caused by low oxygen. It has been suggested that metabolic reprogramming occurs in renal fibrosis and that pyruvate dehydrogenase kinase 1 (PDK1) plays a crucial role in metabolic reprogramming as an important node between glycolysis and the tricarboxylic acid cycle. The aim of this study was to investigate the effects of hypoxia on the renal tissues and renal interstitial fibroblasts of yaks. We found that, at the tissue level, HIF-1α, PDK1, TGF-β1, Smad2, Smad3, and α-SMA were mainly distributed and expressed in tubular epithelial cells but were barely present in the renal mesenchymal fibroblasts of healthy cattle and yak kidneys. Anoptical density analysis showed that in healthy cattle kidneys, TGF-β1, Smad2, and Smad3 expression was significantly higher than in yak kidneys (p < 0.05), and HIF-1α and PDK1 expression was significantly lower than in yak kidneys (p < 0.05). The results at the protein and gene levels showed the same trend. At the cellular level, prolonged hypoxia significantly elevated PDK1 expression in the renal mesangial fibroblasts of cattle and yak kidneys compared with normoxia (p < 0.05) and was proportional to the degree of cellular fibrosis. However, PDK1 expression remained stable in yaks compared with renal interstitial fibroblast-like cells in cattle during the same hypoxic time period. At the same time, prolonged hypoxia also promoted changes in cellular phenotype, promoting the proliferation, activation, glucose consumption, lactate production, and anti-apoptosis in the both of cattle and yaks renal interstitial fibroblasts The differences in kidney structure and expression of PDK1 and HIF-1α in kidney tissue and renal interstitial fibroblasts induced by different oxygen concentrations suggest that there may be a regulatory relationship between yak kidney adaptation and hypoxic environment at high altitude. This provides strong support for the elucidation of the regulatory relationship between PDK1 and HIF-1α, as well as a new direction for the treatment or delay of hypoxic renal fibrosis; additionally, these findings provide a basis for further analysis of the molecular mechanism of hypoxia adaptation-related factors and the adaptation of yaks to plateau hypoxia. Full article

(This article belongs to the Special Issue Production, Breeding and Disease Management of Plateau Animals)

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23 pages, 9129 KiB

Open AccessArticle

Virtual Screening, Molecular Dynamics, and Mechanism Study of Homeodomain-Interacting Protein Kinase 2 Inhibitor in Renal Fibroblasts

by Xinlan Hu, Yan Wu, Hanyi Ouyang, Jiayan Wu, Mengmeng Yao, Zhuo Chen and Qianbin Li

Pharmaceuticals 2024, 17(11), 1420; https://doi.org/10.3390/ph17111420 - 23 Oct 2024

Viewed by 775

Abstract

Background/Objectives: Homeodomain-interacting protein kinase 2 (HIPK2) is critically involved in the progression of renal fibrosis. This study aims to identify and characterize a novel HIPK2 inhibitor, CHR-6494, and investigate its therapeutic potential. Methods: Using structure-based virtual screening and molecular dynamics simulations, we identified [...] Read more.

Background/Objectives: Homeodomain-interacting protein kinase 2 (HIPK2) is critically involved in the progression of renal fibrosis. This study aims to identify and characterize a novel HIPK2 inhibitor, CHR-6494, and investigate its therapeutic potential. Methods: Using structure-based virtual screening and molecular dynamics simulations, we identified CHR-6494 as a potent HIPK2 inhibitor with an IC₅₀ of 0.97 μM. The effects of CHR-6494 on the phosphorylation of p53 in Normal Rattus norvegicus kidney cells (NRK-49F) induced by transforming growth factor-β (TGF-β) were assessed, along with its impact on TGF-β signaling and downstream profibrotic markers. Results: CHR-6494 significantly reduces p53 phosphorylation induced by TGF-β and enhances the interaction between HIPK2 and seven in absentia 2 (SIAH2), facilitating HIPK2 degradation via proteasomal pathways. Both CHR-6494 and Abemaciclib inhibit NRK-49F cell proliferation and migration induced by TGF-β, suppressing TGF-β/Smad3 signaling and decreasing profibrotic markers such as Fibronectin I (FN-I) Collagen I and α-smooth muscle actin (α-SMA). Additionally, these compounds inhibit nuclear factor kappa-B (NF-κB) signaling and reduce inflammatory cytokine expression. Conclusions: The study highlights the dual functionality of HIPK2 kinase inhibitors like CHR-6494 and Abemaciclib as promising therapeutic candidates for renal fibrosis and inflammation. The findings provide new insights into HIPK2 inhibition mechanisms and suggest pathways for the design of novel HIPK2 inhibitors in the future. Full article

(This article belongs to the Special Issue Small-Molecule Inhibitors for Novel Therapeutics)

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