Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway.

BACKGROUND: Airway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma. METHODS: The datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor. RESULTS: Both bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-ß1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression. CONCLUSIONS: Taken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Neural Wiskott-Aldrich syndrome protein (N-WASP) promotes distant metastasis in pancreatic ductal adenocarcinoma via activation of LOXL2.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis has not been established. In this study, we investigated whether Neural Wiskott-Aldrich syndrome protein (N-WASP) plays a role in distant metastasis of PDAC. We found that N-WASP is markedly expressed in clinical patients with PDAC. Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group. N-WASP was noted to be a novel mediator of epithelial-mesenchymal transition (EMT) via gene expression profile studies. Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion, migration, and EMT. We also observed positive association of lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) with the N-WASP-mediated response, wherein EMT and invadopodia function were modulated. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer. These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function, with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.

A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase.

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

LOXL2-mediated chromatin compaction is required to maintain the oncogenic properties of triple-negative breast cancer cells.

Oxidation of histone H3 at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase homolog 2 (LOXL2). This histone modification is enriched in heterochromatin in triple-negative breast cancer (TNBC) cells and has been linked to the maintenance of compacted chromatin. However, the molecular mechanism underlying this maintenance is still unknown. Here, we show that LOXL2 interacts with RuvB-Like 1 (RUVBL1), RuvB-Like 2 (RUVBL2), Actin-like protein 6A (ACTL6A), and DNA methyltransferase 1associated protein 1 (DMAP1), a complex involved in the incorporation of the histone variant H2A.Z. Our experiments indicate that this interaction and the active form of RUVBL2 are required to maintain LOXL2-dependent chromatin compaction. Genome-wide experiments showed that H2A.Z, RUVBL2, and H3K4ox colocalize in heterochromatin regions. In the absence of LOXL2 or RUVBL2, global levels of the heterochromatin histone mark H3K9me3 were strongly reduced, and the ATAC-seq signal in the H3K9me3 regions was increased. Finally, we observed that the interplay between these series of events is required to maintain H3K4ox-enriched heterochromatin regions, which in turn is key for maintaining the oncogenic properties of the TNBC cell line tested (MDA-MB-231).

Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Lysine Deacetylation Is a Key Function of the Lysyl Oxidase Family of Proteins in Cancer.

Mammalian members of the lysyl oxidase (LOX) family of proteins carry a copper-dependent monoamine oxidase domain exclusively within the C-terminal region, which catalyzes ε-amine oxidation of lysine residues of various proteins. However, recent studies have demonstrated that in LOX-like (LOXL) 2-4 the C-terminal canonical catalytic domain and N-terminal scavenger receptor cysteine-rich (SRCR) repeats domain exhibit lysine deacetylation and deacetylimination catalytic activities. Moreover, the N-terminal SRCR repeats domain is more catalytically active than the C-terminal oxidase domain. Thus, LOX is the third family of lysine deacetylases in addition to histone deacetylase and sirtuin families. In this review, we discuss how the LOX family targets different cellular proteins for deacetylation and deacetylimination to control the development and metastasis of cancer.

LncRNA LOXL1-AS1 promotes proliferation and invasion and inhibits apoptosis in retinoblastoma by regulating the MAPK signaling pathway.

Retinoblastoma (RB) is an intraocular malignancy that is most common in children and rare in adults. Addressing novel biomarkers and therapeutic targets for RB to modulate tumor progression has become a challenge. The aim of the present study was to investigate the function of long non-coding RNAs (LncRNAs) LOXL1-AS1 in RB cell proliferation and metastasis. It was found that LOXL1-AS1 was overexpressed in RB tissues and cells. In order to evaluate cell viability and colony formation potential, the knockdown of LOXL1-AS1 has been established. Knockdown of LOXL1-AS1 was also inhibited cells migration and invasion. In addition, the proportion of cells in the G2/M phase of the sh-LOXL1-AS1 group increased significantly, and the proportion of cells in the sh-NC group decreased significantly. In the xenograft model of RB, the tumors in the sh-LOXL1-AS1 group grow slowly compared to the sh-NC group. Western blot analysis revealed that LOXL1-AS1 can regulate the progression of RB cells through MAPK signaling pathway in vitro and in vivo. These results indicated that LncRNA LOXL1-AS1 promotes proliferation, invasion and inhibits apoptosis of retinoblastoma by regulating MAPK signaling pathway, and might be expected to be a novel basis for clinical diagnosis and treatment.

Reshaping the Substrate Binding Pocket of ß-Amino Acid Dehydrogenase for the Synthesis of Aromatic ß-Amino Acids.

By reshaping the substrate-binding pocket of ß-amino acid dehydrogenase (ß-AADH), some variants were obtained with up to 2560-fold enhanced activity toward the model substrates (S)-ß-homophenylalanine and (R)-ß-phenylalanine. A few aromatic ß-amino acids were prepared with >99% ee and high isolated yields via either kinetic resolution of racemates or reductive amination of the corresponding ß-keto acids. This work expands the catalytic capability of ß-AADHs and highlights their practical application in the synthesis of pharmaceutically relevant ß-amino acids.

Interactions between BRD4S, LOXL2, and MED1 drive cell cycle transcription in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) often develops resistance to single-agent treatment, which can be circumvented using targeted combinatorial approaches. Here, we demonstrate that the simultaneous inhibition of LOXL2 and BRD4 synergistically limits TNBC proliferation in vitro and in vivo. Mechanistically, LOXL2 interacts in the nucleus with the short isoform of BRD4 (BRD4S), MED1, and the cell cycle transcriptional regulator B-MyB. These interactions sustain the formation of BRD4 and MED1 nuclear transcriptional foci and control cell cycle progression at the gene expression level. The pharmacological co-inhibition of LOXL2 and BRD4 reduces BRD4 nuclear foci, BRD4-MED1 colocalization, and the transcription of cell cycle genes, thus suppressing TNBC cell proliferation. Targeting the interaction between BRD4S and LOXL2 could be a starting point for the development of new anticancer strategies for the treatment of TNBC.

The possibilities of LOXL4 as a prognostic marker for carcinomas.

Lysyl oxidase-like 4 (LOXL4), a member of lysyl oxidase family, is a copper and lysine tyrosylquinone-dependent amine oxidase that serves the role of catalyzing the cross-linking of elastin and collagen in the extracellular matrix. Numerous studies have shown a significant association between LOXL4 expression levels and tumor proliferation, migration, invasion and patients' prognosis and overall survival in different types of tumors. Here we review their relationship and the molecular pathogenesis behind them, aiming to explore the possibilities of LOXL4 as a prognostic marker for diverse carcinomas and provide some indications for further research in this field.

Genetic background determines severity of Loxl1-mediated systemic and ocular elastosis in mice.

Pseudoexfoliation syndrome (PEX) is a systemic, age-related disorder characterized by elastosis and extracellular matrix deposits. Its most significant ocular manifestation is an aggressive form of glaucoma associated with variants in the gene encoding lysyl oxidase-like 1 (LOXL1). Depending upon the population, variants in LOXL1 can impart risk or protection for PEX, suggesting the importance of genetic context. As LOXL1 protein levels are lower and the degree of elastosis is higher in people with PEX, we studied Loxl1-deficient mice on three different genetic backgrounds: C57BL/6 (BL/6), 129S×C57BL/6 (50/50) and 129S. Early onset and high prevalence of spontaneous pelvic organ prolapse in BL/6 Loxl1-/- mice necessitated the study of mice that were <2 months old. Similar to pelvic organ prolapse, most elastosis endpoints were the most severe in BL/6 Loxl1-/- mice, including skin laxity, pulmonary tropoelastin accumulation, expansion of Schlemm's canal and dilation of intrascleral veins. Interestingly, intraocular pressure was elevated in 50/50 Loxl1-/- mice, depressed in BL/6 Loxl1-/- mice and unchanged in 129S Loxl1-/- mice compared to that of control littermates. Overall, the 129S background was protective against most elastosis phenotypes studied. Thus, repair of elastin-containing tissues is impacted by the abundance of LOXL1 and genetic context in young animals.

A protein complex of LCN2, LOXL2 and MMP9 facilitates tumour metastasis in oesophageal cancer.

During malignant tumour development, the extracellular matrix (ECM) is usually abnormally regulated. Dysregulated expression of lysyl oxidase-like 2 (LOXL2), matrix metalloproteinase 9 (MMP9) and lipocalin 2 (LCN2) are associated with ECM remodelling. In this study, protein-protein interaction assays indicated that LCN2 and LOXL2 interactions and LCN2 and MMP9 interactions occurred both intracellularly and extracellularly, but interactions between LOXL2 and MMP9 only occurred intracellularly. The LCN2/LOXL2/MMP9 ternary complex promoted migration and invasion of oesophageal squamous cell carcinoma (ESCC) cells, as well as tumour growth and malignant progression in vivo, while the iron chelator deferoxamine mesylate (DFOM) inhibited ESCC tumour growth. Co-overexpression of LCN2, LOXL2 and MMP9 enhanced the ability of tumour cells to degrade fibronectin and Matrigel, increased the formation and extension of filopodia, and promoted the rearrangement of microfilaments through upregulation of profilin 1. In addition, the LCN2/LOXL2/MMP9 ternary complex promoted the expression of testican-1 (SPOCK1), and abnormally activated the FAK/AKT/GSK3ß signalling pathway. In summary, the LCN2/LOXL2/MMP9 ternary complex promoted the migration and invasion of cancer cells and malignant tumour progression through multiple mechanisms and could be a potential therapeutic target.

Computational Insights on the Hydride and Proton Transfer Mechanisms of D-Arginine Dehydrogenase.

D-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is an amine oxidase which catalyzes the conversion of D-arginine into iminoarginine. It contains a non-covalent FAD cofactor that is involved in the oxidation mechanism. Based on substrate, solvent, and multiple kinetic isotope effects studies, a stepwise hydride transfer mechanism is proposed. It was shown that D-arginine binds to the active site of enzyme as α-amino group protonated, and it is deprotonated before a hydride ion is transferred from its α-C to FAD. Based on a mutagenesis study, it was concluded that a water molecule is the most likely catalytic base responsible from the deprotonation of α-amino group. In this study, we formulated computational models based on ONIOM method to elucidate the oxidation mechanism of D-arginine into iminoarginine using the crystal structure of enzyme complexed with iminoarginine. The calculations showed that Arg222, Arg305, Tyr249, Glu87, His 48, and two active site water molecules play key roles in binding and catalysis. Model systems showed that the deprotonation step occurs prior to hydride transfer step, and active site water molecule(s) may have participated in the deprotonation process.

Multiple Roles of LOXL2 in the Progression of Hepatocellular Carcinoma and Its Potential for Therapeutic Targeting.

LOXL2, a copper-dependent amine oxidase, has emerged as a promising therapeutic target in hepatocellular carcinoma (HCC). Increased LOXL2 expression in HCC has been linked with an aggressive phenotype and represents a poor prognostic factor. Here, we focus on the mechanisms through which LOXL2 orchestrates multiple oncogenic functions in HCC development. We performed a review of the current knowledge on the roles LOXL2 performs in the modulation of the HCC tumor microenvironment, formation of premetastatic niches, and epithelial-mesenchymal transition. We also highlighted the complex interplay between LOXL2 and hypoxia, angiogenesis, and vasculogenic mimicry in HCC. At the end of the review, we summarize the current LOXL2 inhibitors and discuss their potential in HCC precision treatment.

Denatured collagen inhibits neuroblastoma tumor-sphere migration and growth via the LOX/LOXL2 - FAK signaling pathway.

A complex interplay exists within the tumor microenvironment and extracellular matrix, which could contribute to solid tumor progression. Collagen, a major component of the extracellular matrix, may correlate with cancer prognosis. While thermal ablation has shown promise as a minimally invasive treatment of solid tumors, its impact on collagen is still unknown. In this study, we demonstrate that thermal ablation, but not cryo-ablation, induces irreversible collagen denaturation in a neuroblastoma sphere model. Prolonged collagen denaturation resulted in a significant reduction in sphere stiffness, migration, and proliferation, and an increase in apoptosis. Mechanistic analysis revealed that collagen denaturation inhibited collagen cross-linking, reduced extracellular LOX/LOXL2 expression, and resulted in decreased phosphorylation of FAK. Downstream of FAK, we observed reduced epithelial to mesenchymal transition, attenuated CDC42 expression, and decreased migration. Collectively, these results suggest that denatured collagen presents a novel target for modulating the tumor microenvironment and treating solid cancers via the LOX1/LOXL2-FAK signaling pathway.

New Functions of Intracellular LOXL2: Modulation of RNA-Binding Proteins.

Lysyl oxidase-like 2 (LOXL2) was initially described as an extracellular enzyme involved in extracellular matrix remodeling. Nevertheless, numerous recent reports have implicated intracellular LOXL2 in a wide variety of processes that impact on gene transcription, development, differentiation, proliferation, migration, cell adhesion, and angiogenesis, suggesting multiple different functions for this protein. In addition, increasing knowledge about LOXL2 points to a role in several types of human cancer. Moreover, LOXL2 is able to induce the epithelial-to-mesenchymal transition (EMT) process-the first step in the metastatic cascade. To uncover the underlying mechanisms of the great variety of functions of intracellular LOXL2, we carried out an analysis of LOXL2's nuclear interactome. This study reveals the interaction of LOXL2 with numerous RNA-binding proteins (RBPs) involved in several aspects of RNA metabolism. Gene expression profile analysis of cells silenced for LOXL2, combined with in silico identification of RBPs' targets, points to six RBPs as candidates to be substrates of LOXL2's action, and that deserve a more mechanistic analysis in the future. The results presented here allow us to hypothesize novel LOXL2 functions that might help to comprehend its multifaceted role in the tumorigenic process.

Application of reductive amination by heterologously expressed Thermomicrobium roseumL-alanine dehydrogenase to synthesize L-alanine derivatives.

Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, α-ketobutyrate, α-ketovalerate and α-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.

Inhibition of N-glycosylation by glucosamine hydrochloride inhibits TGF-ß1-induced LOXL2 secretion.

Kidney fibrosis is closely associated with the progression of chronic kidney disease (CKD). Furthermore, copper-containing secretory amine oxidases, such as lysyl oxidase (LOX) and LOX-like 1-4 (LOXL1-4), play pivotal roles in the regulation of extracellular components and facilitate fibrosis. In this study, we investigated the regulation of LOX enzymes in human tubular epithelial HK2 cells to help clarify the role of LOX enzymes in kidney fibrosis. Among 5 LOX enzymes, LOXL2 expression is abundantly expressed in HK2 cells. LOX enzymes inhibitor, ß-aminopropionitrile, suppressed transforming growth factor-ß1 (TGF-ß1)-promoted epithelial-to-mesenchymal transition processes in HK2 cells, indicating that LOX enzymes are involved in TGF-ß1-mediated fibrotic processes. Recent studies suggest that LOX enzymes are secreted into the extracellular spaces by exosomes and promote fibrotic processes. Similar to the previous reports, we observed that exosomes secreted from HK2 cells carry LOXL2 into the extracellular spaces. Furthermore, we determined that N-glycosylation on the asparagine residues plays a key role in LOXL2 secretion. Amino acid mutations in three asparagine residues, which can be glycosylated, suppressed the secretion of mutated LOXL2. Moreover, N-acetylglucosaminyltransferase 5, an enzyme used for the biosynthesis of ß1,6N-acetylglucosamine-branched N-glycans, participated in LOXL2 secretion, and the N-glycosylation inhibitor, glucosamine hydrochloride (GS), inhibited TGF-ß1-mediated LOXL2 secretion and fibrotic processes. Overall, TGF-ß1 promotes LOXL2 secretion and may participate in kidney fibrosis. Our results provide novel insight into the antifibrotic properties of GS that contribute to the inhibition of CKD progression.