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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2024 Sep 3;36(1):19–33. doi: 10.1681/ASN.0000000000000488

Glycolysis in Peritubular Endothelial Cells and Microvascular Rarefaction in CKD

Yujie Huang 1, Ansheng Cong 1, Jinjin Li 1, Zhanmei Zhou 1,✉, Hong Zhou 1,✉, Cailing Su 1, Zuoyu Hu 1, Fan Fan Hou 1,✉, Wei Cao 1,✉
PMCID: PMC11706556  PMID: 39226371

Visual Abstract

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Keywords: CKD

Abstract

Key Points

  • Peritubular endothelial cells have a hypoglycolytic metabolism in CKD.

  • Restoration of glycolysis in CKD peritubular endothelial cells by overexpressing 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase attenuates microvascular rarefaction and kidney fibrosis.

  • Strategies targeting the metabolic defect in glycolysis in peritubular endothelial cells may be effective in the treatment of CKD.

Background

Peritubular endothelial cell dropout leading to microvascular rarefaction is a common manifestation of CKD. The role of metabolism reprogramming in peritubular endothelial cell loss in CKD is undetermined.

Methods

Single-cell sequencing and metabolic analysis were used to characterize the metabolic profile of peritubular endothelial cells from patients with CKD and from CKD mouse models. In vivo and in vitro models demonstrated metabolic reprogramming in peritubular endothelial cells in conditions of CKD and its contribution to microvascular rarefaction.

Results

In this study, we identified glycolysis as a top dysregulated metabolic pathway in peritubular endothelial cells from patients with CKD. Specifically, CKD peritubular endothelial cells were hypoglycolytic while displaying an antiangiogenic response with decreased proliferation and increased apoptosis. The hypoglycolytic phenotype of peritubular endothelial cells was recapitulated in CKD mouse models and in peritubular endothelial cells stimulated by hydrogen peroxide. Mechanically, oxidative stress, through activating a redox sensor kruppel-like transcription factor 9, downregulated the glycolytic activator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase expression, thereby reprogramming peritubular endothelial cells toward a hypoglycolytic phenotype. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase overexpression in peritubular endothelial cells restored hydrogen peroxide–induced reduction in glycolysis and cellular ATP levels and enhanced the G1/S cell cycle transition, enabling peritubular endothelial cells to improve proliferation and reduce apoptosis. Consistently, restoration of peritubular endothelial cell glycolysis in CKD mice, by overexpressing endothelial Pfkfb3, reversed the antiangiogenic response in peritubular endothelial cells and protected the kidney from microvascular rarefaction and fibrosis. By contrast, suppression of glycolysis by endothelial Pfkfb3 deletion exacerbated microvascular rarefaction and fibrosis in CKD mice.

Conclusions

Our study revealed a disrupted regulation of glycolysis in peritubular endothelial cells as an initiator of microvascular rarefaction in CKD. Restoration of peritubular endothelial cell glycolysis in CKD kidney improved microvascular rarefaction and ameliorated fibrotic lesions.

Introduction

CKD is a progressive condition that affects approximately one tenth of the global population.1–3 Patients with CKD are at high risk of kidney failure and death.4 Poor outcomes are attributed to a delay in diagnosis and a lack of therapeutic options to halt or reverse kidney injury.5 Uncovering new therapeutic targets mediating early-stage CKD is of importance.

Microvascular rarefaction, reflected by decreased peritubular microvessel density, is commonly observed in all forms of CKD.6,7 It disrupts oxygen and nutrient delivery, creating a hostile environment that accelerates CKD.8 The extent of microvascular rarefaction is inextricably linked to clinical outcomes.9–11 Targeting cellular mechanisms that facilitate microvessel rarefaction after injury may provide avenues for novel therapies.7

Peritubular endothelial cell dysfunction, detachment, or loss are key cellular events underlying microvascular rarefaction,12 but their cellular phenotype, function and regulation in CKD remain poorly defined. Earlier proangiogenic medicine mainly targets angiocrine signals in an attempt to increase vessel growth. These therapies, however, have been limited by inadequate efficacy and side effects.13–16 More recently, an emerging paradigm is to normalize blood vessels to inhibit CKD progression, necessitating a fundamentally different approach.

Endothelial cells rely on specific metabolic pathways to survive.17 They are addicted to anaerobic glycolysis, generating approximately 85% of their ATP glycolytically even under oxygen-replete conditions.18 Endothelial cells are also highly heterogeneous among organs and can adopt a range of functional characteristics in response to local stimuli.19 Endothelial cell metabolism has gained attention as a therapeutic target for pathological angiogenesis. We and others have reported that blocking glycolysis activator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3) inhibits angiogenesis and normalizes microvessels in tumor and peritoneal dialysis.19–21 Despite intense investigation of endothelial metabolism in pathological angiogenesis, very little is known about how peritubular endothelial cells adapt their metabolism to the unique CKD environment and contribute to microvascular rarefaction.

In this study, we hypothesized that peritubular endothelial cells in CKD develop hypoglycolysis, a metabolic alteration that induced an antiangiogenic response in these cells, leading to microvascular rarefaction and fibrosis. Targeting this metabolic defect in glycolysis in peritubular endothelial cells may protect the kidney from microvascular rarefaction and fibrotic lesions.

Methods

Detailed methods used in this study were provided in Supplemental Methods.

Human Samples

Protocols using human samples were approved by the Institute Ethics Committee (NFEC-2020-254) and performed according to the Declaration of Helsinki.

Normal kidney tissue adjacent to the site of a renal carcinoma was used as normal controls (n=3). Kidney samples of patients with CKD were obtained from diagnostic kidney biopsies (n=3). The collected human kidney samples were used for histological and immunostaining analyses. Eligible patients were 18 years or older and met the Kidney Disease Improving Global Outcomes criteria for stage 3 CKD.22 Exclusion criteria included urinary tract infection and kidney tumor or metastases.

Experimental CKD Model

All animal procedures were performed in accordance with the animal use protocol approved by the Institute Animal Ethics Committee (IACUC-LAC-20220610-002). Male mice aged 8–10 weeks (20–24 g) were used. Model of kidney ischemia–reperfusion injury (IRI) was induced by clamping both kidney pedicles for 35 minutes.23 Model of unilateral ureteral obstruction (UUO) was created by clamping the left ureter.24

Generation of Endothelial-Specific Pfkfb3 Knockout Mice

To obtain endothelial-specific Pfkfb3 knockout (Pfkfb3ΔEC) mice, Pfkfb3flox/flox (Pfkfb3WT) mice were intercrossed with Cdh5-Cre transgenic mice, an endothelial cell–selective Cre-driver line (all from Cyagen Bioscience).25–27

Generation of Endothelial-Specific Pfkfb3 Overexpression Mice

Full-length mouse Pfkfb3 cDNA was inserted into a Tie2-3xflag-WPREs vector and then packaged with adeno-associated virus (AAV serotype 1) by GeneChem (Shanghai, China). AAV harboring Pfkfb3 or control sequence was injected into the mouse kidney.28,29

Cell Culture and Isolation

Isolation of Mouse Peritubular Endothelial Cells by Flow Cytometry

Mouse kidney cortex was harvested, digested, and passed through a 70- and 40-µm mesh to remove glomeruli. Peritubular endothelial cells were enriched by a CD31 MicroBeads kit (130-097-418, Miltenyi, Bergisch Gladbach, Germany) and isolated by fluorescence-activated cell sorting (BD Biosciences, Franklin Lakes, NJ).30

Culture of Isolated Mouse Peritubular Endothelial Cells

Mouse peritubular endothelial cells were enriched, resuspended in the endothelial cell growth medium 2 (CC-3202, LONZA, Basel, Switzerland), and plated onto a dish. Adherent endothelial cells were grown to confluence and used for experiments.

Culture of Human Primary Peritubular Endothelial Cells

Human primary peritubular endothelial cells were purchased from Procell (CP-H261, Wuhan, China) and cultured in a cell medium from the same company (CM-H261, Procell). Passage 2 and 3 cells were used for experiments.

Statistical Analyses

Quantitative data were expressed as mean and SD. Normality tests were assessed by Shapiro–Wilk statistics. Differences among groups were determined by one-way ANOVA or unpaired t test with Bonferroni correction for multiple testing. When normality was rejected, nonparametric Mann–Whitney test or Kruskal–Wallis test was used. A P value of < 0.05 was considered statistically significant.

Results

Peritubular Endothelial Cells Were Hypoglycolytic in Human CKD

To define the metabolic landscape of human peritubular endothelial cells (HPECs) during homeostasis and CKD, we analyzed a single-cell RNA sequencing (scRNA-seq) dataset31 of interstitium-enriched cells from normal kidneys (normal, n=4) and from kidneys of patients with CKD (CKD, n=5). The dataset contained 43,080 single-cell transcriptomes spanning epithelial, mesenchymal, immune, and endothelial compartments31,32 (Supplemental Figure 1, A and B, and Supplemental Table 1).

Subclustering of endothelial cells revealed seven subsets (Figure 1A). Peritubular endothelial cells were identified as the major component of endothelial cells (64.5%) on the basis of previously reported markers,32 including regulator of cell cycle, endothelial cell specific molecule-1, and plasmalemma vesicle–associated protein (Figure 1B; Supplemental Figure 1C). We then performed gene set enrichment analysis on 15,992 peritubular endothelial cells, including 12,772 from normal samples and 3220 from CKD samples (Supplemental Table 2). As shown in Figure 1C, the genes decreased by CKD were related to angiogenesis, supporting an antiangiogenic state in these cells. We also detected a robust signal indicating downregulation of metabolism in CKD, including glycolysis, ATP generation, and nucleotide metabolism (Figure 1C). Specifically, glycolysis had the highest proportion of downregulated genes among differentially expressed metabolic pathways (Supplemental Table 3). By heatmap analysis, CKD peritubular endothelial cells downregulated transcripts of glycolysis genes (including PFKFB3) while lowering transcripts of genes involved in ATP generation, nucleotide synthesis, and antiapoptosis pathways (Figure 1D and Supplemental Figure 1D). In addition, glycolysis was also reduced by CKD in descending and ascending vasa recta subsets, but not significantly affected in the other four endothelial subsets (Supplemental Figure 1E).

Figure 1.

Figure 1

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Peritubular endothelial cells were hypoglycolytic in human CKD. (A) UMAP showing the endothelial cell subsets in the integrated single-cell transcriptomes derived from the interstitium-enriched kidney samples from normal controls (normal, n=4) and from patients with CKD (CKD, n=5) using the human scRNA-seq data (https://www.zenodo.org/record/4059315). (B) Heatmap of scaled expression of the specific marker genes for each endothelial cell subset. (C) GSEA of peritubular endothelial cells from normal and CKD groups. The graph showing the NES for the enrichment of a specific pathway. All P < 0.05. (D) Heatmap showing the expression of key genes of glycolysis, nucleotide metabolism, cell cycle, angiogenesis, and antiapoptosis pathways in peritubular endothelial cells from normal and CKD groups. (E) Monocle trajectories of peritubular endothelial cells colored by pseudotime (up) and group (down). (F) Ordering of scRNA-seq expression data of peritubular endothelial cells according to the pseudotime produced by Monocle 2 package. Expression of glycolysis, angiogenesis, and cell cycle genes demonstrated along the trajectory. (G) Immunofluorescence staining for CD31 (green) and PFKFB3 (red) or CD31 (green) and HK1 (red) in human kidney tissues. Scale bar: 10 μm. (H) The peritubular microvessel density presented by immunofluorescence staining for CD31 (green, scale bar: 50 μm) and the area of kidney fibrosis presented by Masson's trichrome staining (Masson, scale bar: 50 μm). Unpaired t test; *P < 0.05 versus normal group. Data expressed as mean±SD in (H) (n=3 in each group). AEA, afferent/efferent arteriole; AVR, ascending vasa recta; BCL2, B-cell lymphoma-2; DVR, descending vasa recta; ESM1, endothelial cell specific molecule-1; GC, glomerular capillary; GSEA, gene set enrichment analysis; iEC, injured endothelial cell; KDR, kinase insert domain receptor; LEC, lymphatic endothelial cell; NES, normalized enrichment score; PFKFB3, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase; PLVAP, plasmalemma vesicle–associated protein; PTEC, peritubular endothelial cell; RGCC, regulator of cell cycle; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection; VEGF, vascular endothelial growth factor.

Pseudotime analysis showed that normal peritubular endothelial cells resided at the beginning of the trajectory of a deconvoluted pseudotime plot, representing a quiescent state (Figure 1E). The CKD peritubular endothelial cells settled at the end of the trajectory (Figure 1E), displaying a significant shift toward an antiangiogenic state, with downregulation of angiogenesis-related genes kinase insert domain receptor, CDH5, and CCND3 (Figure 1F). A reduction in glycolysis genes coincided with the decrease in angiogenesis-related genes (Figure 1F).

Immunostaining analysis in human kidney specimens further demonstrated that CKD (versus normal) peritubular endothelial cells had downregulated expression of key glycolytic enzymes (including PFKFB3), accompanied by reduced microvessel density and increased fibrosis (Figure 1, G and H, and Supplemental Table 4). Thus, we demonstrate an association between hypoglycolysis and development of an antiangiogenic state in peritubular endothelial cells, suggesting that hypoglycolysis is involved in microvascular rarefaction in CKD. Among the glycolytic enzymes downregulated in CKD peritubular endothelial cells, PFKFB3 is an enzyme that does not directly affect glycolytic flux, but regulates activity of the glycolysis rate–limiting enzyme phosphofructokinase-1 by producing fructose-2,6-bisphosphate. Manipulation of PFKFB3 can partially affect glycolytic flux in endothelial cells and contribute to cell normalization.18,33,34 These render glycolysis, and its regulator PFKFB3, in CKD peritubular endothelial cells an attractive target.

Peritubular Endothelial Cells Were Hypoglycolytic in Mouse Models of CKD

Next, we investigated the metabolic changes of peritubular endothelial cells in mouse models of CKD. We performed scRNA-seq analyses on IRI mouse kidneys35 (Supplemental Figure 2, A and B, and Supplemental Table 5). Annotation of endothelial cell transcriptomes revealed broad mouse–human conservation of marker genes and transcriptional programs of peritubular endothelial cell cluster (Figure 2, A and B, and Supplemental Figure 2, C and D). We obtained 1180 peritubular endothelial cell transcriptomes, including 549 from normal mice (normal, n=2), 304 from IRI mice with CKD progression (IRI-CKD, n=6), and 327 from IRI mice with recovery (IRI-recovery, n=6). Peritubular endothelial cells from IRI-CKD mice had sustained reduction in glycolysis and angiogenesis, accompanied by persistently increased apoptosis from day 7 to 30 (Figure 2, C–E). However, peritubular endothelial cells from IRI-recovery mice had only a transition alteration in these pathways that almost returned to normal levels by day 30 (Supplemental Figure 2, E and F).

Figure 2.

Figure 2

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Peritubular endothelial cells were hypoglycolytic in mouse models of CKD. (A) UMAP showing the endothelial cell subsets from kidneys of normal mice (normal, n=2), IRI mice with CKD progression (IRI-CKD, n=6), and IRI mice with recovery (IRI-recovery, n=6) using the mouse scRNA-seq data GSE197626. (B) Heatmap of scaled expression of the specific marker genes for each endothelial cell subset. (C) GSVA of peritubular endothelial cells from normal and IRI-CKD groups. (D) GSEA plots demonstrating NESs of glycolysis gene set in the scRNA-seq data of peritubular endothelial cells. Genes in the gene set are ranked by signal-to-noise ratio according to their differential expression between the IRI-CKD and normal peritubular endothelial cells. (E) Heatmap showing the expression of key genes of glycolysis, nucleotide metabolism, ATP generation, angiogenesis, and apoptosis pathways in peritubular endothelial cells from normal and IRI-CKD groups. (F) To prepare a CKD model of IRI, mice were subjected to bilateral clamping of renal pedicles for 35 minutes. Animals were followed up for 28 days. Sham-operated mice (sham) were subjected to exposure of kidneys without the induction of ischemia. The peritubular microvessel density presented by immunofluorescence staining for CD31 (green, scale bar: 100 μm) and the area of kidney fibrosis presented by Masson's trichrome staining (Masson, scale bar: 50 μm). One-way ANOVA followed by Bonferroni test. *P < 0.05 versus sham. (G) Protein levels of PFKFB3 in isolated mouse peritubular endothelial cells. One-way ANOVA followed by Bonferroni test. *P < 0.05 versus sham. (H) Metabolite analysis in isolated mouse peritubular endothelial cells. Mann–Whitney U. *P < 0.05 versus sham. Data expressed as mean±SD in (F–H) (n=6 in each group). APAF1, apoptotic protease activating factor-1; FDR, false discovery rate; GSVA, gene set variation analysis; IRI, ischemia–reperfusion injury.

We further generated a CKD model of IRI that had progressive kidney injury within 28 days, characterized by a persistently decreased glomerular filtration rate and increased cellular inflammation, tubular injury, and kidney fibrosis (Figure 2F, Supplemental Figure 3, A–C, and Supplemental Table 6). Serum creatinine and albuminuria, as insensitive measures of kidney function, were comparable between IRI and sham mice on day 28 (Supplemental Table 6). Importantly, microvessel density, identified by the endothelial marker CD31, was significantly reduced in IRI mice from 14 to 28 days (Figure 2F and Supplemental Figure 3D). The reduced microvascular density coincided with increased hypoxia (indicated by pimonidazole staining) (Supplemental Figure 3E), confirming development of microvascular rarefaction in this model.

We then isolated peritubular endothelial cells from IRI mice (Supplemental Figure 4, A–D) and investigated their glycolysis alterations. Transcript levels of key glycolysis genes (including Pfkfb3) were markedly reduced in IRI peritubular endothelial cells from 7 to 28 days (Figure 2G and Supplemental Figure 4E). The reduced glycolysis gene expression was associated with downregulation of nucleotide synthesis–related genes and upregulation of apoptosis genes from 14 to 28 days (Supplemental Figure 4E). Consistently, levels of intermediate metabolites of glycolysis and nucleotide synthesis pathways were reduced in IRI peritubular endothelial cells (Figure 2H). Glucose consumption and lactate excretion were also decreased in the medium of these IRI cells (Supplemental Figure 4F), reinforcing a hypoglycolytic and less proliferative state of peritubular endothelial cells in CKD. The time course experiments indicate that the reduced glycolysis in peritubular endothelial cells precedes the development of microvascular rarefaction, supporting a causal role.

We observed similar changes in peritubular endothelial cells from mice with UUO-induced CKD. UUO kidneys (versus sham kidneys) had decreased microvessel density and increased fibrosis (Supplemental Figure 5A). Levels of key glycolysis enzymes (including Pfkfb3) and nucleotide synthesis–related genes were reduced, whereas levels of apoptosis genes were increased in UUO peritubular endothelial cells (Supplemental Figure 5, B and C). Thus, consistent with findings in patients with CKD, peritubular endothelial cells from CKD models have a hypoglycolytic metabolism associated with their evolution into an antiangiogenic response, contributing to microvascular rarefaction.

Hydrogen Peroxide–Induced Hypoglycolysis and Antiangiogenic Response in Peritubular Endothelial Cells

Several mediators have been identified to reduce glycolysis in endothelial cells, including oxidative stress and laminar shear stress.36–38 Among these, oxidative stress accompanies CKD39 and is reportedly associated with microvascular rarefaction.40,41 Our immunoblot analysis showed upregulation of NADPH oxidase subunits Nox2 and Nox4 in IRI kidneys (Figure 3A), confirming enhanced oxidative stress in CKD niche. We therefore examined the effect of oxidative stress on peritubular endothelial cell glycolysis.

Figure 3.

Figure 3

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H2O2-induced hypoglycolysis and antiangiogenic response in peritubular endothelial cells. (A) Protein levels of Nox2 and Nox4 in kidney homogenates of IRI mice. Unpaired t test. *P < 0.05 versus sham. (B) Transcript levels of key glycolysis enzymes in HPECs treated with H2O2 at different time points. One-way ANOVA followed by Bonferroni test. *P < 0.05 versus PBS. (C) Protein levels of PFKFB3 in HPECs treated with H2O2 at different time points. One-way ANOVA followed by Bonferroni test. *P < 0.05 versus PBS. (D) Glycolytic rate (termed GlycoPER) measured by glycolytic rate assay in HPECs. Unpaired t test. *P < 0.05 versus PBS. (E) ATP level in HPECs. Unpaired t test. *P < 0.05 versus PBS. (F) Flow cytometry showing distribution of different cell cycle phases in HPECs: flow cytometry histograms and quantitative data (n=3 in each group). Unpaired t test. *P < 0.05 versus PBS. (G) Percentage of EdU-positive HPECs presented by EdU incorporation staining. Scale bar: 100 μm. Unpaired t test. *P < 0.05 versus PBS. (H) Flow cytometry showing percentage of cell apoptosis in HPECs: flow cytometry histograms and quantitative data (n=3 in each group). Unpaired t test. *P < 0.05 versus PBS. (I) Transcript levels of apoptosis-related gene in HPECs. Unpaired t test. *P < 0.05 versus PBS. Data expressed as mean±SD (n=6 in each group except [F] and [H]). BAX, bcl-2–associated X protein; DAPI, 4′,6-diamidino-2-phenylindole; Edu, 5-ethynyl-2′- deoxyuridine; H2O2, hydrogen peroxide; HPEC, human peritubular endothelial cell.

Hydrogen peroxide (H2O2) treatment (150 µM for 24 hours) in HPECs downregulated expression of glycolysis enzymes (including PFKFB3) and lowered glycolytic rate (termed GlycoPER) and glucose-induced extracellular acidification rate (ECAR), indicating a lower glycolysis activity (Figure 3, B–D, and Supplemental Figure 6, A–E).

H2O2 treatment further reduced cellular ATP contents (Figure 3E) and dissociated the PFKFB3-cyclin dependent kinase 4 (CDK4) complex42,43 that regulated the ubiquitination and expression of G1/S transition dominator CDK4 (Supplemental Figures 6, F and G, and 8C). Consequently, H2O2-treated peritubular endothelial cells had a reduced percentage of cells in S-phase of the cell cycle, accompanied by decreased 5-ethynyl-2′-deoxyuridine-positive nuclei ratio and reduced migration and formation of tube-like structures (Figure 3, F and G, and Supplemental Figure 6, H and I). These H2O2-treated cells also exhibited increased apoptosis (as illustrated by flow cytometry), elevated apoptosis marker expression (cleaved caspase 3, bcl-2 associated X protein, and apoptotic protease activating factor-1), and decreased antiapoptosis marker B-cell lymphoma-2 expression (Figure 3, H and I, and Supplemental Figure 6J). Thus, excessive oxidative stress induces metabolic reprogramming in peritubular endothelial cells, specifically lowering glycolysis and inducing an antiangiogenic response.

We then analyzed the molecular mechanism responsible for H2O2-induced glycolysis blockade. Conjoint analysis of a human kidney scRNA-seq dataset, Chromatin Immunoprecipitation Enrichment Analysis database, and oxidative stress–related genes44 identified three transcript factors that are targeted by oxidative stress and meanwhile differentially expressed between CKD and normal peritubular endothelial cells (Supplemental Figure 7A). Among them, kruppel-like transcription factor 9 (KLF9) has been reported to function as a critical regulator of PFKFB3.45 Immunostaining analysis in the human kidney demonstrated upregulation of KLF9 in CKD peritubular endothelial cells (Supplemental Figure 7B). H2O2 treatment in peritubular endothelial cells upregulated KLF9 expression, whereas knockdown of KLF9 reversed the ability of H2O2 to reduce PFKFB3 expression, ECAR, and ATP levels (Supplemental Figure 7, C–E). In accordance, KLF9-deficient peritubular endothelial cells were protected from H2O2-induced antiangiogenic response, including cell proliferation (as indicated by 5-ethynyl-2′-deoxyuridine-positive nuclei ratio) and apoptosis (as indicated by expression of apoptosis markers) (Supplemental Figure 7, F–H). Together, oxidative stress–induced and KLF9-mediated suppression of glycolysis are crucial participants in the development of antiangiogenic response in peritubular endothelial cells, suggesting a role for defective peritubular endothelial cell glycolysis in microvascular rarefaction.

Ectopic Expression of PFKFB3 in Peritubular Endothelial Cells Reversed H2O2-Induced Glycolysis Blockade and Antiangiogenic Response

We further established stable HPECs overexpressing PFKFB3 by transfection with lentivirus-PFKFB3. Overexpression of PFKFB3 in peritubular endothelial cells reversed H2O2-induced decrease in ECAR and restored ATP levels (Figure 4, A and B, and Supplemental Figure 8, A and B), supporting that PFKFB3 controls peritubular endothelial cell glycolysis under CKD condition.

Figure 4.

Figure 4

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Ectopic expression of PFKFB3 in peritubular endothelial cells reversed H2O2-induced glycolysis blockade and antiangiogenic response. HPECs were transfected with LV-PFKFB3 or NC (vector). (A) Immunoblots showing upregulation of PFKFB3 in HPECs transfected with LV-PFKFB3. Unpaired t test. *P < 0.05. (B–F) Transfection of HPECs with LV-PFKFB3 restored the H2O2-induced reduction in ECAR (B), increased percentage of cells in S-phase of the cell cycle (C; n=3 in each group) and EdU-positive cells (D; scale bar: 100 μm), decreased protein levels of C-caspase-3 (E), and reduced percentage of cell apoptosis (F; n=3 in each group). One-way ANOVA followed by Bonferroni test. *P < 0.05. Data expressed as mean±SD (n=6 in each group except [C] and [F]). ECAR, extracellular acidification rate; LV-PFKFB3, lentivirus-PFKFB3; NC, negative control.

Furthermore, overexpression of PFKFB3 reversed the ability of H2O2 to induce CDK4 ubiquitination, thereby upregulating CDK4 expression (Supplemental Figure 8, C and D). Consequently, H2O2-induced reductions in S-phase cell population, and cell proliferation, migration, and tube formation were improved by PFKFB3 overexpression (Figure 4, C and D, and Supplemental Figure 8, E and F). Overexpression of PFKFB3 also suppressed H2O2-induced cleaved-caspase 3 expression and apoptosis (Figure 4, E and F). Thus, the defective PFKFB3-mediated glycolysis entails the development of an antiangiogenic response in peritubular endothelial cells in the CKD setting.

Overexpression of Endothelial PFKFB3 in CKD Kidney Attenuated Microvascular Rarefaction and Reduced Fibrotic Lesions

To determine the contribution of peritubular endothelial cell glycolysis to microvascular rarefaction and fibrosis in vivo, we generated endothelial-specific Pfkfb3 knockout mice (Pfkfb3ΔEC)25–27 by intercrossing Cdh5-Cre mice with Pfkfb3flox/flox mice (Pfkfb3WT) (Supplemental Figure 9A). PFKFB3 expression was barely detectable in peritubular endothelial cells from Pfkfb3ΔEC mice, but was maintained in other kidney cells and bone marrow cells (Figure 5A and Supplemental Figure 9B). The Pfkfb3-deficient peritubular endothelial cells exhibited reduced glucose-induced ECAR (Figure 5B), supporting a key role for PFKFB3 in regulating glycolysis of these cells.

Figure 5.

Figure 5

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Endothelial-specific deletion of PFKFB3 exacerbated microvascular rarefaction and fibrosis in a CKD kidney. Endothelial-specific PFKFB3 knockout mice (Pfkfb3ΔEC) were generated by intercrossing Cdh5-Cre mice with Pfkfb3flox/flox (Pfkfb3WT) mice. (A) Protein levels of PFKFB3 in isolated mouse peritubular endothelial cells. Unpaired t test. *P < 0.05. (B) ECAR in isolated mouse peritubular endothelial cells detected by glycolytic stress assay. Unpaired t test. *P < 0.05. (C) The peritubular microvessel density presented by immune-fluorescence staining for CD31 (green): representative images (scale bar: 100 μm) and quantitative data. One-way ANOVA followed by Bonferroni test. *P < 0.05. (D) The area of kidney fibrosis presented by Masson's trichrome staining: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA followed by Bonferroni test. *P < 0.05. (E) Protein levels of FN and Co I in kidney homogenates. One-way ANOVA followed by Bonferroni test. *P < 0.05. (F) Transcript levels of FN and Co I in kidney homogenates. One-way ANOVA followed by Bonferroni test. *P < 0.05. (G) Tubular injury assessed by HE staining: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA with Bonferroni correction. *P < 0.05. (H) Kidney inflammation assessed by immunostaining of F4/80: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA with Bonferroni correction. *P < 0.05. Data expressed as mean±SD (n=6 in each group). Co, collagen; FN, fibronectin; HE, hematoxylin-eosin; HPF, high-power field; WT, wild type.

Pfkfb3ΔEC mice were then subjected to IRI and sacrificed on day 28 after IRI. Peritubular endothelial cells from IRI-treated Pfkfb3ΔEC mice (versus IRI-treated Pfkfb3WTmice) had downregulation of proliferation genes and upregulation of apoptosis genes (Supplemental Figure 9, C and D). As expected, IRI-treated Pfkfb3ΔEC mice (versus IRI-treated Pfkfb3WTmice) exhibited reduced kidney microvessel density and oxygenation, accompanied by worsened kidney function and increased tubular injury, inflammation, and fibrosis (Figure 5, C–H, Supplemental Figure 9E, and Supplemental Table 6). Microvessel density in medulla was also reduced in IRI-treated Pfkfb3ΔEC mice, but to a greater extent than in the cortex (Supplemental Figure 9F).

We also generated kidney endothelial–specific Pfkfb3-overexpressing mice by intrarenal injection28,29 of AAV carrying Flag, Pfkfb3 and a Tie2 promoter (AAV-Pfkfb3) (Supplemental Figure 10A). Pfkfb3 mRNA levels were significantly increased in peritubular endothelial cells on days 15–21 after AAV-Pfkfb3 injection (Supplemental Figure 10B). This effect was specific to kidney endothelial cells, as Pfkfb3 levels were maintained in other kidney cells, bone marrow cells, and endothelial cells of the liver, heart, and lung (Supplemental Figure 10, B and C). Immunostaining analysis in mouse kidney confirmed that PFKFB3 was mainly expressed in endothelial cells, with a transfection rate of approximately 60% on day 21 (rare expression observed in the liver, heart, or lung) (Supplemental Figure 10, D and E).

To highlight the therapeutic potential of PFKFB3, mice were injected with AAV-Pfkfb3 on day 5 after IRI and sacrificed on day 28 (Figure 6A). Peritubular endothelial cells from IRI mice transfected with AAV-Pfkfb3 (versus negative control) exhibited upregulation of proliferation genes and downregulation of apoptosis genes (Supplemental Figure 10, F and G). This endothelial Pfkfb3 overexpression attenuated IRI-induced reduction in kidney microvessel density and oxygenation, and improved kidney function, tubular injury, inflammation, and fibrosis (Figure 6, B–G, Supplemental Figure 10H, and Supplemental Table 6). Microvessel density in the medulla was also improved by endothelial Pfkfb3 overexpression, but to a lesser extent than in the cortex (Supplemental Figure 10I).

Figure 6.

Figure 6

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Overexpression of endothelial PFKFB3 in the CKD kidney attenuated microvascular rarefaction and reduced fibrotic lesions. (A) Outline of the experimental protocol: restoration of PFKFB3 in peritubular endothelial cells in IRI mice was achieved by injecting AAV-Tie2-Pfkfb3-Flag (Pfkfb3 OEEC) or NC into the right kidney of mice 5 days after IRI. (B) The peritubular microvessel density presented by immunofluorescence staining for CD31 (green): representative images (scale bar: 100 μm) and quantitative data. One-way ANOVA followed by Bonferroni test. *P < 0.05. (C) The area of kidney fibrosis presented by Masson's trichrome staining: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA followed by Bonferroni test. *P < 0.05. (D) Protein levels of FN and Co I in kidney homogenates. One-way ANOVA followed by Bonferroni test. *P < 0.05. (E) Transcript levels of FN and Co I in kidney homogenates. One-way ANOVA followed by Bonferroni test. *P < 0.05. (F) Tubular injury assessed by HE staining: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA with Bonferroni correction. *P < 0.05. (G) Kidney inflammation assessed by immunostaining of F4/80: representative images (scale bar: 50 μm) and quantitative data. One-way ANOVA with Bonferroni correction. *P < 0.05. Data expressed as mean±SD (n=6 in each group). AAV, adeno-associated virus.

We then injected mice with AAV-Pfkfb3 10 days before UUO (Supplemental Figure 11A). Endothelial Pfkfb3 overexpression ameliorated microvascular rarefaction and fibrosis in this model (Supplemental Figure 11, B-D). Thus, our results concur in demonstrating that hypoglycolysis in peritubular endothelial cells is a critical factor leading to microvascular rarefaction and fibrosis during CKD. Maintaining proper glycolysis in peritubular endothelial cells by PFKFB3 overexpression helps to impede the development of microvascular rarefaction and fibrotic lesions.

Discussion

Microvascular rarefaction represents a common event leading to CKD progression. A fundamental unanswered question in understanding pathogenesis of microvascular rarefaction is the nature of metabolic switches that mediate peritubular endothelial cell dropout during CKD. Our results indicated a novel role for disrupted glycolysis in peritubular endothelial cells in microvascular rarefaction (illustrated in Supplemental Figure 12). We identified a hypoglycolytic metabolism in CKD peritubular endothelial cells. This was mediated by oxidative stress and induced an antiangiogenic response in these cells, inhibiting their proliferation and enhancing their apoptosis. Restoration of peritubular endothelial cell glycolysis in CKD by overexpressing PFKFB3 corrected the antiangiogenic response, thereby protecting the kidney from microvascular rarefaction and fibrotic lesions. Our data for the first time establish a fundamental role for metabolic reprogramming in peritubular endothelial cells in dictating microvessel fate during CKD. Targeting glycolysis in peritubular endothelial cells may be therapeutically beneficial in CKD.

Previous studies of microvascular rarefaction in CKD have focused on angiogenic signals.13–16 In this study, we revealed the importance of an additional pathway initiated by defective glycolysis in peritubular endothelial cells. We first presented kidney scRNA-seq data from individuals with CKD and from a mouse model of CKD.31,35 Our unbiased analysis revealed a strong correlation between downregulated glycolysis and an antiangiogenic response in peritubular endothelial cells. Because endothelial cells derive most of their energy from glycolysis,17 peritubular endothelial cells may decrease glycolysis to inhibit angiogenesis, thereby promoting microvascular rarefaction in CKD. In support, mouse peritubular endothelial cells exhibited reduced glycolysis by 7 days of IRI, preceding detectable microvascular rarefaction that were seen from day 14. Endothelial Pfkfb3 deficiency in IRI mice downregulated peritubular endothelial cell glycolysis and exacerbated microvascular rarefaction. By contrast, improvement of glycolysis by endothelial Pfkfb3 overexpression in CKD models improved microvascular rarefaction and reduced fibrosis. In the kidney, the presence of metabolic reprogramming in tubular cells as a contributor to CKD progression has been described recently.46 However, less is known about the metabolic contribution in other cell types. In this study, we identify a novel mechanism by which a reduced glycolysis in peritubular endothelial cells promotes microvascular rarefaction after injury.

Recovered microvessel density and oxygenation are hallmarks of microvascular normalization.7 Restoring glycolysis in peritubular endothelial cells promotes microvessel recovery in CKD by at least three mechanisms. First, endothelial PFKFB3 overexpression upregulated expression of CDK4, a known regulator of G1/S cell cycle transition,43,47 reversing CKD-associated cell cycle arrest. Next, endothelial PFKFB3 overexpression increased glycolysis and thereby ATP generation in CKD settings, providing energy for ATP demand processes including angiogenesis. Notably, PFKFB3 overexpression increased glycolysis in peritubular endothelial cells by approximately 14%. This enhancement in glycolysis did not affect baseline microvessel density, supporting that PFKFB3 transfection may not lead to overproliferation, but rather to normalization of peritubular endothelial cells. Finally, restoration of glycolysis increased endothelial survival by reducing apoptosis, leading to improved microvessel integrity. The combined effects of glycolysis on peritubular endothelial cell survival and proliferation make it a promising target for preventing or delaying microvascular rarefaction in CKD. This restored microvascular density improved kidney oxygen delivery, thereby orchestrating a beneficial milieu that promotes adaptive kidney repair.

The major factor investigated here to modulate peritubular endothelial cell glycolysis in CKD is oxidative stress. Oxidative stress, which is reflected by activation of NADPH oxidase in the kidney microenvironment, accompanies CKD and is an established driver of endothelial injury.12 Here, prolonged incubation of peritubular endothelial cells with H2O2, a scenario similar to CKD, impaired glycolysis and initiated an antiangiogenic response. The H2O2-induced detrimental effects were at least partially mediated by PFKFB3 because rescue of PFKFB3 expression upregulated glycolysis and reversed the antiangiogenic response. We further assigned the intracellular oxidative stress signaling underlying reduced glycolysis in these cells to a redox sensor, KLF9,48 which was upregulated by H2O2 and capable of regulating PFKFB3 gene transcription and glycolysis. Interruption of KLF9 reversed the H2O2-induced hypoglycolysis and antiangiogenic response. Thus, enhanced oxidative stress in CKD impairs PFKFB3-mediated glycolysis in peritubular endothelial cells, contributing to microvascular rarefaction.

The study has limitations. First, we did not test our findings in other public scRNA-seq datasets because few datasets have sufficient peritubular endothelial cells for analysis.49–51 Second, we overexpressed Pfkfb3 in peritubular endothelial cells by intrarenal injection of AAV-Pfkfb3 with a Tie2 promoter because of the lack of kidney/peritubular endothelial-specific Pfkfb3 transgenic mice. Third, we cannot exclude a role for myeloid PFKFB3 in our endothelial Pfkfb3 deletion or overexpression experiments. However, neither Cdh5-Cre–mediated Pfkfb3 deletion nor AAV-Tie2-Pfkfb3–mediated Pfkfb3 overexpression significantly altered the Pfkfb3 expression in bone marrow cells and kidney CD45+ immune cells (Supplemental Figures 9B and 10, B and C). Therefore, a dominant effect of myeloid PFKFB3 does not seem to play a role in our model. Finally, only male mice were used in our study. Thus, gender-specific differences in metabolic reprogramming of CKD peritubular endothelial cells cannot be addressed, and caution should be used when extrapolating to female patients.

Our studies have important clinical implications. Restoration of glycolysis in peritubular endothelial cells might be of therapeutic benefit for patients with CKD. Targeted peritubular endothelial Pfbkb3 overexpression could help to promote microvessel normalization and thereby prevent CKD progression. On the other hand, interruption of endothelial glycolysis has gained attention for its benefits in hypervascular diseases, including tumor and ocular abnormalities. Our finding that endothelial Pfkfb3 deficiency exacerbates microvascular rarefaction and fibrosis in CKD warrants future studies to determine the long-term consequences of glycolysis blockade in patients with multiple complications.

In sum, our study demonstrated a disrupted regulation of glycolysis in peritubular endothelial cells in CKD that contributed to microvascular rarefaction. Restoring the glycolysis in peritubular endothelial cells improved microvascular rarefaction and reduced fibrosis. Our findings provide a translation of the normal role of glycolysis in peritubular endothelial cells to CKD and identify this metabolic element as a candidate therapeutic target to improve microvascular rarefaction.

Supplementary Material

SUPPLEMENTARY MATERIAL
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Footnotes

Y.H., A.C., and J.L. contributed equally to this work.

See related editorial, “Metabolic Breakdown: The Hidden Role of Endothelial Glycolysis in Kidney Fibrosis,” on pages 1–3.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/E841.

Funding

F F. Hou: the National Natural Science Foundation of China (Key Program) (82330020 and 82030022), National Key R&D Program of China (2020B1111170013), 111 Plan (D18005), and Guangdong Key R&D Program (2023B1111030004). W. Cao: National Natural Science Foundation of China (82270776 and 82470768), Guangzhou Science and Technology Plan project (2024B01J1326), Natural Science Foundation of Guangdong Province (2024A1515010661), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2020J002).

Author Contributions

Conceptualization: Wei Cao, Fan Fan Hou.

Data curation: Ansheng Cong, Cailing Su, Zuoyu Hu, Yujie Huang, Jinjin Li, Hong Zhou, Zhanmei Zhou.

Formal analysis: Ansheng Cong, Yujie Huang, Jinjin Li.

Funding acquisition: Wei Cao, Fan Fan Hou.

Methodology: Ansheng Cong, Zuoyu Hu, Yujie Huang, Jinjin Li, Cailing Su, Hong Zhou, Zhanmei Zhou.

Project administration: Wei Cao, Fan Fan Hou.

Resources: Wei Cao, Fan Fan Hou, Yujie Huang, Hong Zhou, Zhanmei Zhou.

Supervision: Wei Cao, Fan Fan Hou.

Validation: Wei Cao, Ansheng Cong, Yujie Huang, Jinjin Li.

Visualization: Ansheng Cong, Zuoyu Hu, Cailing Su.

Writing–original draft: Wei Cao.

Writing–review & editing: Wei Cao, Fan Fan Hou.

Data Sharing Statement

Previously published data were used for this study. Previously published data used for this study have been referenced with their original source, which are available at https://www.zenodo.org/record/4059315 and GSE197626.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/JSN/E837, http://links.lww.com/JSN/E838, http://links.lww.com/JSN/E839, http://links.lww.com/JSN/E840.

Supplemental Methods

Supplemental Figure 1. Peritubular endothelial cells were hypoglycolytic in human CKD.

Supplemental Figure 2. Peritubular endothelial cells were hypoglycolytic in mouse models of CKD.

Supplemental Figure 3. The CKD model of IRI had progressive kidney injury within 28 days.

Supplemental Figure 4. Isolated mouse peritubular endothelial cells were hypoglycolytic in a CKD model of IRI.

Supplemental Figure 5. Isolated mouse peritubular endothelial cells were hypoglycolytic in a CKD model of UUO.

Supplemental Figure 6. H2O2-induced hypoglycolysis and antiangiogenic response in human peritubular endothelial cells.

Supplemental Figure 7. H2O2 downregulated PFKFB3 by a KLF9 pathway in human peritubular endothelial cells.

Supplemental Figure 8. Ectopic expression of PFKFB3 reversed H2O2-induced reduction in ATP levels and promoted angiogenic response in human peritubular endothelial cells.

Supplemental Figure 9. Endothelial-specific deletion of PFKFB3 exacerbated antiangiogenic response in IRI peritubular endothelial cells and reduced kidney oxygenation in a IRI kidney.

Supplemental Figure 10. Overexpression of endothelial PFKFB3 in a IRI kidney attenuated antiangiogenic response in peritubular endothelial cells and improved kidney oxygenation.

Supplemental Figure 11. Overexpression of endothelial PFKFB3 in a UUO kidney attenuated microvascular rarefaction and reduced fibrotic lesions.

Supplemental Figure 12. Schematic diagram summarizing a novel role for disrupted glycolysis of peritubular endothelial cells in the development of microvascular rarefaction in the CKD kidney.

Supplemental Table 1. Gene markers of cell clusters in scRNA-seq data of human kidney interstitium-enriched cells (https://zenodo.org/record/4059315).

Supplemental Table 2. GSEA results of pathways differentially regulated in CKD peritubular endothelial cells compared with normal peritubular endothelial cells (https://zenodo.org/record/4059315).

Supplemental Table 3. GSEA results of metabolic pathways in CKD peritubular endothelial cells compared with normal peritubular endothelial cells (https://zenodo.org/record/4059315).

Supplemental Table 4. Patients' characteristics in Figure 1, G and H.

Supplemental Table 5. Gene markers of cell clusters in scRNA-seq data of mouse kidney cells (GSE197626).

Supplemental Table 6. Kidney function in IRI mice.

Supplemental Table 7. List of primers used for real-time PCR.

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Associated Data

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Supplementary Materials

SUPPLEMENTARY MATERIAL
jasn-36-019-s001.xlsx (24.5KB, xlsx)
jasn-36-019-s002.xlsx (1,015.8KB, xlsx)
jasn-36-019-s003.xlsx (18.6KB, xlsx)
jasn-36-019-s004.pdf (6.3MB, pdf)

Data Availability Statement

Previously published data were used for this study. Previously published data used for this study have been referenced with their original source, which are available at https://www.zenodo.org/record/4059315 and GSE197626.

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