Dicarbonyl-modified lipoproteins contribute to proteinuric kidney injury

Research ArticleNephrology Open Access | 10.1172/jci.insight.161878

Jianyong Zhong,1,2 Hai-Chun Yang,1,2 Elaine L. Shelton,1 Taiji Matsusaka,3 Amanda J. Clark,1 Valery Yermalitsky,4 Zahra Mashhadi,4 Linda S. May-Zhang,4 MacRae F. Linton,5 Agnes B. Fogo,1,2,5 Annet Kirabo,4,6 Sean S. Davies,4 and Valentina Kon1

1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

Find articles by May-Zhang, L. in: JCI | PubMed | Google Scholar

1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

Find articles by Fogo, A. in: JCI | PubMed | Google Scholar

1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

Find articles by Kirabo, A. in: JCI | PubMed | Google Scholar |

1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

Find articles by Davies, S. in: JCI | PubMed | Google Scholar |

1Department of Pediatrics and

2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Institute of Medical Sciences and Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan.

4Department of Pharmacology, Division of Clinical Pharmacology,

5Department of Medicine, and

6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Valentina Kon, Vanderbilt University Medical Center, Medical Center North C-4204, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.322.7416; Email: valentina.kon@vumc.org. Or to: Hai-Chun Yang, Vanderbilt University Medical Center, Medical Center North C-4209, 1161 21st Avenue South, Nashville, Tennessee 37232-2584, USA. Phone: 615.343.0110; Email: haichun.yang@vumc.org.

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Published September 20, 2022 - More info

Published in Volume 7, Issue 21 on November 8, 2022
JCI Insight. 2022;7(21):e161878. https://doi.org/10.1172/jci.insight.161878.
© 2022 Zhong et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 20, 2022 - Version history
Received: May 31, 2022; Accepted: September 13, 2022 View PDF Abstract

Lipoprotein modification by reactive dicarbonyls, including isolevuglandin (IsoLG), produces dysfunctional particles. Kidneys participate in lipoprotein metabolism, including tubular uptake. However, the process beyond the proximal tubule is unclear, as is the effect of kidney injury on this pathway. We found that patients and animals with proteinuric injury have increased urinary apolipoprotein AI (apoAI), IsoLG, and IsoLG adduct enrichment of the urinary apoAI fraction compared with other proteins. Proteinuric mice, induced by podocyte-specific injury, showed more tubular absorption of IsoLG-apoAI and increased expression of lipoprotein transporters in proximal tubular cells compared with uninjured animals. Renal lymph reflects composition of the interstitial compartment and showed increased apoAI and IsoLG in proteinuric animals, supporting a tubular cell-interstitium-lymph pathway for renal handling of lipoproteins. IsoLG-modified apoAI was not only a marker of renal injury but also directly damaged renal cells. IsoLG-apoAI increased inflammatory cytokines in cultured tubular epithelial cells (TECs), activated lymphatic endothelial cells (LECs), and caused greater contractility of renal lymphatic vessels than unmodified apoAI. In vivo, inhibition of IsoLG by a dicarbonyl scavenger reduced both albuminuria and urinary apoAI and decreased TEC and LEC injury, lymphangiogenesis, and interstitial fibrosis. Our results indicate that IsoLG-modified apoAI is, to our knowledge, a novel pathogenic mediator and therapeutic target in kidney disease.

Graphical Abstractgraphical abstract Introduction

The beneficial effects of HDL have been extensively studied in cells involved in atherosclerosis, such as macrophages and endothelial cells. Recent emphasis has shifted from the quantity of HDL to the functionality of the HDL particles, which is affected by structural and chemical changes transforming the lipoproteins into harmful molecules (19). A key mechanism in the transformation involves reactive carbonyls formed from peroxidation of arachidonic acid that selectively form adducts with primary amines such as lysyl residues of proteins. Members of this family include malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), 4-oxo-neonenal (4-ONE), and isolevuglandin (IsoLG). HDL modified by these reactive dicarbonyls causes toxicity that impairs the antiatherogenic and the beneficial extracardiac properties of HDL (1013). Kidney disease alters the composition and functionality of HDL thought to contribute to increased atherosclerotic cardiovascular risk but also to progressive kidney damage (14).

Because of their hydrophobic nature, lipids associate with proteins, most often albumin, which in the kidney disease setting can lead to lipid deposition throughout the renal parenchyma. While albuminuria has long been viewed as a mechanism of kidney damage, accumulating evidence indicates it is not albumin itself, but rather the fatty acid cargo that is the critical factor driving albuminuric cytotoxicity (1517). Little is known about other lipids and proteins that may be involved in kidney damage. Recently, we found that albuminuric children with various types of kidney injuries have increased urinary apolipoprotein AI (apoAI), the most abundant protein in the HDL particle, and biopsy samples showed prominent expression of apoAI in the proximal tubules (18). These findings are consistent with the understanding that proximal tubules reabsorb apoAI and HDL (19); however, what is currently unknown is the disposition of lipoproteins beyond the proximal tubule. This is an important biological concern since lipoprotein clearance from the interstitium throughout the body occurs primarily through the lymphatic network, which is increasingly recognized to be a significant modulator in development and progression of many diseases (12, 2022). Whether this occurs in the kidney is unknown. Also unknown is whether disease-modified lipoproteins modulate lymphatic vessel dynamics. Finally, although kidney disease is a high-lipid peroxidation condition that disrupts the circulating levels and composition of HDL (13), the role of reactive carbonyls in injury-induced lymphatic vessel response has not been examined. Our data identify apoAI modified by reactive dicarbonyls as a potentially novel mechanism of kidney damage. This injury involves stimulation of inflammatory factors by the proximal tubule, oxidative stress in lymphatic endothelial cells (LECs), and impaired contractility of lymphatic vessels that can promote stagnation of harmful molecules and cells in the kidney interstitium.

Results

Patients and animals with kidney injury have increased urinary apoAI and IsoLG. Kidney injury increases oxidant stress and generation of lipid aldehydes, e.g., IsoLG. ApoAI is particularly susceptible to covalent modification by this aldehyde (23). Therefore, to determine if kidney injury affects urinary IsoLG in humans, we examined urine samples from our previously characterized cohort of patients with a spectrum of kidney injuries and high urinary excretion of apoAI and compared them with healthy control (CTL) participants (Figure 1A) (18). The clinical demographics for the participants have been reported and are shown in Supplemental Table 1 (supplemental material available online with this article; https://doi.org/10.1172/jci.insight.161878DS1). Subjects with elevated levels of urinary apoAI have higher urinary IsoLG-Lys than matched CTLs, assessed by mass spectrometry (Figure 1B). IP experiments confirmed that the apoAI fraction was markedly enriched with IsoLG adducts compared with the fraction containing all other urinary proteins (Figure 1C).

Kidney injury patients and animals have increased urinary apoAI and IsoLG.Figure 1

Kidney injury patients and animals have increased urinary apoAI and IsoLG. (A) The IsoLG adducts in urine and the IP apoAI fraction of patients (PTs) and matched CTLs were measured by mass spectrometry. (B) Compared with CTLs (n = 19), PTs (n = 50) had higher urinary IsoLG. (C) Urinary apoAI contained more IsoLG adducts versus total urinary proteins (n = 8). (D) Transgenic mice (NEP25) with albuminuria (ACR) following injection of toxin (LMB2) had significantly increased urinary apoAI and IsoLG versus WT mice. (E) Albuminuric puromycin-injected rats (PAN) had higher urinary apoAI and IsoLG versus CTL. n = 6–12 mice or rats/group. Data represented with box-and-whisker plot. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Statistical significance determined by Wilcoxon rank sum test. *P < 0.05, **P < 0.01, ***P < 0.001.

To determine how kidney injury affects renal handling of apoAI, we investigated several models of kidney injury in mice and rats. First, we studied transgenic mice that express human CD25 selectively in podocytes (NEP25). These mice develop primary podocyte injury and progressive albuminuria upon injection of anti-Tac (Fv)-PE38 (LMB2), an immunotoxin with specific binding to human CD25 (24). After injury was induced in NEP25 mice, confirmed by increased albuminuria (albumin/creatinine ratio [ACR]), urinary apoAI was increased compared with normal WT mice (Figure 1D). Paralleling the results in patients, urinary levels of IsoLG adducts were increased in albuminuric NEP25 mice compared with WT mice (Figure 1D). We also studied the puromycin aminonucleoside nephropathy (PAN) model, a well-known rat model of minimal change disease and focal and segmental glomerulosclerosis in humans (25). Albuminuric PAN rats had higher urinary apoAI and significant elevation in urinary IsoLG-Lys versus CTL rats (Figure 1E).

Kidneys from albuminuria NEP25 mice showed greater apoAI within the glomerular capillary tuft versus WT mice (Figure 2A). Further, apoAI expression was more prominent in proximal tubules (containing brush border) than in distal tubular cells (Figure 2A). To examine renal lipoprotein handling, we also studied tubular injury models without glomerular injury. Diphtheria toxin+ (DT+) transgenic mice express the DT receptor (DTR) only on proximal TECs (26) and develop acute kidney injury with proximal tubular injury following DT injection with increased ACR, urinary kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) (Figure 2B). These DT+ mice showed significantly higher urinary apoAI versus DT– after DT injection (Figure 2B). Urinary IsoLG of DT+ mice was not significantly increased compared with DT– mice (6.72 ± 1.12 vs. 5.12 ± 0.70 pmol/creatinine, P NS), indicating urinary IsoLG is derived primarily from the glomerular filtrate. In contrast, the folic acid (FA) model of distal tubular injury evidenced by increased NGAL, but not increased KIM-1, showed no change in urinary apoAI (Figure 2C). The critical features of human and animal models are shown in Supplemental Table 2. These results suggest that kidney injury including glomerular and proximal, but not distal, tubular injury results in increased urinary apoAI and associated IsoLG.

Distinct tubular handling of apoAI.Figure 2

Distinct tubular handling of apoAI. (A) Proteinuric NEP25 kidneys showed greater apoAI within the glomerular capillary tuft (blue arrow) versus WT. ApoAI costaining with periodic acid–Schiff showed greater apoAI expression in proximal tubular cells (red arrow) than distal tubular cells (red arrowhead). Scale bar: 50 μm. (B) Proximal tubular injury following injection of DT+ increased ACR, urinary KIM-1, NGAL, and urinary apoAI versus DT– mice. (C) FA-injected distal tubular injury mice had increased ACR and NGAL, but not KIM-1, and no change in urinary apoAI excretion. n = 6–12 mice/group. Data represent box-and-whisker plot. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Statistical significance determined by Wilcoxon rank sum test. *P < 0.05, **P < 0.01, ***P < 0.001.

Renal accumulation of IsoLG-apoAI reflects avid tubular uptake and compromised removal by renal lymphatics. We next assessed tubular handling of apoAI and IsoLG-apoAI. ApoAI colocalized with megalin-positive cells (Figure 3A), corroborating megalin’s established role in apoAI reabsorption. However, while megalin is key in tubular uptake of apoAI, NEP25/megalin KO mice also showed apoAI in the proximal tubules (Figure 3A). Furthermore, NEP25/megalin mosaic KO mice exhibited apoAI in the megalin-deficient portions of proximal tubules (Figure 3A) (27). These findings suggest a megalin-independent pathway for apoAI reabsorption. Examination of the pathways involved in apoAI transport is illustrated in Figure 3B. Quantitation showed increased expression of ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B member 1 (SRBI) in tubules of proteinuric NEP25 mice versus WT mice. ATP-binding cassette subfamily G member 1 (ABCG1) expression was subtle and appeared unaffected by the proteinuric injury (Figure 3B).

Pathways for tubular handling of apoAI.Figure 3

Pathways for tubular handling of apoAI. (A) Proximal tubular cells (megalin-positive, green) of NEP25 proteinuric kidneys absorbed apoAI (red, top panel). Uptake of apoAI (red) also occurred independently of megalin, illustrated in NEP25/megalin KO (middle panel) and NEP25/megalin mosaic KO mice (bottom panel). (B) IHC staining showed greater expression of lipoprotein transporters ABCA1 and SRBI, but not ABCG1, in NEP25 mice versus WT. Scale bar: 50 μm.

Cultured human renal proximal tubule cells (TECs) with low megalin expression, exposed to IsoLG-apoAI versus apoAI, showed upregulated expression of ABCA1 and SRBI mRNA (ABCA1: 1.98 ± 0.13 vs. 1.21 ± 0.13; and SRBI: 1.44 ± 0.09 vs. 1.12 ± 0.10, both, P < 0.05). In parallel, both ABCA1 and SRBI proteins were significantly elevated by IsoLG-apoAI versus apoAI (Figure 4A). Functional assessment revealed that TECs accumulated more IsoLG-apoAI than apoAI (Figure 4B). To confirm ABCA1 and SRBI contribution to apoAI reabsorption, we used siRNA to knock down TEC expression of ABCA1 and SRBI. Knockdown of ABCA1 and SRBI was successful, with a greater than 80% reduction in ABCA1 and SRBI protein levels. Knockdown of ABCA1 or SRBI reduced TEC uptake of apoAI but not IsoLG-apoAI (Figure 4, C and D). To examine the possibility of a compensatory response, we knocked down 1 pathway and checked the effects on the other pathway. Unlike apoAI, IsoLG-apoAI increased SRBI expression after ABCA1 KD. Similarly, IsoLG-apoAI caused increased ABCA1 expression after SRBI KD (Figure 4, E and F). Knockdown of both ABCA1 and SRBI reduced TEC uptake of IsoLG-apoAI (Figure 4G). Together, these in vivo and in vitro studies suggest that proteinuric injury upregulates tubular expression of ABCA1 and SRBI, which augments reabsorption of filtered IsoLG-apoAI versus apoAI.

Distinct tubular uptake of apoAI versus IsoLG-apoAI.Figure 4

Distinct tubular uptake of apoAI versus IsoLG-apoAI. (A) Cultured TECs exposed to IsoLG-apoAI showed higher expression of ABCA1 and SRBI versus apoAI. (B) TECs took up more IsoLG-apoAI versus apoAI. Scale bar: 50 μm. Knockdown of either ABCA1 or SRBI (C and D) reduced TEC uptake of apoAI but not IsoLG-apoAI. (E) In TECs exposed to apoAI, knockdown of ABCA1 or SRBI decreased expression of the other transporter. (F) In TECs exposed to IsoLG-apoAI, ABCA1 knockdown significantly increased SRBI expression. SRBI siRNA significantly increased ABCA1 expression. (G) Knockdown of both ABCA1 and SRBI reduced cellular uptake IsoLG-apoAI. In vitro, experiments were performed independently 3 times with 3 wells per treatment. Data represent mean ± SEM. Overall statistical difference determined by Kruskal-Wallis test and pairwise difference by Wilcoxon rank sum test followed by Bonferroni correction on P values. *P < 0.05, **P < 0.01, ***P < 0.001.

Since lymphatic vessels are the primary pathway for transporting lipoproteins from the peripheral interstitial space, we next examined the role of renal lymphatics in conveying lipoproteins taken up by tubules into the renal interstitium (28). ApoAI localized in podoplanin-positive lymphatic vessels, which were more prominent in NEP25 mice compared with WT mice (Figure 5A). To directly ascertain the composition of renal lymph, we used our rat proteinuric injury model where lymph collection is more accessible. Renal lymph of PAN rats had higher levels of apoAI versus CTL (Figure 5B), complementing the conspicuous colocalization of apoAI within renal lymphatic vessels observed in NEP25 mice (Figure 5A). IsoLG levels were higher in renal lymph of PAN rats (Figure 5C). The lymph nodes downstream from the kidney in PAN rats contained higher IsoLG versus CTL animals (Figure 5D). Next, we compared the renal lymphatic vessel function in PAN versus CTL rats. PAN rats had increased renal lymph flow (Figure 5E).

Renal transport of apoAI and IsoLG-apoAI involves lymphatic vessels.Figure 5

Renal transport of apoAI and IsoLG-apoAI involves lymphatic vessels. (A) ApoAI (green) immunostaining was increased in podoplanin-positive (LEC marker, red) lymphatic vessels of proteinuric NEP25 versus WT mice. Scale bar: 50 μm. (B) Renal lymph of proteinuric PAN rats had increased apoAI versus CTL. (C and D) Proteinuric PAN rats had increased IsoLG adducts in renal lymph and renal lymph nodes versus CTL. (E) Renal lymphatic flow in PAN was higher versus CTL rats. n = 5–8 rats/group. Data represent box-and-whisker plot. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Statistical significance determined by Wilcoxon rank sum test. * P < 0.05, **P < 0.01.

To determine how normal versus injured kidneys handle IsoLG-modified apoAI versus apoAI, we injected fluorescent apoAI (labeled with red dye) and IsoLG-apoAI (labeled with green dye) into WT and proteinuric NEP25 mice. In all kidneys, the renal apoAI change between 30 and 180 minutes was greater than the change in IsoLG-apoAI, indicating more rapid clearance of apoAI versus IsoLG-apoAI (Figure 6A). Comparison of WT and proteinuric kidneys revealed similar clearance of apoAI. In contrast, kidney clearance of IsoLG-apoAI was delayed in both WT and NEP25 kidneys versus apoAI (Figure 6B). The clearance of IsoLG-apoAI was especially delayed in NEP25 kidneys, which showed accumulated IsoLG-apoAI 3 hours after injection (Figure 6, B and C). These results demonstrate that IsoLG-apoAI is cleared more slowly than unmodified apoAI and that the delay in renal clearance is accentuated in proteinuric kidneys.

Proteinuric NEP25 mice show accumulation of IsoLG-apoAI versus apoAI.Figure 6

Proteinuric NEP25 mice show accumulation of IsoLG-apoAI versus apoAI. (A) Kidney clearance of fluorescence-labeled apoAI (red) was more rapid compared with IsoLG-apoAI (green). (B) Fluorescence-labeled apoAI (red) was similarly cleared by NEP25 and WT mice. In contrast, fluorescence-labeled IsoLG-apoAI (green) persisted in NEP25 kidneys versus WT 3 hours after injection. (C) Representative kidney images of fluorescent apoAI (red) and IsoLG-apoAI (green). Scale bar: 50 μm. n = 4 mice/group. Data represent box-and-whisker plot. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Statistical significance determined by Kruskal-Wallis test and Wilcoxon rank sum test on P values. * P < 0.05, ***P < 0.001.

Differential effects of apoAI versus IsoLG-apoAI on TECs, LECs, and lymphatic vessels. To determine whether apoAI/IsoLG-apoAI is a contributor to kidney injury, TECs were exposed to apoAI or IsoLG-apoAI. IsoLG-apoAI increased TEC gene expression of KIM-1 (HAVCR1), a marker of tubular injury, compared with apoAI (Figure 7A). IsoLG-apoAI also increased markers of inflammation, including NLR family pyrin domain containing 3 (NLRP3), IL-1, and IL-6 gene expression (Figure 7A). Cultured LECs exposed to IsoLG-apoAI showed higher sphingosine kinase 2 (SPHK2) and decreased sphingolipid transporter 2 (SPNS2) expression (Figure 7B), 2 key regulators of sphingosine-1-phosphate (S1P) production, which in turn stimulates proinflammatory cytokines (29). S1P levels in LECs and cellular supernatant were also elevated by IsoLG-apoAI versus apoAI (cells: 55.84 ± 8.90 vs. 32.74 ± 3.11; and supernatant: 146.35 ± 25.01 vs. 64.40 ± 1.18 ng/mg, respectively, both P < 0.05). LECs expressed more inflammatory factor genes, NLRP3, IL-1, and IL-6, when exposed to IsoLG-apoAI versus apoAI (Figure 7C). Thus, absorption of IsoLG-apoAI by epithelial cells promoted TEC/LEC injury and inflammation.

ApoAI and IsoLG-apoAI exert distinct effects on proximal TECs and LECs.Figure 7

ApoAI and IsoLG-apoAI exert distinct effects on proximal TECs and LECs. (A) Cultured TECs exposed to IsoLG-apoAI increased KIM-1 (HAVCR1), NLRP3, IL-1, and IL-6 gene expression versus unmodified apoAI. (B) Cultured LECs exposed to IsoLG-apoAI increased SPHK2 mRNA and reduced SPNS2 mRNA versus apoAI. (C) LECs exposed to IsoLG-apoAI increased NLRP3, IL-1, and IL-6 gene expression versus apoAI. In vitro, experiments were performed independently 3 times with 3 wells per treatment. Data represent mean ± SEM. For more than 2 groups, statistical significance was determined by Kruskal-Wallis test followed by Wilcoxon rank sum tests and Bonferroni correction on P values. *P < 0.05, **P < 0.01, ***P < 0.001.

Renal TECs produce VEGF-C, a growth factor that promotes lymphangiogenesis (30). IsoLG-apoAI significantly increased expression of VEGF-C (VEGFC) gene and protein in cultured TECs (Figure 8A). In LECs, IsoLG-apoAI increased proliferation compared with apoAI (Figure 8B). While apoAI decreased LEC migration, IsoLG-apoAI restored this parameter (Figure 8C). Complementing these results, in vivo studies showed greater immunostaining of VEGF-C in TECs of NEP25 mice versus WT mice (Figure 8D). PAN proteinuric rats had increased VEGF-C in renal lymph versus CTL animals (Figure 8E). In line with greater tubular production in VEGF-C, the renal lymphatic network of injured animals expanded. Lymphangiogenesis, quantitated by podoplanin staining, was significantly increased in both NEP25 mice and PAN rats compared with normal animals (Figure 8, F and G). Another LEC marker, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), also increased (WT: 1.31 ± 0.25 vs. NEP25: 2.40 ± 0.37 no./mm2, P < 0.05). These results demonstrate that IsoLG-apoAI promotes lymphangiogenesis by directly stimulating LEC proliferation and indirectly by activation of TEC generation of VEGF-C.

Distinct effects of apoAI versus IsoLG-apoAI on lymphangiogenesis.Figure 8

Distinct effects of apoAI versus IsoLG-apoAI on lymphangiogenesis. (A) Cultured TECs exposed to IsoLG-apoAI expressed more VEGFC mRNA and produced more VEGF-C protein versus apoAI. (B) IsoLG-apoAI increased LECs proliferation versus apoAI. (C) IsoLG-apoAI increased LECs migration versus apoAI. (D) VEGF-C expression by immunostaining was increased in TECs in NEP25 versus WT mice. Scale bar: 50 μm. (E) VEGF-C in PAN renal lymph was higher versus CTL. (F) NEP25 kidneys showed increased interstitial podoplanin expression versus WT mice. Scale bar: 50 μm. (G) PAN injured rats showed increased interstitial podoplanin expression versus CTL. Scale bar: 100 μm. In vitro, experiments were performed independently 3 times with 3 wells per treatment. n = 4–8 mice or rats/group. Data represent mean ± SEM for in vitro study and box-and-whisker plot for in vivo study. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). For 2 independent groups, statistical significance determined by Wilcoxon rank sum test. For more than 2 groups, statistical significance determined by Kruskal-Wallis test followed by Wilcoxon rank sum tests and Bonferroni correction on P values. *P < 0.05, **P < 0.01, ***P < 0.001.

In ex vivo studies, isolated renal lymphatic vessels from rats showed that IsoLG-apoAI caused a more prominent change in contraction frequency, end-diastolic diameter (EDD), end-systolic diameter (ESD), and amplitude of contraction versus unmodified apoAI (Figure 9). Taken together, these results support the concept that proteinuric injury-driven renal accumulation of IsoLG-apoAI activates TECs, LECs, and lymphatic dynamics. The activated TECs and LECs produce more potentially harmful inflammatory cytokines and lymphangiogenic growth factors.

Distinct effects of apoAI versus IsoLG-apoAI on renal lymphatic vessel dynaFigure 9

Distinct effects of apoAI versus IsoLG-apoAI on renal lymphatic vessel dynamics. (A) Renal lymphatic vessels were isolated, cannulated, and mounted in a perfusion chamber for pressure myography assays. Vasodynamic changes caused by unmodified apoAI (orange) are shown by the interrupted lines to the left of the baseline value, and changes caused by IsoLG-apoAI (purple) are shown by the solid lines to the right of the baseline value. Compared with the modest change with apoAI, IsoLG-apoAI caused a more prominent change in contraction frequency (B), EDD (C), ESD (D), and contraction amplitude (E). n = 8 rats/group. Statistical significance determined by Wilcoxon signed rank test. *P < 0.05, **P < 0.01, ***P < 0.001.

Scavenging IsoLG lessens kidney injury. Results in TEC

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