Oxysterol-binding protein-like 7 deficiency leads to ER stress-mediated apoptosis in podocytes and proteinuria

Abstract

Chronic kidney disease (CKD) is associated with renal lipid dysmetabolism among a variety of other pathways. We recently demonstrated that oxysterol-binding protein-like 7 (OSBPL7) modulates the expression and function of ATP-binding cassette subfamily A member 1 (ABCA1) in podocytes, a specialized type of cell essential for kidney filtration. Drugs that target OSBPL7 lead to improved renal outcomes in several experimental models of CKD. However, the role of OSBPL7 in podocyte injury remains unclear. Using mouse models and cellular assays, we investigated the influence of OSBPL7 deficiency on podocytes. We demonstrated that reduced renal OSBPL7 levels as observed in two different models of experimental CKD are linked to increased podocyte apoptosis, primarily mediated by heightened endoplasmic reticulum (ER) stress. Although as expected, the absence of OSBPL7 also resulted in lipid dysregulation (increased lipid droplets and triglycerides content), OSBPL7 deficiency-related lipid dysmetabolism did not contribute to podocyte injury. Similarly, we demonstrated that the decreased autophagic flux we observed in OSBPL7-deficient podocytes was not the mechanistic link between OSBPL7 deficiency and apoptosis. In a complementary zebrafish model, osbpl7 knockdown was sufficient to induce proteinuria and morphological damage to the glomerulus, underscoring its physiological relevance. Our study sheds new light on the mechanistic link between OSBPL7 deficiency and podocyte injury in glomerular diseases associated with CKD, and it strengthens the role of OSBPL7 as a novel therapeutic target.

NEW & NOTEWORTHY OSBPL7 and ER stress comprise a central mechanism in glomerular injury. This study highlights a crucial link between OSBPL7 deficiency and ER stress in CKD. OSBPL7 deficiency causes ER stress, leading to podocyte apoptosis. There is a selective effect on lipid homeostasis in that OSBPL7 deficiency affects lipid homeostasis, altering cellular triglyceride but not cholesterol content. The interaction of ER stress and apoptosis supports that ER stress, not reduced autophagy, is the main driver of apoptosis in OSBPL7-deficient podocytes.

INTRODUCTION

Chronic kidney disease (CKD) is a growing public health concern, with an increasing incidence and prevalence rate globally (1). CKD refers to the gradual loss of kidney function over time that can lead to a number of serious health complications and is associated with high healthcare costs (2, 3).

Oxysterol-binding proteins (OSBPs) are a family of proteins that bind and exchange phosphatidylinositol-4-phosphate (PI4P) and cholesterol between cellular membranes such as the ER and the Golgi apparatus (4, 5). Their function is highly dependent on PI4P concentration (6, 7). OSBPs extract PI4P from the trans-Golgi network (TGN) and deliver it to the ER, where a specific phosphatase, Sac1, hydrolyses PI4P into phosphatidylinositol (PI) (8). This transfer establishes a PI4P gradient across the ER-Golgi interface, driving the cholesterol/PI4P exchange and hence facilitating lipid transport (9, 10). Research suggests that the disruption of this PI4P gradient or balance can have a significant impact on cellular processes. For example, depletion of PI4P can impair the formation of autophagosomes (11) and it can prevent proper folding of proteins at the ER membrane (12–14). These observations suggest that impaired OSBP function interrupts crucial cellular processes such as autophagy and proper lipid homeostasis and may cause ER stress, thereby contributing to pathological conditions such as various podocytopathies, CKD, liver disease, diabetic kidney disease, and acute kidney injury (AKI) (15–27).

OSBPs can also bind lipids other than oxysterols, including phosphoinositide and phosphatic acid (28). In Chinese Hamster Ovary (CHO) cells, overexpression of OSBP leads to decreased cholesteryl ester synthesis and acyl-CoA:cholesterol acyltransferase (ACAT) activity (29). Thus, OSBPs also play an essential role in cholesterol homeostasis and regulation of the lipid composition of cellular membranes, including ER membranes (5). In support, we recently demonstrated that in podocytes, oxysterol-binding protein-like 7 (OSBPL7) modulates the expression and function of ATP-binding cassette subfamily A member 1 (ABCA1), an enzyme important in cholesterol efflux (4), and that small molecule inducers of ABCA1 binding to OSBPL7 can protect from experimental models of CKD. Others demonstrated that the presence of OSBPs at the ER is necessary for ER protein quality control (30), suggesting that OSBPL7 dysfunction or deficiency could play a role in ER stress (31).

The ER is a key organelle involved in the synthesis, folding, and posttranslational modification of proteins, as well as in lipid metabolism and calcium homeostasis. ER stress arises when the protein-folding capacity of the ER is overwhelmed, leading to the accumulation of misfolded proteins within the ER lumen (32). This triggers a complex adaptive response, known as the unfolded protein response (UPR), which aims to restore ER homeostasis by enhancing the protein-folding capacity of the ER, promoting the degradation of misfolded proteins, and reducing overall protein synthesis. Growing evidence suggests that prolonged or severe ER stress can lead to podocyte apoptosis, contributes to the loss of podocytes, and the disruption of the glomerular filtration barrier (20, 23, 32–38). The goal of this study was to investigate if OSBPL7 dysfunction or deficiency causes ER stress and subsequent podocyte damage, thereby contributing to the progression of kidney disease.

MATERIALS AND METHODS

Collection and Analysis of Kidney Cortex Samples From Col4a3^−/− and db/db Mice

Archival tissue from two mouse models of progressive kidney disease were used for this study: Col4a3^−/− mice (RRID:IMSR_JAX:002908), a mouse model for progressive glomerular disease of nonmetabolic origin and db/db mice (RRID:IMSR_JAX:000697), a mouse model of glomerular disease of metabolic origin. Mice were housed, maintained, and cared for in strict adherence to the guidelines stipulated by the University of Miami Medical School’s Institutional Animal Care and Use Committee. This protocol ensures ethical and humane treatment of the animals, reflecting a commitment to responsible conduct throughout the duration of the study. The alignment with these principles reinforced the scientific integrity of the research and the validity of the results derived from these animal models. Col4a3^−/− and Col4a3^+/+ mice (two male and one female mice/group) were euthanized at 8 wk, a timepoint at which these mice developed signs of renal failure, and kidney cortices were collected. Similarly, kidney cortices (db/db and db/+) were collected at 18 wk (two male and two female mice/group) when db/db mice had persistent proteinuria. The characterization of these mouse models has been previously reported by our laboratory (39, 40).

Generation of Stable OSBPL7-Deficient Podocytes

Stable OSBPL7-deficient mouse podocytes were generated using a lentiviral vector containing siRNA targeting OSBPL7, purchased from Abm (Cat. No. iV035620). As previously described, podocytes were cultured in RPMI-1640 medium supplemented with 10% FBS (41). Immortalized mouse podocytes were then infected with the virus according to manufacturer’s instructions. Western blot analysis was performed to confirm OSBPL7 knockdown.

Overexpression of OSBPL7 and Generation of Deletion Constructs

A plasmid containing the full-length human OSBPL7 cDNA was obtained from Addgene (Plasmid No. 127235). Deletion of the FFAT motif of OSPBL7 was accomplished by PCR mutagenesis, using two primers with the following sequence: 5′- TCATCAGTATCTCGGCCTGCCATGCAG-3′ (OSBPL7-FP-deletion) and 5′- CCGAGATACTGATGAAGCGGAAGCCTCG-3′ (OSBPL7-RP-deletion) and Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific, F531L) Undifferentiated podocytes were transfected with either full length or OSBPL7, in which the FFAT motif [FFAT is an acronym for two phenylalanines (FF) in an acidic tract] was deleted when they reached 60% confluency. Transfection was performed according to published protocols (42). In brief, podocytes were transfected with 3-μg plasmid using Fugene 6 transfection reagent in OptiMEM. Podocytes were thermoshifted to 37°C to induce differentiation for 5 days before further experimental procedures.

Western Blot Analysis

Protein lysates of scOSBPL7 and siOSBPL7 podocytes were prepared and subjected to Western blot analysis using primary antibodies against OSBPL7 (Sigma, HPA036076, 1:1,000), and ER stress markers, including protein kinase RNA-like ER kinase (PERK; Cell Signaling, No. 5683, 1:1,000), inositol-requiring enzyme 1α (IRE1α; Cell Signaling, No. 3294, 1:1,000), protein disulfide isomerase (PDI; Cell Signaling, No. 3501, 1:1,000), phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK; Cell Signaling, No. 4668, 1:1,000), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK; Cell Signaling, No. 67096, 1:1,000), binding immunoglobulin protein (BiP; Cell Signaling, No. 3177, 1:1,000), microtubule-associated protein 1 A/1B-light chain 3-II and microtubule-associated protein 1 A/1B-light chain 3-I (LC3-II/LC3-I; Novus Bio, NB600-1384SS, 1:1,000), Golgi-associated ATPase enhancer of 16 kDa (GATE-16; Cell Signaling, No. 26632, 1:1,000), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma, G8795, 1:3,000) and secondary antibodies for the rabbit (Cell Signaling, No. 7074, 1:1,000) or mouse (Cell Signaling, No. 7076, 1:1,000) conjugated to HRP. Levels were determined in SiOSBPL7 and scOSBPL7 podocytes.

Apoptosis Assessment in Podocytes

Apoptosis (caspase-3/7 activity) in podocytes was measured using the ApoTox-Glo Triplex Assay (Promega) according to the manufacturer’s instructions. Briefly, differentiated podocytes were treated as indicated, and caspase-3 activity was determined after 2 h at excitation = 470 nm and emission = 520 nm for the former or excitation = 400 nm and emission = 505 nm for viability and luminescence for 1 s for caspase-3 activity for the latter. In addition, cytotoxicity was measured using the ApoTox-Glo Triplex Assay according to the manufacturer’s instructions, with excitation = 485 nm and emission = 520 nm.

Immunofluorescence Imaging of OSBPL7 in Immortalized Mouse Podocytes and Mouse Kidney Cortex Sections

Kidney cortex sections (4 μm thick) from mice were prepared for immunofluorescence analysis. These sections underwent fixation using 4% PFA and sucrose in PBS and antigen retrieval in 10 mM sodium citrate buffer (pH 6.0), heated to 100°C for 10 min. After cooling to room temperature, the sections were blocked in 5% FBS in PBS for 1 h. Overnight incubation at 4°C with primary antibodies, goat anti-OSBPL7 (Novusbio, 1:100) and rabbit anti-WT1 (Abcam, 1:100), was performed. Following PBS washes, sections were incubated with fluorochrome-conjugated secondary antibodies. The sections were then mounted using Prolong Gold with DAPI. Imaging was performed using a Dragonfly confocal microscope at ×63 magnification. The number of WT1+ cells (podocytes) and the number of cells co-expressing WT1 and OSBPL7 within the glomeruli were quantified. The proportion of WT1+ OSBPL7+ cells over the total WT1+ cells in each glomerulus was calculated to assess the relative expression of OSBPL7 in podocytes. Data analysis was performed using ImageJ software (National Institutes of Health), and statistical significance was determined by appropriate statistical tests. Negative control samples prepared without primary antibodies were also imaged.

Immortalized mouse podocytes expressing OSBPL7^FL (full-length) or OSBPL7^FFAT− (deletion of the FFAT motif) were fixed in 4% paraformaldehyde (PFA) and sucrose in phosphate-buffered saline (PBS) for 20 min at room temperature. Permeabilization was achieved with 0.3% Triton X-100 in PBS for 15 min, and blocking was performed using 5% fetal bovine serum (FBS) in PBS for 1 h. The cells were incubated with a rabbit anti-OSBPL7 primary antibody (Sigma, 1:100 dilution) followed by incubation with appropriate fluorochrome-conjugated secondary antibodies. After washing in PBS, the cells were incubated in ER-ID green dye (Enzo Life Sciences), washed again in PBS, and mounted using Prolong Gold with DAPI. Imaging was conducted using a Dragonfly confocal microscope at a magnification of ×63.

Inhibitor Studies in Podocytes

In our exploration of the efficacies of KIRA6 and STF62247 in podocytes, dose-response experiments were conducted. siOSBPL7 podocytes, after being starved in RPMI-1640 medium with 0.2% FBS for 18 h, were treated for 1 h with either vehicle (DMSO), KIRA6 at concentrations of 0.25 µM and 0.5 µM, or STF62247 at concentrations of 0.625 µM and 1.25 µM. The choice of 0.5 µM for KIRA6 and 1.25 µM for STF62247 was informed by their demonstrated efficacy in these dose-response studies. The impact of these inhibitors on ER stress and autophagy was evaluated through Western blot analysis, with a specific focus on the expression levels of IRE1α and LC3. GAPDH was used as the loading control to ensure equal protein loading across all samples.

In addition, treatments with methyl-β-cyclodextrin (CD) were performed. Serum-starved human podocytes were treated with 5 mM methyl-β-cyclodextrin for 1 h, a dose established to be effective in reducing lipid droplets (LDs) in cultured podocytes (43). This treatment was crucial for investigating the impact of LD reduction on podocyte health and disease mechanisms.

Evaluation of Lipid Properties

LD numbers were determined in SiOSBPL7 and scOSBPL7 podocytes using BODIPY staining followed by confocal microscopy. LD levels were imaged and quantified using the Opera High-Content Screening System (Perkin-Elmer). The cholesterol and cholesterol ester levels were determined using the Amplex Red Cholesterol Assay Kit (Invitrogen, No. 12216) according to the manufacturer’s instructions.

Free fatty acid (FFA) content was determined using the ab65341 Free Fatty Acid Assay Kit from Abcam. The assay procedure itself was conducted with strict adherence to the kit’s instructions. The colorimetric signal was read on a SpectraMax L microplate reader (Molecular Devices) at a wavelength of 570 nm, as recommended by the kit. The concentration of FFAs in the test samples was calculated based on the standard curve data obtained from the assay, ensuring the reliability and accuracy of the results.

Triglyceride content in the podocytes was quantified using Abcam’s Triglyceride Assay Kit (ab65336). An initial cell count of 1 × 10⁷ cells was used for the assay. The assay was performed according to the manufacturer’s instructions. The resulting color change was measured on a SpectraMax L microplate reader (Molecular Devices) at a wavelength of OD of 570 nm.

Evaluation of Cell Membrane Fluidity

Cell membrane fluidity was analyzed using the Membrane Fluidity Kit (ab189819, Abcam), designed to detect changes influenced by lipid composition. Briefly, podocytes were incubated for 1 h in labeling solution (5 µM fluorescent lipid reagent with perfusion buffer, supplemented with 0.08% Pluronic F127) at 25°C in the dark. Postincubation, cells were washed twice with perfusion buffer to eliminate any unincorporated PDA. Monomer and excimer fluorescence were subsequently assessed using the SpectraMax L microplate reader (Molecular Devices) at an excitation wavelength = 350 nm and emission wavelength = 450 nm.

Assessing Glomerular Filtration and Proteinuria in Transgenic Zebrafish Embryos Using Fluorescence and Transmission Electron Microscopy

Our study used a transgenic zebrafish model, Tg[l-fabp:DBP:eGFP], that expresses a vitamin D-binding protein fused with green fluorescent protein (GFP), allowing us to assess the integrity of glomerular filtration in zebrafish. The zebrafish were bred at a temperature of 28.5°C and raised in a standard E3 solution.

Upon fertilization, zebrafish embryos were immediately injected with three types of morpholino oligonucleotides (Mo), namely, osbpl7 ATG blocker (osbpl7-Mo; Gene Tools, 5′- GATCCACTGAGTCCATTGTGAAAGT-3′), osbpl7 splice donor (osbpl7-SD; Gene Tools, 5′- GTGCCAACTGTAAAATGACTTACTT-3′), and a control group that received a scrambled morpholino (control-Mo). We obtained grayscale images of the pupils of the zebrafish and analyzed these using ImageJ software, which allowed us to quantify the fluorescence intensity in relative units of brightness.

Zebrafish Phenotype Scoring and Proteinuria Measurement

To assess the impact of osbpl7 knockdown on renal function, we used a comprehensive phenotype scoring system alongside proteinuria measurement techniques in our transgenic zebrafish model, Tg[l-fabp:DBP:eGFP]. The scoring system was based on specific criteria including the presence of edema, alterations in body shape, and changes in swimming behavior, which were indicators of potential renal impairment. Each zebrafish was scored on a scale from 1 to 4, with 1 indicating no visible phenotype and 4 indicating severe phenotype presentation.

For proteinuria measurement, we capitalized on the fluorescent properties of the transgenic model. The GFP-tagged vitamin D-binding protein expressed in the zebrafish allowed us to visually assess protein leakage into the urine by measuring the decrease in eye fluorescence. A decrease in fluorescence intensity, observed using a fluorescence microscope and quantified with Image J software, indicated a loss of high-molecular weight proteins from circulation due to a compromised glomerular filtration barrier.

Transmission Electron Microscopy Image Acquisition

For transmission electron microscopy (TEM) image acquisition, zebrafish larvae were fixed in solution D and embedded in EPON according to the manufacturer’s protocol (recipe/protocol from EMS, Hatfield, PA). Semithin (300 nm) and ultrathin (90 nm) sectioning was performed with a microtome (Reichert Austria Ultracut) and transferred to copper slit grids (EMS). Grids were stained with uranyl acetate (2%) for 30 min, then lead citrate for 15 min with three washing steps in between. The resulting TEM images were then thoroughly analyzed, focusing particularly on the glomerular structures such as podocytes, the glomerular basement membrane (GBM), and endothelium. The detailed examination aimed to identify any morphological changes, abnormalities, or signs of damage to these specific glomerular components.

The entire zebrafish component of the research project was conducted in line with the regulations and guidelines of the Mount Desert Island Biological Laboratory’s animal care committee (Institutional Animal Care and Use Committee protocol 0804), ensuring the ethical treatment of the zebrafish subjects throughout all procedures. Table 1 contains a list of commercially obtained reagents used in the study.

Table 1.

Commercially obtained reagents and kits

Experiment	Catalog Number	Description
Apoptosis assay	G809x	Caspase-Glo 3/7 Assay (Promega) was used to measure caspase activation, a hallmark of apoptosis.
Evaluation of membrane fluidity	ab189819	Membrane Fluidity Kit (Abcam) was used to analyze cell membrane fluidity.
Free fatty acid quantification	ab65341	Free Fatty Acid Assay Kit (ab65341, Abcam) was used to measure free fatty acid concentration.
Triglyceride quantification	ab65336	Triglyceride Assay Kit (ab65336, Abcam) was used to quantify triglyceride levels.
Cholesterol and cholesterol ester measurement	A12216	Amplex Red Cholesterol Assay Kit (Invitrogen) was used to measure cholesterol and cholesterol ester levels.
Lentiviral transfection	E269x	Lentiviral transfection by Fugene 6 (Promega) standard protocol was used to introduce full-length OSBPL7 into cells.
ER-ID Green assay kit	ENZ-51025-K500	ER-ID Green assay kit was used for selective endoplasmic reticulum staining.

FITC Injection in Zebrafish

To assess proteinuria, FITC-labeled 70-kDa dextran was injected into the cardiac venous sinus of zebrafish at 48 h postfertilization (hpf). The FITC solution was prepared at a concentration suitable for visible fluorescence without toxicity. The volume of injection was calibrated to ensure consistent delivery into the circulatory system. Measurements of eye fluorescence were taken at designated time points postinjection.

Assessment of Pronephros Development in Zebrafish

Transgenic WT1-gfp-expressing zebrafish were bred and raised under standard conditions. Selection of transgenic embryos was based on GFP expression indicative of pronephros development. Embryos were monitored for normal developmental milestones, with particular attention to pronephric structure and function.

RESULTS

Decreased OSBPL7 Expression in Mouse Models of Experimental CKD Is Associated With Podocyte Injury

OSBPs including OSBPL7 play an important role in regulating cholesterol homeostasis (44, 45), ER stress (46), and autophagy (47), all mechanisms that have been described to contribute to kidney disease progression (4, 17–20, 22, 24, 36–38, 48–52). We first examined OSBPL7 expression in kidney cortices of Col4a3^−/− and db/db mice, two mouse models of CKD. A significant reduction in OSBPL7 expression was observed in both Col4a3^−/− and db/db mice compared with their respective controls (Fig. 1, A and B). The immunofluorescent staining presented in Fig. 1, C and D, shows a podocyte-specific reduction of OSBPL7 expression in WT1-positive podocytes within the glomeruli of Col4a3^−/− and db/db mice compared with their respective controls. Therefore, to explore the functional implications of OSBPL7 deficiency specifically in podocytes, we subsequently developed a podocyte-specific model, leveraging siOSBPL7 podocytes. This targeted approach allows us to precisely dissect the role of OSBPL7 within podocytes, circumventing the limitations posed by erythrocyte staining and thereby providing a clearer understanding of its contribution to kidney disease pathology. We generated podocytes with stable knockdown of OSBPL7 (siOSBPL7) (Fig. 1E). These siOSBPL7 podocytes exhibited significantly increased apoptosis compared with scrambled control (scOSBPL7)-generated podocytes (Fig. 1F). Restoration of OSBPL7 levels through transfection of full-length OSBPL7 into siOSBPL7 podocytes normalized apoptosis to levels seen in scOSBPL7 podocytes. However, transfection with an OSBPL7 construct lacking the FFAT motif, which is crucial for ER binding, did not prevent apoptosis in siOSBPL7 podocytes but also did not change the localization of OSBPL7 from the ER as demonstrated by costaining with an ER marker (Fig. 1G).

Figure 1.

Decreased OSBPL7 expression in mouse models of experimental CKD is associated with podocyte injury. A and B: representative Western blot images (A) and quantification showing OSBPL7 levels in the kidney cortex of Col4a3^−/− and db/db mice compared with Col4a3^+/+ (n = 3, *P < 0.05) and db/+ (n = 4, **P < 0.001) littermates (B). C: immunofluorescence staining showcasing OSBPL7 (green) and WT1 (red) in glomeruli, with the merged image highlighting their colocalization, indicating OSBPL7's association with podocytes. D: quantification of OSBPL7 and WT1 colocalization, presented as the ratio of OSBPL7/WT1 double-positive signals to total WT1-positive cells, showing the significant decrease in OSBPL7 in the glomeruli of CKD models compared with controls (n = 10, ****P < 0.0001). E: stable OSBPL7-deficient (SiOSBPL7) podocytes were generated using siRNA. F: apoptosis levels increased in OSBPL7-deficient podocytes and returned to control levels with transfection of full-length OSBPL7 (OSBPL7^FL) but not with transfection of OSBPL7 plasmid containing a deletion of the FFAT domain (OSBPL7^FFAT−). n = 3 technical replicates. *P < 0.05 and **P < 0.01. G: immunofluorescent images of OSBPL7 (red), ER stain (green), and DAPI (blue) in siOSBPL7 cells with and without OSBPL7^FL or OSBPL7^FFAT− expressing plasmid. CKD, chronic kidney disease; OSBPL7, oxysterol-binding protein-like 7.

OSBPL7 Deficiency in Podocytes Is Associated With Increased ER Stress

The inability of an OSBPL7 construct lacking the FFAT motif to prevent injury in siOSBPL7 podocytes reinforces the essential role of OSBPL7-ER binding in podocyte function. We examined ER stress markers in siOSBPL7 and scOSBPL7 podocytes and observed a significant increase in the expression of IRE1α and BiP, two key markers of the UPR, in siOSBPL7 compared with scOSBPL7 podocytes (Fig. 2, A and B). This suggests that OSBPL7 deficiency contributes to ER stress. Interestingly, levels of PERK and PDI remained unchanged, indicating that the effects predominantly involve IRE1α activation rather than other UPR pathways. In addition, siOSBPL7 podocytes exhibited elevated levels of phosphorylated SAPK/JNK compared with scOSBPL7 (Fig. 2, A and B), aligning with the known role of IRE1α in activating the SAPK/JNK pathway (53, 54), which is implicated in apoptosis initiation.

Figure 2.

OSBPL7 deficiency leads to ER stress and decreased autophagic flux. A and B: representative Western blot images (A) of PERK, IRE1α, PDI, phosphorylated and total SAPK/JNK, BiP, and GAPDH with quantification from three independent experiments (B). C and D: representative Western blot images (C) of LC3I, LC3II, and GATE16 levels with GAPDH loading control with quantification from three independent experiments (D). n = 3. *P < 0.05, **P < 0.01, and ***P < 0.005. BiP, binding immunoglobulin protein; ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α; OSBPL7, oxysterol-binding protein-like 7; PERK, protein kinase RNA-like ER kinase; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase.

OSBPL7 Deficiency in Podocytes Is Associated With Decreased Autophagic Flux

Furthering our investigation, we evaluated the impact of OSBPL7 on autophagic flux in podocytes by measuring LC3 and GATE16, integral to autophagosome biogenesis at different stages. LC3 facilitates phagophore membrane elongation, whereas GATE16 is crucial for autophagosome maturation (55, 56). Our findings reveal a notable decrease in autophagic flux in siOSBPL7 podocytes, evidenced by altered LC3I to LC3II conversion and GATE16 levels (Fig. 2, C and D). These results align with previous studies indicating the necessity of lipid transfer proteins like OSBPL7 for LC3-I to LC3-II lipidation during autophagy (47). The decrease in autophagic flux mediated by OSBPL7 deficiency thus appears to play a significant role in podocyte injury.

OSBPL7 Deficiency in Podocytes Alters Lipid Homeostasis

As OSBPL7 was shown to modulate LD content in other cell types, and we demonstrated important role of LD as a modulator of podocyte function, we next investigated if and how OSBPL7 deficiency may affect podocyte lipid metabolism. We observed a significant increase in LD in siOSBPL7 podocytes compared with the scOSBPL7 controls (Fig. 3, A and B). Given that LDs mainly comprise esterified cholesterol and triglycerides, we then evaluated cholesterol content in siOSBPL7 and scOSBPL7 podocytes. Despite the increase in LDs, we did not observe any significant differences in free, esterified, or total cholesterol content between siOSBPL7 and scOSBPL7 podocytes (Fig. 3, C and D). As the esterified cholesterol content remained the same in scOSBPl7 and siOSBPL7 podocytes, we next quantified the triglyceride levels to determine the composition of the increased LDs. As anticipated, we found that triglyceride content was significantly higher in siOSBPL7 than in scOSBPL7 podocytes (Fig. 3E). To better understand the source of increased triglyceride, we measured total FFA content. However, the FFA content was similar in siOSBPL7 and scOSBPL7 podocytes, suggesting that the elevated triglycerides were not a result of increased metabolism of FFAs in the siOSBPL7 podocytes (Fig. 3F) but may result from an altered subcellular distribution of lipids. Given that OSBPL7 is a lipid-trafficking protein, changes in lipid distribution can affect membrane characteristics. Next, we therefore assessed plasma membrane fluidity and demonstrated increased plasma membrane fluidity in siOSBPL7 podocytes when compared with scOSBPL7 (Fig. 3G). Further studies will be needed to determine the role of OSBPL7 in orchestrating the broader alterations in lipid composition across subcellular structures.

Figure 3.

OSBPL7 deficiency in podocytes alters lipid homeostasis. A: LD quantification per cell in siOSBPL7 podocytes versus control. B: representative images of LD in scOSBPL7 and siOSBPL7 cells stained for LD (green), cytoskeleton (red), and nucleus (blue). Total cholesterol (C) and cholesterol ester levels (D) were not changed between siOSBPL7 and scOSBPL7 podocytes. Triglycerides were increased in siOSBPL7 podocytes (E), while free fatty acids (F) were not changed as indicated by the fold change from siOSBPL7 compared with scOSBPL7 levels. G: membrane fluidity was increased in siOSBPL7 podocytes compared with scOSBPL7. n = 3. *P < 0.05 and ****P < 0.001. LD, lipid droplet; OSBPL7, oxysterol-binding protein-like 7.

Effectiveness of KIRA6 and STF62247 in Modulating ER Stress and Autophagy in Podocytes

In our study, we first sought to establish the effectiveness of specific compounds in modulating cellular pathways in OSBPL7-deficient podocytes. To this end, we conducted dose-response experiments using KIRA6 and STF62247, as evidenced by Western blots of IRE1α (Fig. 4A) and LC3 (Fig. 4B), respectively. KIRA6, known to inhibit IRE1α-mediated ER stress, effectively reduced IRE1α expression. Similarly, STF62247, which enhances autophagy, resulted in increased levels of LC3. These results demonstrate the targeted actions of these compounds on ER stress and autophagy pathways.

Figure 4.

ER stress and not decreased autophagy or increased LD accumulation is responsible for apoptosis in podocytes. A: Western blot analysis illustrating the effect of KIRA6 on IRE1α expression in siOSBPL7 podocytes, with a marked reduction in IRE1α levels observed. The underlined and bold concentration indicates the specific concentration used in our experiments. B: Western blot showing the impact of STF62247 on LC3 levels in siOSBPL7 podocytes. The increase in LC3 levels upon STF62247 treatment confirmed the promotion of autophagy. The underlined and bold concentration indicates the specific concentration used in our experiments. C: apoptosis assessment in siOSBPL7 podocytes posttreatment with KIRA6, STF62247, and hydroxypropyl β-cyclodextrin (CD). Apoptosis, heightened in untreated cells, was normalized with KIRA6 treatment. STF62247 and CD did not significantly mitigate apoptosis, highlighting ER stress as the central mechanism in apoptosis induction, rather than autophagy or LD content alterations. n = 3. ***P < 0.005. ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α; LC-3, light chain-3; LD, lipid droplet; OSBPL7, oxysterol-binding protein-like 7.

Role of ER Stress in Inducing Apoptosis in OSBPL7-Deficient Podocytes

Building upon the verification of drug efficacy, we explored their impact on apoptosis in OSBPL7-deficient podocytes (Fig. 4C). Notably, we observed a significant increase in apoptosis in these podocytes. Treatment with KIRA6, which targets ER stress pathways, successfully normalized apoptosis levels. In contrast, neither STF62247 nor hydroxypropyl β-cyclodextrin (CD), used to reduce LD content and enhance autophagy, were effective in mitigating apoptosis.

These observations underscore that ER stress, particularly through IRE1α activation, is a primary driver of apoptosis in OSBPL7-deficient podocytes. This finding contrasts with the roles of altered autophagy and LD accumulation, which, despite being contributing factors to podocyte injury in kidney diseases, do not appear to be the central mechanisms in this specific context of OSBPL7 deficiency.

OSBPL7 Knockdown in Zebrafish Leads to Proteinuria and Increased Edema and Glomerular Damage

Building on our understanding of OSBPL7’s role in lipid regulation, autophagy, and ER stress in podocytes, we sought to extend our insights through in vivo examination. The utilization of zebrafish models for this study was underpinned by the notable homology between human and zebrafish osbpl7, which makes the zebrafish a pertinent model organism to investigate the physiological consequences of osbpl7 deficiency.

Our in vivo observations in zebrafish models showed that reduced osbpl7 levels lead to proteinuria, as evidenced by decreased eye fluorescence and increased edema (Fig. 5, A–C). Detailed examination via TEM revealed clear podocyte and glomerular damage, including podocyte effacement and rupture of the GBM (Fig. 5D).

Figure 5.

Proteinuria and glomerular damage in zebrafish with Osbpl7 knockdown. Osbpl7 knockdown by osbpl7-Mo in l-fabp:DBP-eGFP ZF led to decreased eye fluorescence (A) and increased edema (B). C: representative images of individual zebrafish. D: transmission electron microscopy (TEM) images of the zebrafish glomerulus at 5 DPI with either Control-Mo and osbpl7-Mo. Substantial glomerular damage was conspicuous in the osbpl7-Mo-injected larvae. The detailed TEM analysis revealed the loss of endothelial fenestrations (white arrows) and widening and rupture of the glomerular basement membrane (GBM) (star). The distinct alterations in podocyte structures are shown, including podocyte effacement and protrusions of foot processes into the GBM (black arrows), a common hallmark of proteinuric states in fish. These observations point to clear podocyte and glomerular damage, in alignment with the proteinuria documented in the fish. 4 DPI. n = 16. **P < 0.01 and ****P < 0.001. OSBPL7, oxysterol-binding protein-like 7.

To affirm that the observed renal phenotypes were due to specific osbpl7 knockdown, we compared two morpholino approaches: ATG blocking (OSBPL7 ATG) and splice donor (OSBPL7 SD), each leading to a decrease in eye fluorescence and the edema phenotype (Supplemental Fig. S1A). In addition, FITC Cardinal Vein injections in osbpl7 morpholino-injected larvae demonstrated decreased eye fluorescence independent of liver function (Supplemental Fig. S1B), thereby reinforcing the renal-specific consequences of osbpl7 knockdown. Crucially, examination of WT1-gfp transgenic zebrafish confirmed that the pronephros develops normally in osbpl7-Mo-injected embryos (Supplemental Fig. S1C), indicating that proteinuria and glomerular damage were not due to general developmental defects but were directly attributable to the lack of osbpl7.

These findings in zebrafish, reflecting the close genetic correspondence with human OSBPL7, collectively point to pronounced podocyte and glomerular damage in the osbpl7-Mo-injected embryos, consistent with the proteinuria documented in the fish. The morphological insights obtained from this study offer a valuable mechanistic understanding of the effects of OSBPL7 deficiency, further reinforcing its crucial role in maintaining the integrity of the glomerular filtration barrier and the overall function of the kidney. The connection between our in vitro and in vivo findings serves to strengthen our comprehension of osbpl7’s physiological relevance and could pave the way for future therapeutic interventions targeting related kidney disorders.

DISCUSSION

In the intricate landscape of CKD pathophysiology, our study has shed light on the pivotal role of OSBPL7 in the progression of CKD, particularly in podocyte injury. The absence of OSBPL7 initiates a cascade of events, among which prolonged ER stress leading to podocyte apoptosis is especially significant. This finding is consistent with previous studies that have highlighted the role of ER stress and the UPR in kidney diseases (17, 18, 25, 33, 35–38, 50, 57, 58).

The UPR involves three major mediators: ATF6, IRE1α, and PERK. Among these, IRE1α is of particular interest as it catalyzes the unconventional processing of the mRNA encoding X-box binding protein-1 (XBP1), creating a transcriptionally active form known as spliced XBP1 (sXBP1). This sXBP1 is critical for cellular adaptation under stressful conditions, regulating genes that enhance protein folding, transport, and degradation (59, 60). The activation of sXBP1 initially serves as a protective mechanism, prolonged activation can lead to podocyte apoptosis (61, 62), a significant contributor to glomerular injury. In addition, the Ser/Thr kinase domain in Ire1 has been documented to trigger the SAPK-JNK pathway (53, 54). This activation is linked to the initiation of apoptosis, aligning with the elevated levels of apoptosis we observed in siOSBPL7 podocytes.

ER stress has been implicated in both AKI and its progression to CKD (63–67). For instance, angiogenin, a regulator of the stress response integrated into the UPR, plays a critical role in tissue adaptation to AKI (68). Moreover, ER stress can also prompt the development of CKD, as evidenced by genetic studies focusing on reticulon 1 (RTN1), an ER-shaping protein that induces apoptosis of renal epithelial cells through ER stress-induced activation of PERK and downstream induction of CHOP (18).

Beyond ER stress, OSBPL7 deficiency also leads to lipid metabolic changes, including increased LDs and triglycerides, as well as altered membrane fluidity. These lipid alterations could potentially modify the structural components of the kidney, affecting the GBM and foot process effacement (69–71). Decreased autophagy, another consequence of OSBPL7 deficiency, may further exacerbate these effects, compromising the cell’s ability to maintain proper function and integrity.

Our study also extends these insights to zebrafish. Reduced levels of osbpl7 in zebrafish led to proteinuria, edema, and clear signs of glomerular damage, including podocyte effacement and rupture of the GBM. These findings in zebrafish models are consistent with the proteinuria and glomerular injuries observed, reinforcing the crucial role of OSBPL7 in regulating kidney filtration. However, the use of zebrafish models has limitations as they do not fully mimic the complex nephron structure of mammalian kidneys, which can limit the direct applicability of findings to human kidney diseases.

Our initial investigations using mouse models revealed critical aspects of OSBPL7 deficiency in CKD, focusing on both tissue and cultured podocytes. Complementing these mouse model insights, zebrafish were used to demonstrate the impact of osbpl7 knockdown, which led to notable podocyte damage and proteinuria. This approach highlights the zebrafish model’s utility in rapidly identifying key phenotypes and disease mechanisms.

Furthermore, our study observed notable changes in the GBM, a critical focus for future investigations. These preliminary findings in zebrafish will be further explored and expanded upon using mouse models, which offer a more detailed and anatomically relevant representation of GBM and kidney structure. Our study’s dual-model approach demonstrates how combining zebrafish with mouse models can effectively advance our understanding of kidney diseases.

In conclusion, our study suggests that while OSBPL7 serves as a master regulator of ER stress, lipid dysregulation, and autophagy, it is specifically through the ER stress pathway that OSBPL7 expression is linked to podocyte injury in the pathogenesis of CKD and potentially other renal diseases. The role of OSBPL7 deficiency in these processes provides an innovative perspective that could pave the way for the development of novel diagnostic and therapeutic strategies targeting these shared pathological features across diverse kidney diseases. Given the critical role of ER stress in both AKI and CKD, further studies are warranted to explore the therapeutic potential of modulating the UPR pathways in renal diseases.

Perspectives and Significance

These observations build on the existing knowledge of the roles of OSBPL7 as a master regulator of lipid trafficking, ER stress, and autophagy flux in multiple cell types. We provide the first evidence that reduced podocyte expression of OSBPL7 may represent a new early biomarker of podocyte injury. As we have already developed drugs targeting OSBPL7 that are currently being tested in phase II trial, this study is highly significant as it may lead to a better understanding of the mechanism of action of existing drugs and/or to the development of novel specific agents targeting OSBPL7. By bridging the knowledge gap in this area, the study aims to further our understanding of CKD’s underlying molecular pathways and contribute to the broader landscape of kidney disease research.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL MATERIAL

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.25145726.v1.

GRANTS

J.D. is supported by the Miami Clinical and Translational Science Institute, from National Institutes of Health (NIH) Grant 1K12TR004555. A.F. and S.M. are supported by NIH Grant R01DK136679. A.F. is supported by NIH Grants U54DK083912, UM1DK100846, U01DK116101, and UL1TR000460 (Miami Clinical Translational Science Institute). A.F. and S.M. are supported by Aurinia Pharmaceuticals Inc. and Pfizer Inc.

DISCLOSURES

A.F. and S.M. are inventors on pending (PCT/US2019/032215; US 17/057,247; PCT/US2019/041730; PCT/US2013/036484; US 17/259,883; US17/259,883; JP501309/2021, EU19834217.2; CN-201980060078.3; CA2,930,119; CA3,012,773; CA2,852,904) or issued patents (US10,183,038 and US10,052,345) aimed at preventing and treating renal disease. They stand to gain royalties from their future commercialization. A.F. is Vice-President of L&F Health LLC and is a consultant for ZyVersa Therapeutics Inc. ZyVersa Therapeutics Inc. has licensed worldwide rights to develop and commercialize hydroxypropyl-β-cyclodextrin from L&F Research for the treatment of kidney disease. A.F. also holds equities in the Renal 3 River Corporation. S.M. holds indirect equity interest in, and potential royalty from, ZyVersa Therapeutics Inc. by virtue of assignment and licensure of a patent estate. A.F. and S.M. are supported by Aurinia Pharmaceuticals Inc. and Pfizer Inc. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

J.M., M.G., M.S., S.M., and A.F. conceived and designed research; J.D., J.M., M.T., M.G., R.N., and M.G. performed experiments; J.D., M.T., R.N., J.M., M.S., S.M., and A.F. analyzed data; J.D., J.M.D., J.-J.K., A.M., M.G., A.S., M.S., S.M., and A.F. interpreted results of experiments; J.D. and J.M. prepared figures; J.M., M.T., M.S., S.M., and A.F. edited and revised manuscript; S.M. and A.F. approved final version of manuscript.