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. Author manuscript; available in PMC: 2026 Jan 8.
Published before final editing as: J Am Soc Nephrol. 2025 Oct 28:10.1681/ASN.0000000915. doi: 10.1681/ASN.0000000915

Testican-2 Interaction with the Extracellular Matrix and Podocyte Protection

Donghai Wen 1,2, Qian Zhang 1,2,3, Johannes van Agthoven 1, Astrid Weins 4, Ivy A Rosales 4, Wen Zhou 1,2, Taesoo Kim 1, Xavier Vela Parada 5, Robert B Colvin 4, Martin R Pollak 6, Morgan E Grams 7, Insa M Schmidt 8, Sushrut S Waikar 8, M Amin Arnaout 1, Eugene P Rhee 1,2
PMCID: PMC12776367  NIHMSID: NIHMS2133258  PMID: 41177530

Abstract

Background

Testican-2 is a podocyte-derived glycoprotein encoded by SPOCK2. Circulating levels of testican-2 are associated with less glomerulosclerosis and better kidney prognosis, but its biologic function in the podocyte is unknown.

Methods

We studied the protective effect of testican-2 on immortalized cultured human podocytes and in mice treated with adriamycin. We used immunoprecipitation mass spectrometry to identify binding partners of testican-2 and biolayer interferometry to characterize these protein–protein interactions. Using global and podocyte-specific Spock2 knockout mice, we assessed the effect of testican-2 deficiency in models of podocyte injury and also tested whether exogenous testican-2 confers podocyte protection in testican-2–deficient mice. Finally, we analyzed testican-2 expression in human kidney biopsy samples.

Results

Testican-2 reduced adriamycin-induced podocyte injury in cultured human podocytes and mice. Vitronectin was a strong binding partner for testican-2, and testican-2 inhibited the interaction between vitronectin and integrin αVβ3, an effector of podocyte injury. Consistent with this, testican-2 administration reduced activation of integrin β3 in injured podocytes. Furthermore, Spock2 deficiency increased susceptibility to podocyte injury due to adriamycin- and streptozotocin-induced diabetes, as determined by albuminuria, foot process effacement, nephrin expression, and Wilms’ tumor 1-positive podocyte number. Importantly, exogenous testican-2 circulated to the kidney, bound to vitronectin, reduced vitronectin-integrin β3 interaction, and reduced podocyte injury in Spock2-deficient mice. Finally, glomerular testican-2 expression was reduced in human focal segmental glomerulosclerosis and diabetic kidney disease, but not tubulointerstitial nephropathy.

Conclusions

Testican-2 modulated the podocyte’s interaction with its extracellular matrix and had a functional role in kidney protection.

Introduction

Podocyte dysfunction plays an important role in CKD initiation and progression.1,2 Genetic studies have identified several proteins expressed by podocytes that are essential for maintaining normal kidney function, including structural components of the podocyte slit diaphragm, podocyte cytoskeleton, and glomerular basement membrane (GBM).3,4 Moreover, impactful treatments for CKD such as renin-angiotensin and sodium-glucose cotransporter 2 inhibition work, at least in part, by reducing podocyte injury.5,6 Despite these advances, many individuals with CKD will experience disease progression, and CKD and its consequences continue to represent a major public health challenge. Thus, identification of a podocyte-specific mediator of kidney health amenable to therapeutic targeting would meet a major unmet need.7

Using an aptamer-based proteomic screen of samples obtained from the aorta and renal vein of human participants, we identified testican-2 as a novel protein released by the kidney into circulation. Unlike existing blood bio-markers of kidney disease, testican-2 levels are positively correlated with eGFR, and higher baseline levels of testican-2 are associated with less eGFR loss over time in population-based cohorts (N>3500).8 Furthermore, higher levels of circulating testican-2 are associated with reduced risk of incident kidney failure across three large cohorts enriched for CKD and its risk factors (N>8000).9 Most recently, we found that testican-2 had the strongest inverse association with glomerulosclerosis among individuals who had undergone kidney biopsy and plasma proteomic profiling (N=434).10 Importantly, microscopy and single-cell RNA sequencing demonstrated that testican-2 expression is highest in podocytes, and immunogold staining showed that testican-2 is secreted into the GBM.8 Together, these findings advance the concept that testican-2 is a podocyte-derived protein whose circulating levels provide insight on kidney health and prognosis.

Encoded by the SPOCK2 gene, testican-2 is a 424 amino acid–secreted glycoprotein originally cloned in neuronal cells. It has been characterized as a component of the extracellular matrix, but little is known about its function.11–13 A previous study showed that testican-2 may protect lung epithelial cells from influenza virus infection, while others found that it may affect cancer cell migration and invasion.14,15 Our prior work has raised the possibility that it may enhance glomerular endothelial migration and tube formation.8 In this study, we sought to elucidate the biologic function of testican-2 in podocytes, with the hypothesis that it confers podocyte protection through its interaction with other extracellular matrix proteins in the GBM.

Methods

Cell Culture

A conditionally immortalized human podocyte cell line was used.16 In brief, cells were grown in RPMI-1640 media (R8785, Sigma) with insulin, transferrin, and selenium Liquid Media Supplement (I3146, Sigma; 100× insulin, transferrin, and selenium) and 10% FBS. Cells were placed at 33°C for propagation before switching to 37°C and incubated for 10–14 days for differentiation. Cultured human podocytes were treated with adriamycin (D1515, Sigma), testican-2 (2328-PI, R&D Systems), and/or high affinity tenth domain of fibronectin (hFN10) (provided by Dr. M. Amin Arnaout) as indicated in the text.17

Animal Studies

Eight-week-old to 10-week-old male Bagg Albino Lac Bombyx mice were injected with a single dose of adriamycin (10 mg/kg body wt) through the tail vein, together with placebo enhanced green fluorescent protein (eGFP) (1060, Vector Biolabs) or mouse Spock2 adenovirus (BC057324, Vector Biolabs; 109 plaque-forming unit for each mouse). Mice treated with an equal volume injection of normal saline were used as the control group. To generate global Spock2 knockout mice, Spock2+/− mice were obtained from the Mutant Mouse Resource & Research Center at University of California Davis (MMRRC_049849-UCD). These mice were generated on a C57Bl/6N background with targeted deletion of the Spock2 gene using Velocigene (http://velocigene.com/komp/detail/14859). In our laboratory, Spock2+/− mice were bred to generate Spock2−/− mice and Spock2+/+ littermates. Podocyte-specific Spock2 knockout mice were generated by crossing Nphs2-Cre with Spock2-floxed mice (Cyagen) (https://www.cyagen.com/mouseatlas/S-CKO-17958). Since C57Bl/6 mice are relatively resistant to adriamycin, a higher dose of 15 mg/kg body wt was used to induce kidney injury in this background (8–10 weeks old, male).18–20 To induce diabetic kidney disease (DKD), 10- to 12-week-old male mice were given two injections of streptozotocin (100 mg/kg body wt; C8532, Sigma) at 4-day intervals as previously described.21 Three weeks after the adriamycin injection or 10 weeks after the first streptozotocin injection, mice were euthanized and samples were collected for analysis.

Immunoprecipitation

Protein lysates from the whole-cell or extracellular matrix fractions of cultured human podocytes were prepared as previously described.22 Protein lysates of cultured cells were immunoprecipitated with primary goat anti–testican-2 antibody (AF2328, Lot# VYP0213061, R&D Systems) or normal goat IgG (AB-108-C, Lot# ES4119121, R&D Systems) using an immunoprecipitation kit with Protein A/G Sepharose beads (ab206996, Abcam). The pull-down was then sent for mass spectrometry and immunoblot analysis. Protein lysates of kidney homogenates were immunoprecipitated with primary goat anti–testican-2 antibody (AF2328, Lot# VYP0213061, R&D Systems), normal goat IgG (AB-108-C, Lot# ES4119121, R&D Systems), primary mouse antivitronectin (VTN) antibody (sc-74484, Lot# C0918, Santa Cruz), or normal mouse IgG (sc-2025, Lot# L0619, Santa Cruz) using the same immunoprecipitation kit. The pull-down was then used for immunoblot analyses.

Liquid Chromatography/Mass Spectrometry–Based Proteomics

The immunoprecipitation pull-down of testican-2 antibody or normal IgG was analyzed using liquid chromatography/mass spectrometry at the Taplin Biological Mass Spectrometry Facility, Harvard Medical School. In brief, excised gel bands were cut into approximately 1-mm3 pieces and subjected to a modified in-gel trypsin digestion.23 Samples were then placed in a 37°C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The samples were reconstituted in 5–10 ml of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6-μm C18 spherical silica beads into a fused silica capillary (100 μm inner diameter×approximately 30 cm length) with a flame-drawn tip.24 After equilibrating the column, peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid), subjected to electrospray ionization, and then entered into an linear trap quadrupole Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program Sequest (Thermo Fisher Scientific).25 All databases include a reversed version of all the sequences, and the data were filtered to between a 1% and 2% peptide false discovery rate. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRoteomics IDEntifications partner repository with the dataset identifier PXD066935 and 10.6019/PXD066935.

Biolayer Interferometry

Protein–protein interactions among testican-2, vitronectin, and integrin αVβ3 were examined using biolayer interferometry (BLI) with the Octet Red 96 BLI system (Sartorius). The His-tagged protein of interest was immobilized to the nickel-nitrilotriacetic acid or HIS1K biosensors (Sartorius). The biosensors were then placed into solutions containing different concentrations of analyte proteins (non–His-tagged) (Association), followed by blank buffer solutions (Dissociation). All steps were performed in solutions prepared in PBS pH 7.4, 0.05% tween-20, 1 mg/ml BSA, at 25°C. Analysis of the data was performed using the Octet Data Analysis 9.0 (Sartorius). KD was calculated using model 1:1. The following proteins were used: His-tagged human testican-2 (2328-PI, R&D Systems); His-tagged mouse testican-2 (MBS1458329, MyBioSource); non–His-tagged human testican-2 (TP303974, OriGene); human vitronectin (2349-VN, R&D Systems); mouse vitronectin (ab92727, Abcam); His-tagged human integrin αVβ3 (CT098-H08H, Sino Biological); non–His-tagged human integrin αVβ3 (CC1018, Sigma); and His-tagged human soluble urokinase plasminogen activator receptor (suPAR; 807-UK/CF, R&D Systems). Deglycosylation of human testican-2 and vitronectin was performed using the Protein Deglycosylation Mix II kit (P6044S, New England BioLabs).

Human Kidney Biopsy

The Boston Kidney Biopsy Cohort study is a prospective cohort study of adult patients (older than 18 years) who underwent a native kidney biopsy between September 2006 and June 2016 at three tertiary care hospitals in Boston, Massachusetts.10,26 Each biopsy was examined jointly by two experienced kidney pathologists. Patients’ charts were reviewed alongside histopathologic diagnoses to determine clinicopathologic diagnoses. A subset of four diagnostic groups with available paraffin blocks at Massachusetts General Hospital or Brigham and Women’s Hospital were included in this study: control, primary FSGS, DKD, and tubulointerstitial nephropathy.

Statistical Analyses

Based on power analysis performed using preliminary data, we sought to use 4–5 mice per group to be able to detect >30% changes in most of the measured parameters. Statistical analysis was calculated using Prism 6.0 software (GraphPad). All data were evaluated for normality using the Shapiro–Wilk test. For normally distributed data, one-way ANOVA with Tukey multiple comparison test and Student two-tailed unpaired t test were used for continuous variables, and the chi-squared test was used for categorical variables. For non-normally distributed data, Kruskal–Wallis test with Dunn multiple comparison test or Mann–Whitney 𝒰 test were used for continuous variables. Pearson correlation was used to assess correlations. A P value < 0.05 was considered significant.

Study Approval

All animal studies were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and conducted under their guidelines (2011N000138). The Institutional Review Board protocol for the Boston Kidney Biopsy Cohort study was approved by the Institutional Review Board of Mass General Brigham, and all individuals provided informed consent (2024P001020). Additional methods are described in the Supplemental Methods.

Results

Testican-2 Alleviated Adriamycin-Induced Podocyte Injury In Vitro and In Vivo

In cultured human podocytes, we found that adriamycin reduced testican-2 expression in a dose-dependent manner (Figure 1, A and B), whereas the administration of recombinant testican-2 protein attenuated adriamycin-induced actin cytoskeleton disruption (Figure 1, C and D) and reduction in surface focal adhesion kinase expression (Supplemental Figure 1, A and B).27 To test whether testican-2–mediated protection is also observed in vivo, we increased circulating testican-2 levels in mice by administration of adenovirus containing Spock2 cDNA. Adriamycin significantly reduced testican-2 expression in the kidney (Figure 1, E and F) as well as in circulation (Figure 1G). Administration of Spock2 adenovirus increased serum testican-2 levels compared with placebo (eGFP) adenovirus in adriamycin-treated mice (Figure 1G) and attenuated adriamycin-induced podocyte damage, as demonstrated by reduced urine albumin/creatinine ratio (UACR) (Figure 1H) and podocyte foot process effacement (Figure 1, I and J). Increased expression of testican-2 did not have a significant impact on serum creatinine or BUN levels (Supplemental Figure 1, C and D) but did attenuate the loss in body weight (Supplemental Figure 1E), increase in glomerular injury (Supplemental Figure 1, F and G), and reduction of podocyte number and nephrin expression induced by adriamycin (Supplemental Figure 1, H–K).

Figure 1. Testican-2 alleviates adriamycin-induced podocyte injury.

Figure 1.

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(A) Representative immunoblot and (B) quantification analysis of testican-2/GAPDH expression in cultured human podocytes treated with adriamycin at different concentrations for 48 hours (n=3). (C) Representative F-actin staining and (D) quantification analysis of actin fiber number per μm cell diameter in cultured human podocytes treated with adriamycin (2 μg/ml) and testican-2 at different concentrations for 48 hours before staining. For each condition, a total of 15 randomly selected cells were analyzed. (E) Immunoblot and (F) quantification analysis of testican-2/β-actin expression in kidney tissue from BALB/c male mice 3 weeks after a single IV injection of normal saline (control) or adriamycin (10 mg/kg of body wt; n=54). (G) Serum testican-2 concentration (n=5) and (H) UACR (n=4–10) in BALB/c male mice 3 weeks after a single injection of normal saline (control), adriamycin with eGFP adenovirus, or adriamycin with Spock2 adenovirus. (I) Representative transmission electron microscope and (J) quantification analysis of podocyte foot process number per μm of GBM (#FP/μm) in glomeruli of BALB/c male mice 3 weeks after a single injection of adriamycin with eGFP adenovirus or adriamycin with Spock2 adenovirus (n=4). Data are mean±SD; one-way ANOVA with Tukey multiple comparison test (B, D, G, H, and J) and Student two-tailed unpaired t test (F). BALB/c, Bagg Albino Lac Bombyx; eGFP, enhanced green fluorescent protein; FP, foot process; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBM, glomerular basement membrane; IV, intravenous; UACR, urine albumin/creatinine ratio.

Testican-2 Bound to Vitronectin and Inhibited the Interaction between Vitronectin and Integrin αVβ3

Because testican-2 is secreted into the GBM,8 we postulated that it may exert its effect on podocytes by modulating podocyte interactions with this specialized extracellular matrix. To identify testican-2 binding partners, we performed an immunoprecipitation of the whole cell and isolated extracellular matrix fractions from cultured human podocytes using a testican-2 antibody and then analyzed the immunoprecipitation pull-down by mass spectrometry. As presented in Table 1, we found significant enrichment of vitronectin in both whole cell (approximately 21.8-fold) and extracellular matrix (approximately 15.6-fold) fractions in the testican-2 antibody pull-down as compared with normal IgG. This finding was further supported by immunoblot of the immunoprecipitation pull-down (Figure 2A).

Table 1.

Top proteins associated with testican-2 in cultured human podocytes

Symbol Whole Cell Extracellular Matrix
Normal IgG Testican-2 Antibody Testican-2 Antibody/Normal IgG Normal IgG Testican-2 Antibody Testican-2 Antibody/Normal IgG
VTN 8.7×103 1.9×105 21.8 9.6×103 1.5×105 15.6
RPL18 2.9×106 6.5×106 2.2 1.2×105 1.4×106 11.7
IGKV3-15 NA 7.2×106 NA 3.9×106 2.9×107 5.9
RPL15 3.9×104 1.0×105 2.6 2.0×104 1.0×105 5.0
HBA1 9.4×104 1.9×105 2.0 9.2×104 3.4×105 3.7
MDH2 1.9×104 2.4×104 1.3 9.8×103 3.3×104 3.4
PIP 2.3×104 4.7×104 2.0 1.9×104 5.9×104 3.1
GAPDH 9.5×104 1.5×105 1.6 4.4×104 1.3×105 3.0
PRSS2 5.5×105 1.2×106 2.2 5.4×105 1.4×106 2.6
UBA52 3.6×105 1.0×106 2.8 2.4×105 6.0×105 2.5
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Protein fractions were collected from the whole cell or extracellular matrix of cultured human podocytes and then immunoprecipitated by normal IgG or testican-2 antibody. The pull-downs were then sent for mass spectrometry to analyze the binding partners of testican-2. Units are intensity units (a.u.) and ratios as indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBA1, hemoglobin subunit alpha; IGKV3–15, Ig kappa variable 3–15; MDH2, malate dehydrogenase 2; NA, not applicable; PIP, prolactin-inducible protein; PRSS2, trypsin-2; RPL15, 60S ribosomal protein L15; RPL18, 60S ribosomal protein L18; UBA52, ubiquitin-60S ribosomal protein L40; VTN, vitronectin.

Figure 2. Testican-2 binds directly to vitronectin and inhibits the interaction between vitronectin and integrin αVβ3.

Figure 2.

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(A) Immunoblot analysis of testican-2 antibody/normal IgG immunoprecipitation of the extracellular matrix of cultured human podocytes. (B) Schema of BLI. (C) BLI analysis of human testican-2 (sensor) and vitronectin (solution) or (D) testican-2 (sensor) and integrin αVβ3 (solution). (E) BLI analysis of integrin αVβ3 (sensor) and vitronectin with or without testican-2 (solution). For BLI analyses, protein–protein binding capacities are exhibited on the y axis as response, with representative data shown for three independent experiments. (F) Schema of competitive ELISA of testican-2 and vitronectin/integrin αVβ3 interaction using the meso scale discovery assay. Integrin αVβ3 was immobilized onto the 96-well meso scale discovery assay plate. The plate was then washed, and fixed concentrations of vitronectin together with different concentrations of testican-2 were added. The plate was probed with mouse anti-vitronectin antibody and SULFO-TAG anti-mouse detection antibody. (G) Competitive ELISA of testican-2 and vitronectin/integrin αVβ3 (n=6). Data are mean±SD; Kruskal–Wallis test with Dunn multiple comparison test (G). BLI, biolayer interferometry; IB, immunoblot; IP, immunoprecipitation; SULFO-TAG, sulfonated-TAG.

Vitronectin is an extracellular matrix glycoprotein and the major endogenous ligand for integrin αVβ3, an adhesion protein expressed on podocytes.28–30 Vitronectin contains an arginyl-glycyl-aspartic acid (RGD)-motif critical for its binding interaction with integrin αVβ3 that can drive podocyte injury.28,30,31 To further characterize the interactions between testican-2, vitronectin, and the αVβ3 integrin, we used BLI (Figure 2B). As shown in Figure 2C, His-tagged human testican-2 protein was immobilized to the BLI biosensor, and this sensor was placed into solutions containing human vitronectin at different concentrations (association), followed by a blank buffer solution (dissociation). These analyses demonstrated strong binding affinity between human testican-2 and human vitronectin, with a KD=37.4±0.6 nM. By contrast, there was no direct binding between human testican-2 and human integrin αvβ3 (Figure 2D). Mouse testican-2 also exhibited high binding affinity to mouse vitronectin (KD=63.7±2.3 nM, Supplemental Figure 2A). Furthermore, we found that protein glycosylation strengthens, but is not required, for binding between testican-2 and vitronectin (KD=348.5±12.4 nM for deglycosylated testican-2, Supplemental Figure 2, B and C). Finally, we found that testican-2 does not directly interact with suPAR, an immune-derived protein that, like vitronectin, is postulated to mediate podocyte injury through its interaction with integrin αVβ3 (Supplemental Figure 2D).30,31

Next, His-tagged integrin αVβ3 was immobilized to the BLI biosensor, and this sensor was placed into solutions containing a fixed concentration of vitronectin (400 nM) plus different concentrations of testican-2. As shown in Figure 2E, testican-2 demonstrated dose-dependent inhibition of binding between vitronectin and integrin αVβ3. This finding was further supported by competitive ELISA, whereby integrin αVβ3 was immobilized on the surface of a 96-well plate, incubated with mixtures containing a fixed concentration of vitronectin (3 μM) plus different concentrations of testican-2, and assayed with an anti-vitronectin antibody. As with BLI, ELISA demonstrated dose-dependent inhibition of vitronectin-integrin αVβ3 interaction by testican-2 (Figure 2, F and G).

Testican-2 Attenuated Adriamycin-Induced Integrin αVβ3 Activation

In addition to reducing testican-2 expression, adriamycin increased glomerular expression of integrin β3 and vitronectin; integrin β3, but not vitronectin, colocalized with nephrin (Figure 3, A and B). The adriamycin-induced increase in integrin β3 and vitronectin expression was confirmed by immunoblot of kidney lysates (Figure 3, C–E). To investigate the role of testican-2 in adriamycin-induced podocyte injury mediated by vitronectin/integrin αVβ3 activation, the AP5 antibody was used to detect the active conformation of integrin β3.32,33 In cultured human podocytes, adriamycin increased the ratio of AP5 staining relative to total integrin β3, whereas this effect was significantly attenuated by the administration of testican-2 (200 nM) (Figure 3, F and G). No effect for adriamycin or testican-2 was observed for the ratio of active integrin β1 to total integrin β1 (Figure 3, H and I). hFN10 is a pure peptide antagonist of integrin αVβ3 that demonstrates dose-dependent attenuation of integrin αVβ3 activation in adriamycin-treated podocytes (Figure 3, J and K).17 We found that compared with hFN10 alone, the combination of hFN10 and testican-2 did not confer any additional protection in adriamycin-induced actin cytoskeleton disruption (Figure 3, L and M) in podocytes. These findings suggest that the protective effect of testican-2 on podocytes is mediated through its inhibition of integrin αVβ3 activation.

Figure 3. Testican-2 reduces adriamycin-induced integrin αVβ3 activation and podocyte injury.

Figure 3.

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Representative immunofluorescence of (A) integrin β3, nephrin, and merge or (B) Vitronectin, nephrin, and merge of glomeruli and (C) immunoblot and quantification analysis of (D) integrin β3/β-actin and (E) vitronectin/β-actin expression in kidney tissue from BALB/c male mice 3 weeks after a single IV injection of normal saline (control) or adriamycin (10 mg/kg of body wt; n=3). (F) Representative immunofluorescence and (G) quantification analysis of AP5 (red)/integrin β3 (green) of human podocytes treated with adriamycin (2 μg/ml) for 24 hours with or without testican-2 (200 nM). (H) Representative immunofluorescence and (I) quantification analysis of active integrin β1 (green)/total integrin β1 (red) of human podocytes treated with adriamycin (2 μg/ml) for 24 hours with or without testican-2 (200 nM). (J) Representative immunofluorescence of AP5 (red) and (K) quantification analysis of human podocytes treated with adriamycin (2 μg/ml) for 24 hours together with hFN10, a pure integrin αVβ3 antagonist, at different concentrations. (L) Representative immunofluorescence of F-actin (red) and (M) quantification analysis of actin fiber number per μm cell diameter in human podocytes treated with adriamycin (2 μg/ml) for 48 hours, with or without hFN10 (100 nM) and testican-2 (200 nM). For each condition, a total of 15 randomly selected cells were analyzed (G, I, K, and M). DAPI is in blue. Data are mean±SD; Student two-tailed unpaired t test (D and E), one-way ANOVA with Tukey multiple comparison test (G, I, and M), and Kruskal–Wallis test with Dunn multiple comparison test (K). DAPI, 4’,6-diamidino-2-phenylindole; hFN10, high affinity tenth domain of fibronectin.

Deletion of Testican-2 Exacerbated Adriamycin-Induced Podocyte Injury In Vivo

To further verify the protective effect of testican-2 on podocytes, we assessed global Spock2 knockout (Spock2−/−) mice (Figure 4A). Reverse-transcription PCR of Spock2 mRNA, ELISA of serum testican-2, and assessment of glomerular testican-2 expression were used to validate the absence of testican-2 (Supplemental Figure 3, A–C). At baseline, no difference was observed in body weight, serum creatinine, BUN, or UACR between Spock2−/− and Spock2+/+ mice (Figure 4B and Supplemental Figure 3, D–F). As compared with Spock2+/+ mice, adriamycin-treated Spock2−/− mice developed substantially higher albuminuria (Figure 4B), more severe glomerular injury (Figure 4, C and D, and Supplemental Figure 3G), more prominent podocyte foot process effacement (Figure 4, E and F) and lower podocyte number and nephrin expression (Figure 4, G–J). No difference in serum creatinine or BUN was noted between Spock2−/− and Spock2+/+ littermates after a single adriamycin dose (Supplemental Figure 3, E and F).

Figure 4. Testican-2 deficiency exacerbates adriamycin-induced podocyte injury.

Figure 4.

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(A) Schematic experimental design. Wild-type (Spock2+/+) and Spock2−/− mice were treated with a single dose of adriamycin (15 mg/kg) and sacrificed after 3 weeks. (B) UACR and (C) representative PAS stain of glomeruli and (D) quantification analysis of glomerular injury score in Spock2+/+ and Spock2−/− mice treated with adriamycin or control (n=4–5). (E) Representative transmission electron microscope and (F) quantification analysis of podocyte foot process number per μm of GBM (#FP/μm n=3). (G) Representative immunofluorescence of WT1/nephrin and (H) quantification analysis of podocyte number (WT1-positive cells) per glomerulus (n=5). (I) Representative immunoblot and (J) quantification analysis of nephrin/β-actin in the kidney homogenates of Spock2+/+ and Spock2−/− mice treated with adriamycin (n=3). Data are mean±SD; one-way ANOVA with Tukey multiple comparison test (B and D) and Student two-tailed unpaired t test (F, H, and J). PAS, periodic acid–Schiff; WT1, Wilms’ tumor 1.

Testican-2 Attenuated Adriamycin-Induced Podocyte Injury in Spock2−/− Mice

To test the potential protective effect of circulating testican-2, distinct from podocyte-derived testican-2, we administered eGFP placebo or Spock2 adenovirus to adriamycin-treated Spock2−/− mice (Figure 5A). As was observed in wild-type (Spock2+/+) mice, treatment with Spock2 adenovirus significantly improved albuminuria (Figure 5B), glomerular injury (Figure 5, C and D), podocyte foot process effacement (Figure 5, E and F), and podocyte number and nephrin expression (Figure 5, G–J) in the adriamycin-treated Spock2−/− mice compared with eGFP control. Immunofluorescence and coimmunoprecipitation using a testican-2 antibody verified the deposition of circulating testican-2 and its interaction with vitronectin in kidney tissue (Figure 5, K and L). Coimmunoprecipitation using a vitronectin antibody recapitulated these findings and, importantly, also demonstrated that this interaction between testican-2 and vitronectin resulted in reduced binding between vitronectin and integrin β3 and between vitronectin and integrin αV (Figure 5, M–Q). By contrast, the interaction between testican-2 and vitronectin had no effect on the amount of binding between vitronectin and integrin β1 or between vitronectin and integrin β5 (Figure 5, M, R, and S). These findings show that exogenously delivered testican-2 in circulation binds to vitronectin in the kidney, inhibits its interaction with integrin αVβ3, and attenuates adriamycin-induced podocyte injury, even when endogenous kidney testican-2 expression is absent.

Figure 5. Testican-2 attenuates adriamycin-induced podocyte injury in Spock2−/− mice.

Figure 5.

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(A) Schematic experimental design. Spock2−/− mice were treated with a single dose of adriamycin (15 mg/kg) and eGFP or Spock2 adenovirus and sacrificed after 3 weeks. (B) UACR (n=4–5). (C) Representative PAS stain of the glomeruli and (D) quantification analysis of glomerular injury score (n=5). (E) Representative transmission electron microscope and (F) quantification analysis of podocyte foot process number per μm of GBM (#FP/μm; n=4). (G) Representative immunofluorescence of WT1/nephrin and (H) quantification analysis of podocyte number (WT1-positive cells) per glomerulus (n=5). (I) Representative immunoblot and (J) quantification analysis of nephrin/β-actin in the kidney homogenates of Spock2−/− mice treated with adriamycin and eGFP adenovirus or adriamycin and Spock2 adenovirus (n=3). (K) Representative immunofluorescence of testican-2 (red) and nephrin (green) in the glomeruli of Spock2−/− mice treated with adriamycin and eGFP adenovirus or adriamycin and Spock2 adenovirus. (L) Representative immunoblot of testican-2 antibody immunoprecipitation and (M) representative immunoblot and (N–S) quantification analysis of vitronectin antibody immunoprecipitation of kidney homogenates from Spock2−/− mice treated with adriamycin and eGFP adenovirus or adriamycin and Spock2 adenovirus (n=3). Immunoprecipitation pull-down probed for vitronectin, testican-2, integrin β3, integrin αV, integrin β1, and integrin β5. Data are mean±SD; Student two-tailed unpaired t test (B, D, F, H, J, and N–S).

Deletion of Testican-2 Exacerbated Podocyte Injury in Streptozotocin-Induced Diabetic Mice

To evaluate the protective effect of testican-2 in another model of glomerular injury, we administered streptozotocin to Spock2+/+ and Spock2−/− mice (Supplemental Figure 4A). As with adriamycin, we found that streptozotocin reduced testican-2 expression while increasing integrin β3 and vitronectin expression in the kidneys of wild-type (Spock2+/+) mice (Supplemental Figure 4, B–E). No significant difference was found in blood glucose level, body weight, serum creatinine, and BUN between the Spock2+/+ and Spock2−/− mice after streptozotocin injection (Supplemental Figure 4, F–I). As compared with Spock2+/+, Spock2−/− mice had increased albuminuria (Supplemental Figure 5A), increased podocyte foot process effacement (Supplemental Figure 5, B and C), reduced nephrin expression (Supplemental Figure 5, D and E), and increased glomerular matrix deposition (Supplemental Figure 5, F and G) after streptozotocin injection.

Podocyte-Specific Deletion of Testican-2 Exacerbated Adriamycin-Induced Podocyte Injury

To highlight a specific role for podocyte-derived testican-2 in glomerular injury, we administered adriamycin to mice with podocyte-specific Spock2 deletion and control littermates (Supplemental Figure 6, A–E). Notably, podocyte-specific Spock2 deletion resulted in lower circulating levels of testican-2 (Supplemental Figure 6F). Compared with controls, we found that podocyte-specific Spock2 knockout mice had increased albuminuria (Supplemental Figure 6G), glomerular matrix deposition (Supplemental Figure 6, H and I), and podocyte foot process effacement (Supplemental Figure 6, J and K) after adriamycin. Coimmunoprecipitation of kidney lysates with a vitronectin antibody confirmed reduced vitronectin and testican-2 interaction (Supplemental Figure 6, L–N), as well as increased vitronectin interactions with integrin β3 and integrin αV in the kidneys of podocyte-specific Spock2 null mice (Supplemental Figure 6, L, O, and P).

Glomerular Testican-2 Expression Was Reduced in Patients with FSGS and DKD

To assess the potential relevance of our findings to human disease, we obtained archived kidney tissue samples obtained from adult patients with idiopathic FSGS, DKD, and tubulointerstitial nephropathy, as well as individuals with no significant kidney pathology (Table 2). The mean eGFR was 61±38 ml/min per 1.73 m2 among individuals with FSGS, 36±23 ml/min per 1.73 m2 among individuals with DKD, 25±20 ml/min per 1.73 m2 among individuals with tubulointerstitial nephropathy, and 98±24 ml/min per 1.73 m2 among controls. The mean UACR was 3583±2682 mg/g among individuals with FSGS, 2426±1988 mg/g among individuals with DKD, 531±593 mg/g among individuals with tubulointerstitial nephropathy, and 218±291 mg/g among controls. As compared with control samples, the samples obtained from individuals with FSGS had a trend for lower glomerular testican-2 and significantly lower glomerular nephrin immunofluorescence intensities, and the samples from individuals with DKD had significantly lower glomerular testican-2 and nephrin immunofluorescence intensities (Figure 6, A–C). By contrast, no difference in glomerular testican-2 or nephrin immunofluorescence intensities was observed between tubulointerstitial nephropathy and normal control samples. Furthermore, testican-2 immunofluorescence intensity was strongly correlated with nephrin immunofluorescence intensity (r=50.92, P < 0.001) while demonstrating a more modest correlation with UACR (r=−0.30, P = 0.02) (Figure 6, D and E).

Table 2.

Clinical characteristics of kidney biopsy cohort

Characteristic Control (n=11) TIN (n=17) FSGS (n=17) DKD (n=17)
Age, yr 28±9 47±15 47±16 50±17
Female (%) 5 (45) 9 (53) 9 (53) 8 (47)
Black (%) 1 (9) 1 (6) 3 (18) 3 (18)
Hypertension (%) 2 (18) 9 (53) 8 (47) 15 (88)
Diabetes (%) 0 (0) 4 (24) 4 (24) 17 (100)
eGFR, ml/min per 1.73 m2 98±24 25±20 61±38 36±23
UACR, mg/g 218±291 531±593 3583±2682 2426±1988
Open in a new tab

Tubulointerstitial nephropathy, which includes acute interstitial nephritis (n=3); chronic/active interstitial nephritis (n=6); acute tubular injury (n=7); and oxalate nephropathy (n=1). Data are shown as mean±SD. DKD, diabetic kidney disease; TIN, tubulointerstitial nephropathy; UACR, urine albumin/creatinine ratio.

Figure 6. Glomerular testican-2 expression is reduced in patients with FSGS and DKD.

Figure 6.

Open in a new tab

(A) Representative glomerular immunofluorescence of testican-2 (red) and nephrin (green) and (B and C) quantification analyses of immunofluorescence intensities in human kidney biopsies. Scatter plots showing correlations between (D) testican-2 immunofluorescence and nephrin immunofluorescence intensities and (E) testican-2 immunofluorescence intensity and log UACR; n=11 for control, n=17 for other diagnoses. Data are mean±SD; Kruskal–Wallis test with Dunn multiple comparison test (B and C) or Pearson correlation coefficient (r) and corresponding P (D and E). DKD, diabetic kidney disease; IF, immunofluorescence; TIN, tubulointerstitial nephropathy.

Discussion

Building on data spanning >11,000 individuals that identify testican-2 as a circulating marker of kidney health and prognosis,8–10,34 this study identifies testican-2 as a functional mediator of podocyte protection. More specifically, we showed that testican-2 gain-of-function is beneficial, whereas testican-2 loss-of-function is detrimental, in both cultured podocytes and mice subjected to glomerular injury. Furthermore, we identified vitronectin as a key binding partner for testican-2 in the extracellular matrix. In turn, we showed that testican-2 inhibits vitronectin’s interaction with integrin αVβ3 and that its protective effect is associated with reduced integrin αVβ3 activation (Supplemental Figure 7).

Vitronectin has been proposed to play an important role in podocyte injury as a ligand for integrin αVβ3 through its RGD motif.29–31 Wei et al. showed that vitronectin expression is significantly induced in rodent models of podocyte injury, e.g., after puromycin or LPS administration, and in a mouse model of lupus nephritis.30 Furthermore, they found that deletion of integrin β3 or vitronectin attenuates the degree of podocyte injury in mice administered LPS.30 Activation of integrin αVβ3 leads to phosphorylation of its downstream intracellular signaling proteins including Cdc42 and Rac1, which in turn causes actin cytoskeleton disruption and podocyte foot process effacement.35,36 The importance of integrin αVβ3 activation has been independently confirmed, with demonstration that inhibition of integrin αVβ3 signaling reduces podocyte injury in vitro and in vivo.28,37–39 For example, Maile et al. showed that a mAb that inhibits integrin β3 activation attenuates proteinuria and streptozotocin-induced diabetic nephropathy in pigs and rats.38,39 Recently, Koh et al. also found that nonimmune cell–derived inducible costimulator ligand, which contains the RGD motif, binds to integrin αVβ3 and serves as an endogenous αVβ3-selective antagonist that protects against podocyte injury.28

Our study identifies testican-2 as a novel factor that modulates vitronectin and integrin αVβ3 interactions in the extracellular matrix. We note that publicly available single-cell RNA sequencing datasets of mouse and human kidney demonstrate low levels of integrin β3 message in podocytes. In our study, we found that glomerular integrin β3 protein expression was low but detectable at baseline and then increased significantly after glomerular injury. Furthermore, whereas testican-2 reduced the interaction between vitronectin and integrin β3 in the kidney, it had no effect on the interaction between vitronectin and either integrin β1 or integrin β5 (both of which are highly expressed in podocytes). At least in vitro, we also found that testican-2 did not confer any additional podocyte protection in the presence of a pure αVβ3 antagonist. Together, these findings are consistent with our working model whereby testican-2 inhibits vitronectin-mediated integrin αVβ3 activation; however, they do not exclude the possibility that testican-2 also modulates vitronectin’s interaction with additional glomerular proteins in vivo.

Our findings reinforce the critical importance of podocyte interactions with its surrounding extracellular matrix, including the GBM, in maintaining kidney health. Therapeutic targeting of this interface, which spans a number of receptors and adhesion molecules, represents an exciting and as yet untapped approach that has the potential to complement treatments targeting other facets of kidney physiology (e.g., glomerular capillary pressure) and podocyte biology. In our working model, both testican-2 and integrin β3 are expressed by podocytes, with the former secreted into the extracellular matrix. By contrast, the podocyte’s contribution to local glomerular vitronectin content is unclear. We found that glomerular vitronectin protein expression was low at baseline and increased with adriamycin-induced injury but, unlike integrin β3, did not colocalize with nephrin. Thus, the increase in extracellular matrix vitronectin could reflect expression by other cell types or deposition of circulating protein. Importantly, podocyte-specific deletion of testican-2 resulted in reduced interaction between vitronectin and testican-2, and increased interaction between vitronectin and integrin αVβ3, in the kidney.

Because of our specific interest in how testican-2 impacts podocyte health, we used adriamycin as our primary experimental disease model. Adriamycin has been used widely in the literature as it is easy to administer and provides reproducible podocyte injury both in vitro and in vivo.27,40,41 In mice treated with adriamycin, we found that exogenous testican-2 reduces podocyte foot process effacement, glomerular injury, and albuminuria. Importantly, this was accomplished with Spock2 delivery through adenovirus, which increases gene expression in the liver, not the kidney. Thus, increased hepatic expression that results in increased circulating levels of testican-2 was sufficient to confer kidney protection, whether or not there was endogenous testican-2 expression. Consistent with these gain-of-function findings, mice with either global or podocyte-specific Spock2 deletion had greater podocyte foot process effacement, albuminuria, and glomerular injury than control mice after adriamycin, with findings in the global knockout mice extended to a streptozotocin-induced model of diabetic kidney injury.21

Adriamycin-induced injury is often used to model human podocytopathies such as FSGS, while streptozotocin-induced diabetic kidney injury is used to model human DKD.21,27,42 In kidney tissue obtained from individuals with idiopathic FSGS and DKD, we found reductions in glomerular testican-2 expression compared with both controls as well as individuals with significantly impaired kidney function but primarily tubulointerstitial pathology, thus corroborating our mechanistic findings. In addition, glomerular testican-2 expression had a strong positive correlation with nephrin expression and was negatively correlated with albuminuria in this patient cohort. However, we acknowledge that the reduction in testican-2 we observed with human FSGS and DKD is in the context of established disease. Understanding when testican-2 expression begins to fall in relation to disease onset would be of considerable interest. In addition to more detailed studies of human FSGS and DKD, future work in animals should investigate the potential protective role of testican-2 in other disease models.

Several study limitations warrant mention. First, although we provide strong evidence that testican-2 can both interact with vitronectin and attenuate podocyte injury, more work is required to show that its impact on vitronectin-mediated integrin β3 activation underlies its kidney protective effect in vivo. For example, experiments in vitronectin and/or integrin β3–deficient mice could further substantiate the role these molecules play in testican-2–mediated podocyte protection. Second, the detailed binding motifs that govern testican-2 and vitronectin binding are not well understood. Artificial intelligence–powered approaches do not yield high confidence predictions of testican-2 structure that permit modeling of its interaction with vitronectin (not shown).43 Improved understanding of this interaction, for example, using X-ray crystallography or cryo-electron microscopy, could lead to the development of small molecules that recapitulate testican-2’s protective effects. The demonstration that increasing circulating testican-2 levels is beneficial, even when endogenous testican-2 expression is absent, provides strong evidence that an exogenous mimetic could reach its site of action in the kidney, enhancing the feasibility of drug development. Third, little is known about the upstream regulation of testican-2 expression. Additional work is required to understand how testican-2 expression is controlled, why it decreases in response to injury, and whether its expression can be enhanced for therapeutic benefit.

In sum, this study identifies testican-2 as both a marker and mediator of podocyte health, provides new insight on podocyte interactions with the extracellular matrix, and outlines an exciting potential therapeutic pathway in CKD. Future studies will seek to define the testican-2 and vitronectin interaction at higher resolution and to establish the therapeutic potential of modulating testican-2 in other clinically relevant CKD models.

Supplementary Material

Supplement

Supplemental Methods

Supplemental Figure 1. Testican-2 alleviates adriamycin-induced glomerular injury.

Supplemental Figure 2. Mouse testican-2 and deglycosylated human testican-2 bind to vitronectin but not suPAR.

Supplemental Figure 3. Spock2 knockout mice (Spock2−/−).

Supplemental Figure 4. Wild-type and global Spock2 knockout mice treated with streptozotocin.

Supplemental Figure 5. Global deletion of testican-2 exacerbates podocyte injury in streptozotocin-induced diabetic mice.

Supplemental Figure 6. Podocyte-specific deletion of testican-2 exacerbates adriamycin-induced podocyte injury.

Supplemental Figure 7. Proposed model of testican-2–mediated podocyte protection.

Original Immunoblots

This article contains the following supplemental material online at http://links.lww.com/JSN/F511.

Key Points.

  • Testican-2 reduced adriamycin-induced podocyte injury in vitro and in vivo.

  • Testican-2 interacted with vitronectin and reduced vitronectin-mediated integrin αVβ3 activation in vitro.

  • Exogenous testican-2 circulated to the kidney, bound vitronectin, and conferred podocyte protection, even when endogenous testican-2 was absent.

Acknowledgments

Electron microscopy was performed in the Microscopy Core of the Center for Systems Biology/Program in Membrane Biology, which is partially supported by an Inflammatory Bowel Disease Grant DK043351 and a Boston Area Diabetes and Endocrinology Research Center Award DK057521. Because Dr. Eugene P. Rhee is an Associate Editor of JASN, he was not involved in the peer-review process for this manuscript. Another editor oversaw the peer-review and decision-making process for this manuscript.

Funding

D. Wen: National Institute of Diabetes and Digestive and Kidney Diseases (K08DK132411). E.P. Rhee, S.S. Waikar, and M.E. Grams: National Institute of Diabetes and Digestive and Kidney Diseases (R01DK108803). M.A. Arnaout: National Institute of Diabetes and Digestive and Kidney Diseases (R01DK088327).

Footnotes

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F510.

Declarative Statements

This study includes clinical experimentation and received Institutional Review Board or Ethics Committee approval. The need to obtain informed patient consent was waived. This study includes clinical experimentation and complies with the Declaration of Helsinki. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals or an equivalent standard that meets or exceeds the ethical and welfare requirements outlined in the NIH Guide. All protocols were approved by the appropriate institutional animal care and use committee.

Data Availability Statements

Original data generated for the study are available in a public access repository. Data Type: Raw Data/Source Data. Repository Name: PRoteomics IDEntifications database. Linkable Citation: https://www.ebi.ac.uk/pride. All original data, including deidentified patient-level data or individual laboratory data measurements, are included in the manuscript and/or supplemental material.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement

Supplemental Methods

Supplemental Figure 1. Testican-2 alleviates adriamycin-induced glomerular injury.

Supplemental Figure 2. Mouse testican-2 and deglycosylated human testican-2 bind to vitronectin but not suPAR.

Supplemental Figure 3. Spock2 knockout mice (Spock2−/−).

Supplemental Figure 4. Wild-type and global Spock2 knockout mice treated with streptozotocin.

Supplemental Figure 5. Global deletion of testican-2 exacerbates podocyte injury in streptozotocin-induced diabetic mice.

Supplemental Figure 6. Podocyte-specific deletion of testican-2 exacerbates adriamycin-induced podocyte injury.

Supplemental Figure 7. Proposed model of testican-2–mediated podocyte protection.

Original Immunoblots

NIHMS2133258-supplement-Supplement.pdf (1.3MB, pdf)

Data Availability Statement

Original data generated for the study are available in a public access repository. Data Type: Raw Data/Source Data. Repository Name: PRoteomics IDEntifications database. Linkable Citation: https://www.ebi.ac.uk/pride. All original data, including deidentified patient-level data or individual laboratory data measurements, are included in the manuscript and/or supplemental material.

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