The kidney’s contribution to the circulating Testican-2 pool is no longer speculative. Paired renal arterial and venous proteomic sampling demonstrated a net increase in circulating Testican-2 from the arterial to the renal venous compartment, establishing that the kidney releases the protein into the bloodstream. Single-nucleus RNA sequencing of human kidney tissue identified podocytes as the predominant renal cell type expressing SPOCK2, while histological studies localized the protein along the glomerular capillary wall near the podocyte-facing aspect of the glomerular basement membrane. Podocyte-specific Spock2 deletion in mice subsequently reduced circulating Testican-2, providing direct evidence that podocytes contribute to the systemic pool. They can therefore be considered a major renal source, although their fractional contribution to the total circulating pool remains undefined, and possible context-dependent expression by other renal cells has not been ruled out.

Higher circulating levels have generally been associated with better-preserved kidney function, less proteinuria, more favorable renal outcomes, and milder glomerular pathology. In community-based cohorts, higher circulating Testican-2 was linked to higher estimated glomerular filtration rate, slower subsequent loss of kidney function, and a lower risk of incident chronic kidney disease. A transethnic proteomic analysis reproduced the positive relationship with eGFR and examined its direction using Mendelian randomization. The results supported an effect of kidney function on circulating Testican-2 but provided no statistically significant evidence for the reverse direction. Circulating abundance is therefore more appropriately interpreted as a correlate of preserved kidney status than as evidence that the plasma protein directly protects the kidney, an interpretation that remains compatible with a local protective role for matrix-associated Testican-2 at the podocyte–GBM interface.

Across the African American Study of Kidney Disease and Hypertension, the Chronic Renal Insufficiency Cohort, and the Atherosclerosis Risk in Communities study, higher circulating Testican-2 was generally associated with less proteinuria or albuminuria and a lower risk of subsequent kidney failure. After adjustment including proteinuria, the association with kidney failure remained significant in AASK and ARIC but not in CRIC, although the pooled association remained significant. A separate proteome-wide analysis found that Testican-2 was the only protein significantly associated with a lower risk of incident kidney failure in the pooled analysis. Yet when Testican-2 was added to fully adjusted models or the Kidney Failure Risk Equation, it did not improve discrimination or risk reclassification for incident end-stage kidney disease. Clinically usable cutoffs, within-person variability, sample stability, and treatment-related changes also remain undefined.

Biopsy data provide a closer link between circulating Testican-2 and glomerular structure. In the Boston Kidney Biopsy Cohort, higher plasma levels were associated with less glomerulosclerosis after adjustment for eGFR, proteinuria, and demographic factors. The analysis was cross-sectional, however, and cannot determine whether reduced circulating Testican-2 precedes podocyte loss or reflects established chronic damage. Tissue studies suggest the relationship differs among glomerular diseases. Glomerular Testican-2 immunofluorescence intensity was significantly lower in diabetic kidney disease, showed a nonsignificant decrease in focal segmental glomerulosclerosis, and did not differ from controls in tubulointerstitial nephropathy. Membranous nephropathy showed a strikingly different pattern: plasma Testican-2 and glomerular SPOCK2 expression were increased, and the combination of Testican-2 with MGT5A, an enzyme involved in glycoprotein processing, improved diagnostic discrimination of membranous nephropathy from other biopsy diagnoses in the Boston Kidney Biopsy Cohort and the Nephrotic Syndrome Study Network.

The best-supported mechanistic link between Testican-2 and the podocyte–GBM interface runs through vitronectin. Vitronectin binds integrin αVβ3, whose excessive activation in podocytes promotes motility, actin remodeling, loss of adhesion, foot process injury, and proteinuria. Immunoprecipitation followed by mass spectrometry identified vitronectin as a Testican-2-interacting extracellular protein. Direct binding was confirmed by biolayer interferometry, and competition assays showed that Testican-2 reduced vitronectin engagement with integrin αVβ3. In cultured podocytes, this was accompanied by lower integrin activation, better preservation of actin organization and adhesion, and less adriamycin-induced injury. How Testican-2 modifies vitronectin remains unresolved: its binding region has not been mapped, and current evidence does not distinguish whether it restricts access to the arginine–glycine–aspartate motif, changes vitronectin conformation, or alters its presentation within the extracellular matrix. Deglycosylation weakens but does not abolish the interaction, suggesting that carbohydrate chains facilitate rather than solely determine binding.

The protective phenotype was reproduced in vivo. Both global and podocyte-specific Spock2 deletion increased susceptibility to adriamycin-induced glomerular injury, with greater albuminuria and foot process effacement, and global deletion also exacerbated streptozotocin-induced injury. In Spock2-deficient mice, adenoviral expression of Spock2 increased circulating Testican-2; the restored protein reached the glomerulus, bound vitronectin, reduced the vitronectin–integrin αVβ3 interaction, and attenuated podocyte and filtration barrier injury. These findings implicate the vitronectin–integrin αVβ3 pathway in the protective effects of Testican-2, but whether vitronectin binding is required for protection in vivo remains unclear. The mechanistic and rescue evidence comes largely from a single integrated study and requires independent replication. Podocytes are not the only glomerular cells that respond: in primary human glomerular endothelial cells, Testican-2 increased tube formation and migration without increasing proliferation, effects accompanied by increased matrix metalloproteinase-2 and -9 activity in the culture medium, although the role of Testican-2 in the glomerular endothelium in vivo remains unclear.

Current evidence allows three overlapping interpretations of plasma Testican-2. Under a coupled-source model, podocytes contribute to both the matrix-associated glomerular pool and the protein entering renal venous blood, consistent with the renal arteriovenous gradient, podocyte-enriched SPOCK2 expression, lower circulating levels after podocyte-specific deletion, and associations with glomerular pathology. A second interpretation places kidney status upstream of the circulating signal, so higher levels could reflect preserved kidney function and be associated with favorable outcomes without implying that circulating Testican-2 itself mediates protection. Partial uncoupling is also plausible: matrix retention and glycosylation may influence local availability and vitronectin binding, whereas proteolytic processing, urinary loss, extrarenal production, systemic clearance, and epitope accessibility may alter what reaches plasma or is detected by current assays. The higher plasma Testican-2 levels and glomerular SPOCK2 expression reported in membranous nephropathy highlight a disease-specific pattern, though whether increased glomerular expression or glomerular basement membrane remodeling contributes to the higher circulating levels remains unclear.

Practical uncertainties compound these interpretive questions. Most kidney cohort studies measured relative Testican-2 abundance using aptamer-based platforms, and whether these measurements are equivalent to antibody-based or mass-spectrometry measurements remains unknown. Affinity-based protein measurements can show substantial inter-platform discordance and may be affected by epitope accessibility. Testican-2 can undergo N-linked glycosylation and cell-dependent glycosaminoglycan modification, and proteolytic processing and matrix association may further influence local retention and release, but kidney-specific cleavage products have not been systematically characterized. Cross-platform analysis of the same samples and molecularly defined reference preparations are needed before absolute thresholds can be compared. The intended use must also be specified: a marker for long-term kidney failure risk and a diagnostic marker for membranous nephropathy require different study populations, comparators, and performance criteria. Anti-phospholipase A2 receptor testing was not included in the models assessing Testican-2 and MGT5A, so the combination’s added diagnostic value beyond anti-PLA2R testing remains unclear, and its performance in anti-PLA2R-negative patients was not evaluated separately.

Therapeutic development remains at the proof-of-concept stage. Published restoration experiments increased Testican-2 through adenoviral Spock2 expression rather than a clinically deployable protein or targeted formulation. A Testican-2 preparation would need a defined active form, adequate exposure at the podocyte-facing glomerular basement membrane, measurable engagement of vitronectin, and an acceptable safety profile. Raising the plasma concentration alone may not reproduce a matrix-dependent local effect, and nonrenal distribution, immunogenicity, durability, and interference with physiological vitronectin–integrin signaling will all require evaluation. The most informative next studies would combine longitudinal plasma and tissue sampling with quantitative tracing of podocyte-derived Testican-2 and molecularly resolved proteomic or glycoproteomic analysis. Rescue using a Testican-2 variant that retains secretion and glomerular localization but lacks vitronectin-binding activity would directly test whether this interaction is required in vivo. Until such evidence is available, circulating Testican-2 is best regarded as a candidate marker of preserved renal status rather than a direct readout of local glomerular activity, a distinction that will determine its potential as a context-specific biomarker, a locally acting extracellular therapeutic candidate, or both.

Subject of Research: A new review examines how Testican-2, a podocyte-derived proteoglycan, links glomerular biology to circulating biomarker potential in kidney disease.

Article Title: Testican-2: from glomerular activity to circulating biomarker

Article References: Luo, J., Sun, H., Jiang, L.-Y., & Xiao, X.-H. (2026). Testican-2: from glomerular activity to circulating biomarker. Frontiers in Physiology, 17, Article 1953293. https://doi.org/10.3389/fphys.2026.1953293

Image Credits: AI Generated

DOI: 10.3389/fphys.2026.1953293

Keywords: kidney disease, podocytes, glomerulus, biomarker, proteomics, SPOCK2, vitronectin, integrin αVβ3, membranous nephropathy, chronic kidney disease, Testican-2, glomerular