Synthetic peptides block a kidney-damage molecule and protect renal function in mice

Synthetic peptides modeled on two short sequences within the extracellular matrix protein biglycan reduced inflammation and preserved kidney function in a mouse model of renal ischemia–reperfusion injury, according to a study published in Cellular & Molecular Biology Letters.

Renal ischemia–reperfusion injury arises when blood flow to the kidney is temporarily restricted and then restored, a sequence common to kidney transplantation, cardiac surgery, sepsis, and trauma. The reperfusion phase triggers hypoperfusion damage followed by reoxygenation, driving inflammation, immune cell infiltration, cytokine and chemokine secretion, apoptosis, and necroptosis. The result is tubular damage, reduced glomerular filtration rate, and elevated serum creatinine. The authors note that management of acute kidney injury and chronic kidney disease remains limited to dialysis or transplantation, with no curative drug therapies, which motivates the search for new targets.

Biglycan, a small leucine-rich proteoglycan of the extracellular matrix, normally contributes to cell–matrix interactions and tissue structure through its 42-kilodalton core protein, twelve tandem leucine-rich repeats, and two covalently attached glycosaminoglycan chains. When proteolytically cleaved from the matrix, however, soluble biglycan behaves as a damage-associated molecular pattern, or DAMP: an endogenous danger signal that binds TLR2 and TLR4 and activates the transcription factor NF-κB, inducing proinflammatory cytokines and chemokines such as TNFα, CXCL1, and CCL2. Elevated serum biglycan correlates with the progression of ischemic acute kidney injury and chronic kidney disease, and previous work by the same group showed that biglycan–TLR2/TLR4 signaling worsens outcomes in a murine model of ischemic kidney injury.

To locate the receptor-binding region, the team used PepSpot peptide arrays with overlapping 15-amino-acid fragments spanning the human biglycan sequence, incubated with recombinant human TLR2 and TLR4. The screen revealed that sequences in leucine-rich repeat 2 (LRR2) and leucine-rich repeat 10 (LRR10) bound both receptors strongly. Sequence alignment pinpointed the shared motifs 113LQHLYA118 in LRR2 and 302LQVVYL307 in LRR10, which fit a consensus pattern L-Q-X1-V/L-Y-X2. Structural visualization of the biglycan dimer placed these peptides on one face of the molecule, consistent with a role in receptor contact. TLR4 also showed strong binding to LRR3 and LRR4 and weaker signals with several other repeats, but LRR2 and LRR10 were the common strong binders for both receptors.

The researchers then tested whether these sequences are functionally required. Using site-directed mutagenesis, they either swapped the motifs with the corresponding stretches of neighboring repeats—generating the mutants swLRR2 and swLRR10—or substituted the exposed polar glutamine and tyrosine residues with nonpolar isoleucine and phenylalanine, producing LRR2ΔF and LRR10ΔF. Microscale thermophoresis, an immobilization-free method for measuring binding in solution, showed that wild-type biglycan bound TLR2 with a dissociation constant of roughly 37–39 nanomolar and TLR4 at about 24–26 nanomolar. The effect was more pronounced for TLR4 than for TLR2.

Weakened binding translated into weakened signaling. In HEK-Blue-TLR2 and HEK-Blue-TLR4 reporter cells, all four biglycan mutants induced lower NF-κB-dependent reporter activity than wild-type biglycan. In primary murine macrophages, stimulation with the mutants produced significantly less ERK phosphorylation and lower mRNA and protein levels of CXCL1, CCL2, and TNFα than wild-type biglycan, measured by quantitative PCR and ELISA. Cell viability assays confirmed that neither the biglycan variants nor the peptides were cytotoxic at the concentrations used, while the kinase inhibitor staurosporine abolished viability as a positive control.

Building on the identified motifs, the team designed two longer synthetic peptides: peptide 1, spanning residues 109–127 and containing the LRR2 sequence LQHLYA, and peptide 2, spanning residues 293–315 and containing the LRR10 sequence LQVVYL. Both peptides bound TLR2 and TLR4 with nanomolar dissociation constants—peptide 1 at 37.8 and 28.1 nanomolar, peptide 2 at 42.0 and 23.4 nanomolar, respectively—although slightly weaker than full-length wild-type biglycan. Albumin, used as a negative control, showed no binding. AlphaFold-Multimer predictions suggested the peptides do not form stable homo- or heterodimers that could complicate their behavior, and in silico estimation with the PlifePred platform predicted short blood half-lives of approximately 6 minutes for peptide 1 and 11.5 minutes for peptide 2.

In cell-based inhibition assays, preincubation with peptides 1 and 2 significantly blocked biglycan-induced NF-κB activation in the TLR2 and TLR4 reporter cells and suppressed ERK phosphorylation, CXCL1, CCL2, and TNFα expression in primary murine macrophages. Crucially for translational relevance, the same inhibition was observed in human monocyte-derived macrophages from four healthy donors, where peptide pretreatment significantly reduced biglycan-induced mRNA and protein expression of all three inflammatory mediators.

The in vivo test used male C57BL/6J mice subjected to renal ischemia–reperfusion injury, with 25 minutes of clamping of the right renal artery after removal of the left kidney. Eighteen mice were divided into four groups: sham-operated controls, sham-operated mice given peptides, untreated IRI mice, and peptide-treated IRI mice, with six animals per IRI group and three per sham group. Peptides 1 and 2 were injected intravenously 30 minutes before surgery. Twenty hours after reperfusion—a time point the authors describe as one of maximal immune cell infiltration—immunohistochemistry showed significantly reduced infiltration of neutrophils and macrophages into kidney tissue of peptide-treated IRI mice compared with untreated IRI mice. Renal tissue expression of Cxcl1, Ccl2, and Tnfα was substantially lower, and serum creatinine, a standard indicator of kidney function, was significantly decreased in the treated group. Peptides alone did not induce inflammatory markers in sham-operated animals, indicating they were nonimmunogenic under these conditions.

The authors frame the approach as an alternative to broad TLR blockade. Because TLR2 and TLR4 signaling is also needed for tissue repair and disease resolution after injury, directly inhibiting the receptors risks interfering with recovery; neutralizing a specific endogenous DAMP ligand instead may blunt the acute inflammatory surge while leaving beneficial receptor signaling intact. They also caution that the findings represent proof of concept for short-term, acute pretreatment only. The peptides’ predicted single-digit-minute blood half-lives, susceptibility to proteolytic degradation, limited bioavailability, and unresolved questions about long-term receptor dynamics, tissue regeneration, and skeletal effects of chronically interfering with biglycan—all biglycan is a structural component of bone and tendon matrix—mean that chemical modifications, improved delivery systems, and further preclinical testing in models such as renal transplantation will be required before clinical translation. The study was conducted under German animal protection law with approval from the Ethics Review Committee for Laboratory Animals of the District Government of Darmstadt.

Subject of Research: Identification of biglycan TLR2/TLR4 binding sequences and development of inhibitory peptides for renal ischemia–reperfusion injury

Article Title: Targeting biglycan–TLR2/TLR4 interaction with synthetic peptides mitigates inflammation and ameliorates renal function in ischemia–reperfusion injury

Article References: Merline, R., Hsieh, L. T.-H., Zeng-Brouwers, J., Vutukuri, R., Briggs, D. C., Day, A. J., Wygrecka, M., Nowak, S., Bartoszewski, R., & Schaefer, L. (2026). Targeting biglycan–TLR2/TLR4 interaction with synthetic peptides mitigates inflammation and ameliorates renal function in ischemia–reperfusion injury. Cellular & Molecular Biology Letters, 31(1), Article 151. https://doi.org/10.1186/s11658-026-01019-2

Image Credits: AI Generated

DOI: 10.1186/s11658-026-01019-2

Keywords: biglycan, TLR2, TLR4, ischemia-reperfusion injury, acute kidney injury, inflammation, damage-associated molecular patterns, NF-kB, macrophages, synthetic peptides, serum creatinine, renal function