Preface

Chronic Kidney Disease (CKD) is a condition characterized by long-term damage to kidney structure, leading to gradual loss of kidney function. Currently, about 10% of adults worldwide are affected by CKD, causing approximately 1.2 million deaths annually. Renal fibrosis is one of the pathological manifestations of CKD, characterized by the activation of fibroblasts and accumulation of extracellular matrix (ECM) in the renal interstitium. Despite significant progress in the treatment of CKD patients, such as the use of renin-angiotensin-aldosterone system inhibitors, novel sodium-glucose cotransporter 2 inhibitors, and non-steroidal mineralocorticoid receptor antagonists, these therapies only have indirect anti-fibrotic effects, and there are no specific anti-fibrotic drugs.

Research Background

Managing renal fibrosis is a highly challenging task, as the complex signal redundancy and diverse cellular responses in kidney diseases significantly increase the difficulty of treatment. Renal fibrosis is often accompanied by a macrophage-dominated immune response, where these macrophages activate fibroblasts at fibrotic sites. However, macrophages are highly heterogeneous, which greatly diminishes their potential as therapeutic targets. Therefore, this study aims to specifically eliminate several macrophage subsets that drive renal fibrosis in a spatiotemporal manner.

Research Team and Publication

This research was conducted by the Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Institute of Nephrology of Chinese PLA, State Key Laboratory of Kidney Diseases, Beijing Key Laboratory of Kidney Diseases, and other collaborative units. The main researchers include Qing Ouyang, Chao Wang, Tian Sang, and others. The paper was published online in the journal “CELL Molecular Immunology” on June 13, 2024.

Research Methods

In this study, researchers first identified the main pro-fibrotic macrophage subsets (FN1+, SPP1+, ARG1+) in the kidney and constructed a 12-peptide glycopeptide named “Bioactivated In Vivo Assembly PK (BIVA-PK)”. This peptide can specifically bind to and internalize into these pro-fibrotic macrophages, thereby reshaping the renal microenvironment by inducing macrophage death and inhibiting pro-fibrotic immune responses.

Sample and Model Establishment

Researchers used a unilateral ischemia/reperfusion injury (uIRI) mouse model to demonstrate the progression of renal fibrosis caused by ischemia-reperfusion injury. Through Masson’s trichrome staining and immunohistochemical analysis, they revealed the process of fibrosis transitioning from acute inflammation to chronic inflammation and developing into fibrosis.

Protein Identification and Functional Analysis

Using techniques such as single-cell RNA sequencing (scRNA-seq) and cytometry by time of flight (CyTOF), researchers successfully identified the main macrophage subgroups associated with renal fibrosis. CD206+ M2-like macrophages were found to be the main contributors to renal fibrosis. Immunofluorescence staining and flow cytometry further confirmed the distribution and expression levels of these macrophages at renal interstitial fibrosis sites.

Site-Specific Treatment

To specifically reduce the number of problematic macrophages and alleviate renal fibrosis, researchers designed a CD206+ macrophage-targeting peptide by modifying BIVA nanotechnology. The terminal was modified with mannose to ensure specific binding to CD206. Subsequently, it was combined with the functional killing peptide LTX-315.

Main Research Findings

According to Western blot analysis and immunostaining, the structurally modified BIVA-PK peptide can specifically enter macrophages through CD206-dependent endocytosis and be cleaved by Cathepsin B in lysosomes. Subsequently, the peptide fragments self-assemble into β-sheet nanofibers intracellularly, pulling the functional peptide PK to trigger macrophage death. BIVA-PK demonstrated high selectivity for pro-fibrotic macrophages and potent anti-fibrotic activity both in vitro and in vivo.

In Vivo Distribution and Safety Testing

In vivo fluorescence imaging data showed that BIVA-PK preferentially accumulated in damaged kidneys for an extended period after injection, while accumulation in healthy kidneys and other major organs was not significant. Fluorescence analysis and histopathological examination of major organs showed that BIVA-PK caused mild systemic toxicity and only mild morphological changes.

Verification of Anti-fibrotic Efficacy

In site-specific treatment experiments, BIVA-PK significantly alleviated renal fibrosis and improved kidney function by inhibiting excessive accumulation of ECM and activation of α-SMA+ fibroblasts. Immunostaining showed a significant reduction in the number of CD206+ macrophages in kidney tissues of mice receiving BIVA-PK treatment.

Conclusion and Significance

BIVA-PK significantly improved renal fibrosis by specifically eliminating pro-fibrotic macrophages, thus providing a potential treatment option for CKD patients. The study shows that BIVA-PK reshapes the microenvironment inducing renal fibrosis, halts the fibrosis process, and protects kidney function, demonstrating high scientific value and application prospects. Future clinical studies are expected to further validate the efficacy and safety of this treatment strategy.

Outlook

Although BIVA-PK has demonstrated its powerful anti-fibrotic ability in the current study, future research is needed to clarify the mechanism of how macrophage death affects immune responses and renal fibrosis. Additionally, considering the species differences between humans and mice, future preclinical studies should use humanized mouse models or large animal models (such as non-human primates) with higher translational potential to replicate human CKD progression.

This study, by proving that FN1+SPP1+MRC1+ macrophages are the main contributors to the pro-fibrotic microenvironment in the kidney, discovered a 12-peptide - BIVA-PK, which can disrupt macrophage membrane and mitochondrial homeostasis and induce cell death, showing powerful efficacy in blocking renal fibrosis and protecting kidney function. This research provides a potential curative method for future CKD patient treatment.