Maritza Guadalupe Verdugo-Molinares

RESUMO Introdução: A doença renal crônica (DRC) em fase inicial é frequentemente assintomática e permanece subdiagnosticada em populações de alto risco. As vesículas extracelulares urinárias (uEVs) constituem uma fonte não invasiva de biomoléculas relacionadas ao rim e podem aprimorar as estratégias de detecção precoce. Avaliamos a interleucina-6 (IL-6), a catepsina D (CTD) e o fator de crescimento transformador-β (TGF-β) associados às uEVs como biomarcadores candidatos para a DRC em fase inicial, incluindo padrões estratificados por sexo. Métodos: Neste estudo transversal, 230 adultos de alto risco (diabetes mellitus tipo 2 e/ou hipertensão arterial sistêmica) foram classificados como tendo função renal normal (FRN) ou DRC nos estágios G1–G4. As uEVs foram isoladas por meio de um protocolo padronizado e caracterizadas por microscopia eletrônica de varredura, espectroscopia Raman, espalhamento dinâmico de luz e Western blot para Alix e flotilina-1. A proteína total associada às uEVs foi quantificada, e a CTD, a IL-6 e o TGF-β foram mensurados nas preparações de uEVs. Resultados: As uEVs apresentaram a morfologia e a expressão de marcadores esperadas. As distribuições de tamanho das partículas diferiram conforme o sexo e o estado da DRC. O conteúdo total de proteína das uEVs não diferiu significativamente entre os estágios da DRC. A IL-6 associada às uEVs foi significativamente mais elevada em G1 em comparação com a FRN (p ≤ 0,05). A CTD apresentou uma tendência decrescente de G1 a G4, com diferenças específicas de estágio e sexo, mais evidentes em mulheres no estágio G1. O TGF-β associado às uEVs não diferiu significativamente entre os estágios. Conclusões: A IL-6 e a CTD derivadas das uEVs mostram potencial como indicadores não invasivos de DRC em fase inicial, com padrões dependentes do sexo clinicamente relevantes, enquanto o TGF-β pode fornecer informação complementar. Fluxos de trabalho padronizados de isolamento e mensuração de uEVs são essenciais para a reprodutibilidade dos biomarcadores e para a futura translação clínica.

Background: Early chronic kidney disease (CKD) is frequently asymptomatic and remains underdiagnosed in high-risk populations. Urinary extracellular vesicles (uEVs) provide a non-invasive source of kidney-related biomolecules and may improve early detection strategies. We evaluated uEV-associated interleukin-6 (IL-6), cathepsin D (CTD), and transforming growth factor-β (TGF-β) as candidate biomarkers for early CKD, including sex-stratified patterns. Methods: In this cross-sectional study, 230 adults at high risk (type 2 diabetes mellitus and/or systemic arterial hypertension) were classified as having normal kidney function (NKF) or CKD stages G1–G4. uEVs were isolated using a standardized protocol and characterized by scanning electron microscopy, Raman spectroscopy, dynamic light scattering, and Western blot for Alix and flotillin-1. Total uEV-associated protein was quantified, and CTD, IL-6, and TGF-β were measured in uEV preparations. Results: uEVs displayed expected morphology and marker expression. Particle size distributions differed by sex and CKD status. Total uEV protein content did not significantly differ across CKD stages. uEV-associated IL-6 was significantly higher in G1 compared with NKF (p ≤ 0.05). CTD showed a decreasing trend from G1 to G4 with stage- and sexspecific differences, most evident in women at G1. uEV-associated TGF-β did not differ significantly across stages. Conclusions: uEV-derived IL-6 and CTD show potential as non-invasive indicators of early CKD, with clinically relevant sex-dependent patterns, while TGF-β may provide complementary information. Standardized uEV isolation and measurement workflows are essential for biomarker reproducibility and future clinical translation.

Renal ischemia reperfusion injury (IRI) is a major driver of acute kidney injury and a critical determinant of long term renal outcomes. Although sex differences in susceptibility to renal injury are well recognized, the hormonal mechanisms underlying these disparities remain incompletely understood. Prolactin (PRL), a pleiotropic hormone, has been implicated in both protective and deleterious renal effects, while Cathepsin D (CTD), a lysosomal protease, cleaves PRL into Vasoinhibins that possess antiangiogenic and immunomodulatory properties. The impact of CTD deficiency on PRL's role in renal IRI has not been defined. Here, we investigated whether CTD regulated prolactin signaling modulates renal responses to IRI in a sex dependent manner. Wild type (WT) and heterozygous CTD mice of both sexes were subjected to unilateral renal IRI. PRL overexpression was achieved via lentiviral vector, and renal function, inflammatory cytokines, and angiogenic factors were assessed. In vitro Prolactin cleavage assays confirmed the enzymatic role of CTD. Proteomic analysis of renal cortex tissue was performed using label free LC MS/MS and pathway enrichment tools. CTD cleaved PRL into Vasoinhibins at physiological pH, an effect blocked by Pepstatin A. PRL effects varied by sex and genotype. In WT females, PRL reduced renal dysfunction and increased IL-6 and TGF beta; expression following IRI. CTD+/– females exhibited protection from injury, with PRL treatment reversing IL-6 induction. In males, PRL alone had modest effects, but CTD deficiency led to pronounced creatinine elevation, which PRL treatment normalized. Proteomic data revealed differential regulation of cytoskeletal and inflammatory proteins. Cathepsin D heterozygosity modifies PRL's biological effects on the injured kidney. The CTD, PRL axis regulates key inflammatory and angiogenic responses after IRI, revealing new mechanistic insights and potential therapeutic targets in acute kidney injury.

This chapter focuses on the emerging field of exosomes in the context of reperfusion injuries. Exosomes, nano extracellular vesicles with diverse cargo, play a crucial role in cell-to-cell communication. Exosome quantity and content changes have been implicated in various pathologies, including kidney, brain, heart, and liver ischemia-reperfusion injury. Particularly, exosomes derived from mesenchymal stem cells (MSCs) have shown promising potential as a treatment approach. This chapter aims to provide a comprehensive understanding of exosome biogenesis, the role of organ-specific exosomes in reperfusion injury pathophysiology, and the advantages and prospects of exosome-based treatments. By exploring the multifaceted aspects of exosomes in reperfusion injuries, this chapter will contribute to the advancement of knowledge in this field.

The targeting of transforming growth factor β (TGF-β) has been shown to reduce complications related to ischemia-reperfusion injury (IRI) post-surgically. Pirfenidone (PFD) specifically inhibits TGF-β expression and has been demonstrated to provide protection from IRI in short-term allograft models, though not yet in long-term models. A chronic unilateral IRI model was established using male Wistar rats. The animals were divided into two groups: one with IRI and a pre-treatment of PFD (0.5 mg/kg) followed by 0.5 mg/kg/day of orally administered PFD for 30 days, and a control group without PFD treatment. A sham group was also included. Kidneys and blood samples were collected after 30 days, and the renal function was evaluated by measuring the serum creatinine and KIM-1 levels. RT-PCR was used to analyze fibrosis-related genes, and Luminex to quantify the pro-inflammatory serum IL-18 cytokine. Renal section staining and histological analysis were used to detect collagen deposits. Comparison within the groups showed an increase in serum creatinine and KIM-1 expression after IRI in the control group, while PFD reduced COLL1A1 and TGF-β expression and demonstrated a reduction in fibrosis through histological stains. The treatment group also showed a reduction in IL-18. Our results suggest that PFD exerts protective effects on chronic renal IRI, reducing fibrosis development and inflammation. This study provides new insights into the treatment and management of chronic renal function loss after IRI.

Renal ischemia reperfusion (IR) presents a common challenge for organ graft and function after transplantation. In the kidney, although there are several mechanisms involved in the IR injury, some studies have pointed to angiogenesis as an important process in the pathophysiology of IR and, therefore, as a possible target mechanism to reduce IR damage. Angiogenesis can be modulated by different molecules and recent evidence has shown that opioids are among these molecules. Angiogenesis preconditioning with opioids is a useful and non-invasive strategy to increase the transplant success rate. Although some results have suggested an interaction between the opioid system and VEGF-mediated angiogenesis, more studies are required to fully elucidate the specific mechanisms involved in these actions. The present review summarizes the recent findings on kidney IR-related mechanisms, with as special emphasis on vascular changes. Finally, the evidence about the modulation of angiogenesis by opioids in a preconditioning scheme will be addressed.