Juliette Hadchouel

Background: Acute kidney injury (AKI) is a frequent complication in hospitalized patients, affecting up to 50% of those in intensive care units (ICU). It is linked to high morbidity and mortality, with severity closely tied to patient outcomes. While early biomarkers like KIM1 and NGAL can detect subclinical AKI, their practical benefit is limited, as treatment must begin at suspicion rather than biomarker confirmation. Conversely, when injury exceeds serum creatinine’s diagnostic capacity, a “blind spot” emerges, highlighting the need for “supra-clinical” AKI biomarkers. The KDIGO staging system, based on urine output and creatinine, lacks granularity: even in stage 3, outcomes range from full recovery to permanent dialysis. Anuric patients, despite complete loss of filtration, also show heterogeneous recovery, suggesting injury severity influences prognosis. This study aims to explore “supra-clinical” AKI and assess whether further injury occurs beyond current clinical markers using a preclinical ischemia-reperfusion model and human single-cell transcriptomic data. Results: We performed unilateral renal ischemia of increased duration (5-30 minutes), associated with contralateral nephrectomy, in mice. Plasma urea and creatinine concentration reached a plateau when ischemia duration exceeded 10 minutes. In contrast, we observed a continuous increase in the severity of the renal lesions with the ischemia duration. Similarly, the expression level of Quinolinate phosphoribosyltransferase (QPRT), an enzyme contributing to the biosynthesis of nicotinamide adenine dinucleotide (NAD), was inversely correlated to the ischemia duration. Using single-nucleus RNA sequencing data on human kidney biopsies from ICU patients, we showed a largely reciprocal distribution of QPRT and KIM1 (Kidney Injury Marker 1), expressed specifically in injured proximal tubule cells. Conclusions: Our data demonstrate the existence of a “supra-clinical” stage in AKI, where more ischemia causes more histological and metabolic injury than serum creatinine, serum urea and KIM1 expression can reflect. This highlights a critical diagnostic blind spot: there are currently no available biomarkers to distinguish several stages of AKI when the plasma creatinine concentration does not increase further, or in patients treated with dialysis.

The roles of the long isoform of the With No Lysine (K) 1 (L-WNK1) kinase have been mainly studied in the distal renal tubule, where it participates in sodium and chloride homeostasis. Yet, little is known about its role in its predominant expression site within the kidney, i.e. the glomerulus. We chose to study the consequences of L-WNK1 inhibition in a mouse model of rapidly progressive glomerulonephritis (RPGN), combining podocyte injury and parietal epithelial cell (PEC) activation. We show that L-WNK1 expression is upregulated in glomerular cells during RPGN in mice. A 50% reduction in L-WNK1 expression mitigates experimental nephrotoxic serum-induced RPGN in mice. Given that L-WNK1 is strongly expressed in podocytes, which play an important role in RPGN pathogenesis, we then studied a model of podocyte-specific knockdown of WNK1 . Even if we could observe some changes in NTS-induced RPGN following this knock-down, the effects are not as marked as in the global inhibition model. These data suggest that L-WNK1 plays an important role in other glomerular cell types. Accordingly, we show in vitro that the inhibition of L-WNK1 activity impairs PECs migration. These results suggest that L-WNK1 could represent a new player in the pathophysiology of RPGN.

Nuclear Protein 1 (NUPR1) is a ubiquitous protein playing an important role in cancer and acute kidney injury. Its specific targeting by natural or synthetic antibodies like molecularly imprinted polymers (MIPs), is therefore of interest. NUPR1 is an intrinsically disordered protein (IDP), such that it displays a high degree of flexibility and an unstable secondary/tertiary structure, resulting in a continuous fluctuation of its conformation in the free state. These characteristics are not in favor of the creation of homogeneous binding sites during molecular imprinting, so that imprinting using peptide epitopes is investigated. Based on an in silico rational approach, two α‐helices from the model structure of NUPR1, as predicted by AlPhaFold, are selected. Two cysteine residues are added at both ends of the epitopes to form a disulfide bond, which provides high stability to the α‐helix. The template peptides possess the same 3D structure as the epitope fragments in NUPR1. Consequently, they are effective in producing MIP nanogels that cross‐react with high affinity (IC₅₀ 1 nm) only with NUPR1. The work indicates that α‐helices, besides the preferred flexible loops, can be considered as viable template epitopes for MIPs, opening new opportunities for the obtention of selective antipeptide MIP nanogels for IDPs.

BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe ^−/− to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe ^−/− mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1 -deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6C ^hi monocytes, and γδ T cells. Sm22Cre+Wnk1 ^lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1 ^lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-β (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-β blockade using neutralizing anti-TGF-β antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1 ^lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

ATP depletion is a hallmark of acute kidney injury. Proliferation is instrumental to kidney repair. We show that ATP levels vary during the cell cycle and that proliferation sensitizes renal epithelial cells to superimposed injuries in vitro. More proliferation and less energy production by the mitochondria are associated with adverse outcomes in injured kidney allografts. This suggests that controlling the timing of kidney repair might be beneficial to mitigate the extent of acute kidney injury.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation.

A recent article described a thickening of the glomerular basement membrane (GBM) along with changes in the expression of key components of the extracellular matrix in 6‐month‐old NPHS2‐Cre transgenic mice, which express the Cre recombinase specifically in podocytes. This transgenic line has been widely used to characterize the implication of candidate genes in glomerular diseases in younger mice. Using a different mouse strain (C57BL/6J) than the previous report (129S6/SvEvTac), we sought to characterize 3‐ and 6‐month‐old NPHS2‐Cre^+/− mice in control and pathological conditions. At baseline, there was no difference in renal function and histology between control and NPHS2‐Cre^+/− mice. Notably, GBM thickness evaluated by transmission electron microscopy was similar between the two groups. We then induced an immune‐mediated severe glomerular insult, the anti‐glomerular basement membrane glomerulonephritis model (anti‐GBM‐GN) in 3‐month‐old control and NPHS2‐Cre^+/− mice. NPHS2‐Cre^+/− mice exhibited the same alterations in renal function and structure as control mice. In summary, our study strongly suggests that NPHS2‐Cre^+/− transgenic mice on a C57BL/6J background can be safely used for podocyte‐specific gene inactivation in control conditions and in the anti‐GBM‐GN model.