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The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.

Endothelial injury and dysfunction: Role in the extension phase of acute renal failure. The pathophysiology of ischemic acute renal failure (ARF) involves a complex interplay between renal hemodynamics, tubular and endothelial cell injury, and inflammatory processes. A growing body of evidence supports the contribution of altered renal vascular function, especially at the microvascular level, in initiating and subsequently extending the initial tubular injury. The extension phase of ischemic ARF involves continued reduction in renal perfusion, ongoing hypoxia, and inflammatory processes that occur during reperfusion and contribute to continued tubular cell injury. Vascular endothelial cell injury and dysfunction play a vital part in this extension phase. With injury, the endothelial cell loses its ability to regulate vascular tone, perfusion, permeability and inflammation/adhesion. This loss of regulatory function has a detrimental impact upon renal function. Vascular congestion, edema formation, diminished blood flow, and infiltration of inflammatory cells have been documented in the corticomedullary junction of the kidney. However, linking their genesis to microvascular endothelial injury and dysfunction has been difficult. New diagnostic and therapeutic approaches to ischemic ARF must incorporate these finding to devise early recognition strategies and therapeutic approaches.

Microvascular endothelial injury and dysfunction during ischemic acute renal failure. The pathophysiology of ischemic acute renal failure (ARF) appears to involve a complex interplay between renal hemodynamics, tubular injury, and inflammatory processes. While the current paradigm of the pathophysiology of ischemic ARF invokes both sublethal and lethal tubular injury as being of paramount importance to diminished renal function, a growing body of evidence supports the contribution of altered renal vascular function in potentially initiating and subsequently extending the initial tubular injury. We propose that the “extension phase” of ischemic ARF involves alterations in renal perfusion, continued hypoxia, and inflammatory processes that all contribute to continued tubular cell injury. Vascular endothelial cell injury and dysfunction play a vital part in this extension phase. In the constitutive state the endothelium regulates migration of inflammatory cells into tissue, vascular tone and perfusion, vasopermeability, and prevents coagulation. Upon injury, the endothelial cell loses its ability to regulate these functions. This loss of regulatory function can have a subsequent detrimental impact upon renal function. Vascular congestion, edema formation, diminished blood flow, and infiltration of inflammatory cells have been documented in the corticomedullary junction of the kidney, but linking their genesis to vascular endothelial injury and dysfunction has been difficult. However, new investigative approaches, including multiphoton microscopy and the Tie2-GFP mouse, have been developed that will further our understanding of the roles endothelial injury and dysfunction play in the pathophysiology of ischemic ARF. This knowledge should provide new diagnostic and therapeutic approaches to ischemic ARF.

STAT3 is a transcriptional regulator that plays an important role in coordinating inflammation and immunity. In addition, there is a growing appreciation of the role STAT3 signaling plays in response to organ injury following diverse insults. Acute kidney injury (AKI) from ischemia-reperfusion injury is a common clinical entity with devastating consequences, and the recognition that endothelial alterations contribute to kidney dysfunction in this setting is of growing interest. Consequently, we used a mouse with a genetic deletion of Stat3 restricted to the endothelium to examine the role of STAT3 signaling in the pathophysiology of ischemic AKI. In a mouse model of ischemic AKI, the loss of endothelial STAT3 signaling significantly exacerbated kidney dysfunction, morphologic injury, and proximal tubular oxidative stress. The increased severity of ischemic AKI was associated with more robust endothelial-leukocyte adhesion and increased tissue accumulation of F4/80+ macrophages. Moreover, important proximal tubular adaptive mechanisms to injury were diminished in association with decreased tissue mRNA levels of the epithelial cell survival cytokine IL-22. In aggregate, these findings suggest that the endothelial STAT3 signaling plays an important role in limiting kidney dysfunction in ischemic AKI and that selective pharmacologic activation of endothelial STAT3 signaling could serve as a potential therapeutic target.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography–mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.

Chen et al. demonstrate endothelial expression of Toll-like receptor 4 (TLR4) in the outer medulla of the kidney early in the course of ischemic acute kidney injury. Furthermore, they provide data that support the hypothesis that activation of endothelial TLR4 in the early extension phase of AKI by damage-associated molecular pattern molecules released from injured tubules results in endothelial activation. This activation can serve to amplify inflammation and tubular damage.

The maintenance of a healthy epithelial-endothelial juxtaposition requires cross-talk within glomerular cellular niches. We sought to understand the spatially-anchored regulation and transition of endothelial and mesangial cells from health to injury in DKD. From 74 human kidney samples, an integrated multi-omics approach was leveraged to identify cellular niches, cell-cell communication, cell injury trajectories, and regulatory transcription factor (TF) networks in glomerular capillary endothelial (EC-GC) and mesangial cells. Data were culled from single nucleus RNA and ATAC sequencing and three orthogonal spatial transcriptomic technologies for correlation with histopathological and clinical trial data. We identified a cellular niche in diabetic glomeruli enriched in a proliferative endothelial cell subtype (prEC) and altered vascular smooth muscle cells (VSMCs). Cellular communication within this niche maintained pro-angiogenic signaling with loss of anti-angiogenic factors. We identified a TF network of MEF2C, MEF2A, and TRPS1 which regulated SEMA6A and PLXNA2, a receptor-ligand pair opposing angiogenesis. In silico knockout of the TF network accelerated the transition from healthy EC-GCs toward a degenerative (injury) endothelial phenotype, with concomitant disruption of EC-GC and prEC expression patterns. Glomeruli enriched in the prEC niche had histologic evidence of neovascularization. MEF2C activity was increased in diabetic glomeruli with nodular mesangial sclerosis. The gene regulatory network (GRN) of MEF2C was dysregulated in EC-GCs of patients with DKD, but sodium glucose transporter-2 inhibitor (SGLT2i) treatment reversed the MEF2C GRN effects of DKD. The MEF2C, MEF2A, and TRPS1 TF network carefully balances the fate of the EC-GC in DKD. When the TF network is \"on\" or over-expressed in DKD, EC-GCs may progress to a prEC state, while TF suppression leads to cell death. SGLT2i therapy may restore the balance of MEF2C activity.

The organizational principles of nephronal segments are based on longstanding anatomical and physiological attributes that are closely linked to the homeostatic functions of the kidney. Novel molecular approaches have recently uncovered layers of deeper signatures and states in tubular cells that arise at various timepoints on the spectrum between health and disease. For example, a dedifferentiated state of proximal tubular cells with mesenchymal stemness markers is frequently seen after injury. The persistence of such a state is associated with failed repair. Here, we introduce a novel analytical pipeline applied to highly multiplexed spatial protein imaging to characterize proximal tubular subpopulations and neighborhoods in reference and disease human kidney tissue. The results were validated and extended through integration with spatial and single cell transcriptomics. We demonstrate that, in reference tissue, a large proportion of S1 and S2 proximal tubular epithelial cells express THY1, a mesenchymal stromal and stem cell marker that regulates differentiation. Kidney disease is associated with loss of THY1 and transition towards expression of PROM1, another stem cell marker shown recently to be linked to failed repair. We demonstrate that the trajectory of proximal tubular cells to THY1 expression is clearly distinct from that of PROM1, and that a state with PROM1 expression is associated with niches of inflammation. Our data support a model in which the interplay between THY1 and PROM1 expression in proximal tubules associates with their regenerative potential and marks the timeline of disease progression.