Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Peritoneal dialysis (PD) employs hypertonic glucose to remove excess water and uremic waste. Peritoneal membrane failure limits its long‐term use. T‐cell cytokines promote this decline. T‐cell differentiation is critically determined by the microenvironment. We here study how PD‐range hypertonic glucose regulates T‐cell polarization and IL‐17 production. In the human peritoneal cavity, CD3+ cell numbers increased in PD. Single cell RNA sequencing detected expression of T helper (Th) 17 signature genes RORC and IL23R. In vitro, PD‐range glucose stimulated spontaneous and amplified cytokine‐induced Th17 polarization. Osmotic controls l‐glucose and d‐mannose demonstrate that induction of IL‐17A is a substance‐independent, tonicity dose‐dependent process. PD‐range glucose upregulated glycolysis and increased the proportion of dysfunctional mitochondria. Blockade of reactive‐oxygen species (ROS) prevented IL‐17A induction in response to PD‐range glucose. Peritoneal mesothelium cultured with IL‐17A or IL17F produced pro‐inflammatory cytokines IL‐6, CCL2, and CX3CL1. In PD patients, peritoneal IL‐17A positively correlated with CX3CL1 concentrations. PD‐range glucose‐stimulated, but neither identically treated Il17a−/−Il17f−/− nor T cells cultured with the ROS scavenger N‐acetylcysteine enhanced mesothelial CX3CL1 expression. Our data delineate PD‐range hypertonic glucose as a novel inducer of Th17 polarization in a mitochondrial‐ROS‐dependent manner. Modulation of tonicity‐mediated effects of PD solutions may improve membrane survival.

Background: The risk of cardiovascular events rises after acute kidney injury. Leukocytes promote atherosclerotic plaque growth and instability. We have established a model of enhanced remote atherosclerosis after renal ischemia reperfusion (IR) injury and investigate the underlying inflammatory mechanisms. Methods: Atherosclerotic lesions and inflammation were investigated in native and bone marrow-transplanted LDL receptor-deficient (LDLr-/-) mice after unilateral renal IR injury using histology, flow cytometry, and gene expression analysis. Results: Aortic root atherosclerotic lesions were significantly larger after renal IR injury than in controls. A gene expression screen revealed enrichment for chemokines and their cognate receptors in aortas of IR-injured mice in early atherosclerosis, and of T cell-associated genes in advanced disease . Confocal microscopy revealed increased aortic macrophage proximity to T cells. Differential aortic inflammatory gene regulation in IR-injured mice largely paralleled the pattern in the injured kidney. Single-cell analysis identified renal cell types that produced soluble mediators upregulated in the atherosclerotic aorta. The analysis revealed a marked early increase in Ccl2, which CCR2+ myeloid cells mainly expressed. CCR2 mediated myeloid cell homing to the post-ischemic kidney in a cell-individual manner. Reconstitution with Ccr2-/- bone marrow dampened renal post-ischemic inflammation, reduced aortic Ccl2 and inflammatory macrophage marker CD11c, and abrogated excess aortic atherosclerotic plaque formation after renal IR. Conclusions: Our data introduce an experimental model of remote proatherogenic effects of renal IR and delineate myeloid CCR2 signaling as a mechanistic requirement. Monocytes should be considered as mobile mediators when addressing systemic vascular sequelae of kidney injury.

Renal immune cells serve as sentinels against ascending bacteria but also promote detrimental inflammation. The kidney medulla is characterized by extreme electrolyte concentrations. We here address how its main osmolytes, NaCl and urea, regulate tubular cell cytokine expression and monocyte chemotaxis. In the healthy human kidney, more monocytes were detected in medulla than cortex. The monocyte gradient was attenuated in patients with medullary NaCl depletion by loop diuretic therapy and in the nephrotic syndrome. Renal tubular epithelial cell gene expression responded similarly to NaCl and tonicity control mannitol, but not urea. NaCl significantly upregulated chemotactic cytokines, most markedly CCL26, CCL2, and CSF1. This induction was inhibited by the ROS scavenger n-acetylcysteine. In contrast, urea, the main medullary osmolyte in catabolism, dampened tubular epithelial CCL26 and CSF1 expression. Renal medullary chemokine and monocyte marker expression decreased in catabolic mice. NaCl-, but not urea-stimulated tubular epithelium or CCL2 and CCL26, promoted human classical monocyte migration. CCL26 improved bactericidal function. In the human kidney medulla, monocyte densities correlated with tubular CCL26 protein abundance. In summary, medullary-range NaCl, but not urea, promotes tubular cytokine expression and monocyte recruitment. This may contribute to the pyelonephritis vulnerability in catabolism but can possibly be harnessed against pathologic inflammation.

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