The aging kidney is characterized by tubuloinflammaging, a phenotype associated with MHC-II gene expression.

Introduction: Even during physiologic aging, the kidney experiences a loss of mass and a progressive functional decline. This is clinically relevant as it leads to an increased risk of acute and chronic kidney disease. The kidney tubular system plays an important role in the underlying aging process, but the involved cellular mechanisms remain largely elusive. Methods: Kidneys of 3-, 12- and 24-month-old male C57BL/6J mice were used for RNA sequencing, histological examination, immunostaining and RNA-in-situ-hybridization. Single cell RNA sequencing data of differentially aged murine and human kidneys was analyzed to identify age-dependent expression patterns in tubular epithelial cells. Senescent and non-senescent primary tubular epithelial cells from mouse kidney were used for in vitro experiments. Results: During normal kidney aging, tubular cells adopt an inflammatory phenotype, characterized by the expression of MHC class II related genes. In our analysis of bulk and single cell transcriptional data we found that subsets of tubular cells show an age-related expression of Cd74, H2-Eb1 and H2-Ab1 in mice and CD74, HLA-DQB1 and HLADRB1 in humans. Expression of MHC class II related genes was associated with a phenotype of tubular cell senescence, and the selective elimination of senescent cells reversed the phenotype. Exposure to the Cd74 ligand MIF promoted a prosenescent phenotype in tubular cell cultures. Discussion: Together, these data suggest that during normal renal aging tubular cells activate a program of 'tubuloinflammaging', which might contribute to age-related phenotypical changes and to increased disease susceptibility.

Induction of ferroptosis selectively eliminates senescent tubular cells.

The accumulation of senescent cells is an important contributor to kidney aging, chronic renal disease, and poor outcome after kidney transplantation. Approaches to eliminate senescent cells with senolytic compounds have been proposed as novel strategies to improve marginal organs. While most existing senolytics induce senescent cell clearance by apoptosis, we observed that ferroptosis, an iron-catalyzed subtype of regulated necrosis, might serve as an alternative way to ablate senescent cells. We found that murine kidney tubular epithelial cells became sensitized to ferroptosis when turning senescent. This was linked to increased expression of pro-ferroptotic lipoxygenase-5 and reduced expression of anti-ferroptotic glutathione peroxidase 4 (GPX4). In tissue slice cultures from aged kidneys low dose application of the ferroptosis-inducer RSL3 selectively eliminated senescent cells while leaving healthy tubular cells unaffected. Similar results were seen in a transplantation model, in which RSL3 reduced the senescent cell burden of aged donor kidneys and caused a reduction of damage and inflammatory cell infiltration during the early post-transplantation period. In summary, these data reveal an increased susceptibility of senescent tubular cells to ferroptosis with the potential to be exploited for selective reduction of renal senescence in aged kidney transplants.

Single cell versus single nucleus: transcriptome differences in the murine kidney after ischemia-reperfusion injury.

The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions.NEW & NOTEWORTHY Single-cell and single-nucleus RNA sequencing technologies provide powerful new tools to examine complex tissues such as the kidney. This research reference paper provides practical information on the differences between the two technologies when examining murine kidneys after ischemia-reperfusion injury. The results will serve those who are debating which protocols to use in their given study.