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After acute kidney injury (AKI), patients either recover or alternatively develop fibrosis and chronic kidney disease. Interactions between injured epithelia, stroma, and inflammatory cells determine whether kidneys repair or undergo fibrosis, but the molecular events that drive these processes are poorly understood. Here, we use single nucleus RNA sequencing of a mouse model of AKI to characterize cell states during repair from acute injury. We identify a distinct proinflammatory and profibrotic proximal tubule cell state that fails to repair. Deconvolution of bulk RNA-seq datasets indicates that this failed-repair proximal tubule cell (FR-PTC) state can be detected in other models of kidney injury, increasing during aging in rat kidney and over time in human kidney allografts. We also describe dynamic intercellular communication networks and discern transcriptional pathways driving successful vs. failed repair. Our study provides a detailed description of cellular responses after injury and suggests that the FR-PTC state may represent a therapeutic target to improve repair.

Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER ᵀ² cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.

Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin-positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC-/-) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC-/- mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.

Acute kidney injury (AKI), commonly caused by ischemia, sepsis, or nephrotoxic insult, is associated with increased mortality and a heightened risk of chronic kidney disease (CKD). AKI results in the dysfunction or death of proximal tubule cells (PTCs), triggering a poorly understood autologous cellular repair program. Defective repair associates with a long-term transition to CKD. We performed a mild-to-moderate ischemia–reperfusion injury (IRI) to model injury responses reflective of kidney injury in a variety of clinical settings, including kidney transplant surgery. Single-nucleus RNA sequencing of genetically labeled injured PTCs at 7-d (“early”) and 28-d (“late”) time points post-IRI identified specific gene and pathway activity in the injury–repair transition. In particular, we identified Vcam1⁺/Ccl2⁺ PTCs at a late injury stage distinguished by marked activation of NF-κB–, TNF-, and AP-1–signaling pathways. This population of PTCs showed features of a senescence-associated secretory phenotype but did not exhibit G₂/M cell cycle arrest, distinct from other reports of maladaptive PTCs following kidney injury. Fate-mapping experiments identified spatially and temporally distinct origins for these cells. At the cortico-medullary boundary (CMB), where injury initiates, the majority of Vcam1⁺/Ccl2⁺ PTCs arose from early replicating PTCs. In contrast, in cortical regions, only a subset of Vcam1⁺/Ccl2⁺ PTCs could be traced to early repairing cells, suggesting late-arising sites of secondary PTC injury. Together, these data indicate even moderate IRI is associated with a lasting injury, which spreads from the CMB to cortical regions. Remaining failed-repair PTCs are likely triggers for chronic disease progression.

Acute kidney injury (AKI) is one of the most common clinical syndromes. AKI is associated with significant morbidity and subsequent chronic kidney disease (CKD) development. Thus, it is urgent to develop a strategy to hinder AKI progression. Renal tubules are responsible for the reabsorption and secretion of various solutes and the damage to this part of the nephron is a key mediator of AKI. As we know, many common renal insults primarily target the highly metabolically active proximal tubular cells (PTCs). PTCs are the most energy-demanding cells in the kidney. The ATP that they use is mostly produced in their mitochondria by fatty acid β-oxidation (FAO). But, when PTCs face various biological stresses, FAO will shut down for a time that outlives injury. Recent studies have suggested that surviving PTCs can adapt to FAO disruption by increasing glycolysis when facing metabolic constraints, although PTCs do not perform glycolysis in a normal physiological state. Enhanced glycolysis in a short period compensates for impaired energy production and exerts partial renal-protective effects, but its long-term effect on renal function and AKI progression is not promising. Deranged FAO and enhanced glycolysis may contribute to the AKI to CKD transition through different molecular biological mechanisms. In this review, we concentrate on the recent pathological findings of AKI with regards to the metabolic reprogramming in PTCs, confirming that targeting metabolic reprogramming represents a potentially effective therapeutic strategy for the progression of AKI.

Chronic kidney disease (CKD) remains one of the leading causes of death in the developed world, and acute kidney injury (AKI) is now recognized as a major risk factor in its development. Understanding the factors leading to CKD after acute injury are limited by current animal models of AKI, which concurrently target various kidney cell types including epithelial, endothelial, and inflammatory cells. Here, we developed a mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian diphtheria toxin (DT) receptor in renal epithelia derived from the metanephric mesenchyme. By adjusting the timing and dose of DT, a highly selective model of tubular injury was created to define the acute and chronic consequences of isolated epithelial injury. The DT-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule rather than being widespread in the metanephric mesenchyme–derived epithelial lineage. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation, leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at 1-week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis, and glomerulosclerosis, which was highly correlated with the degree of interstitial fibrosis. Thus, selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction, and potentially glomerulosclerosis, substantiating a direct role for damaged tubule epithelium in the pathogenesis of CKD.

Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD.

Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-β pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment. Kidney compartment–specific eQTL analysis goes beyond GWAS to reveal causal genes and pathways involved in renal disease development.

Mitochondrial dysfunction has been implicated in the pathogenesis of acute kidney injury due to ischemia and toxic drugs. Methods for imaging mitochondrial function in cells using confocal microscopy are well established; more recently, it was shown that these techniques can be utilized in ex vivo kidney tissue using multiphoton microscopy. We extended this approach in vivo and found that kidney mitochondrial structure and function can be imaged in anesthetized rodents using multiphoton excitation of endogenous and exogenous fluorophores. Mitochondrial nicotinamide adenine dinucleotide increased markedly in rat kidneys in response to ischemia. Following intravenous injection, the mitochondrial membrane potential–dependent dye TMRM was taken up by proximal tubules; in response to ischemia, the membrane potential dissipated rapidly and mitochondria became shortened and fragmented in proximal tubules. In contrast, the mitochondrial membrane potential and structure were better maintained in distal tubules. Changes in mitochondrial structure, nicotinamide adenine dinucleotide, and membrane potential were found in the proximal, but not distal, tubules after gentamicin exposure. These changes were sporadic, highly variable among animals, and were preceded by changes in non-mitochondrial structures. Thus, real-time changes in mitochondrial structure and function can be imaged in rodent kidneys in vivo using multiphoton excitation of endogenous and exogenous fluorophores in response to ischemia–reperfusion injury or drug toxicity.

Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia–reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule–specific autophagy-related gene 7–knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia–reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.