{'fraction_non_alphanumeric': 0.029408956363983576, 'fraction_numerical': 0.042967631051274706, 'mean_word_length': 4.761276127612761, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Immune-mediated tubule atrophy promotes acute kidney injury to chronic kidney disease transition

Incomplete repair after acute kidney injury can lead to development of chronic kidney disease. To define the mechanism of this response, we compared mice subjected to identical unilateral ischemia-reperfusion kidney injury with either contralateral nephrectomy (where tubule repair predominates) or contralateral kidney intact (where tubule atrophy predominates). By day 14, the kidneys undergoing atrophy had more macrophages with higher expression of chemokines, correlating with a second wave of proinflammatory neutrophil and T cell recruitment accompanied by increased expression of tubular injury genes and a decreased proportion of differentiated tubules. Depletion of neutrophils and T cells after day 5 reduced tubular cell loss and associated kidney atrophy. In kidney biopsies from patients with acute kidney injury, T cell and neutrophil numbers negatively correlated with recovery of estimated glomerular filtration rate. Together, our findings demonstrate that macrophage persistence after injury promotes a T cell- and neutrophil-mediated proinflammatory milieu and progressive tubule damage.


48
Acute kidney injury (AKI) is the syndromic term used to describe the abrupt reduction in 49 glomerular filtration rate (GFR) caused by an insult such as ischemia, sepsis, nephrotoxin 50 exposure or urinary obstruction. Based on animal models, these insults often precipitate a rapid 51 innate immune response dominated by neutrophil and proinflammatory macrophage recruitment 52 with significant tubular and endothelial injury, leading to GFR reduction even after correction of 53 the inciting event 1, 2, 3, 4, 5, 6 . Under ideal conditions (e.g., transient insults in young healthy 54 subjects), repair pathways that include neutrophil egress and a switch to reparative macrophage 55 activation then promote clearance of cell debris and proliferation of surviving tubular cells, 56 leading to restoration of nephron structure and GFR 7,8,9,10 . However, in many cases this repair 57 phase is incomplete and includes maladaptive processes such as interstitial fibrosis and tubule 58 atrophy 11 . As a result, people who survive AKI are at an 8.8-fold increase in risk for chronic 59 kidney disease (CKD) and a 3.3-fold increase in risk for end stage renal disease (ESRD) 12, 13, 14 .

60
To date, no therapy is available to improve the post-injury repair process or prevent progression 61 from AKI to CKD. Defining the signals that promote maladaptive kidney repair following AKI may 62 therefore help us identify therapeutic targets to prevent or even reverse tubule atrophy and the 63 progression to CKD. 6 megalin IF staining analyses confirmed that both mRNA expression and protein levels of the 126 differentiated proximal tubular brush border proteins megalin (Lrp2), NaPi-IIa (Scl34a1, mRNA 127 level alone), and NaDC3 (Slc13a3, mRNA level alone) were significantly lower at 14 and 30 days 128 in the U-IRI kidneys as compared to IRI/CL-NX kidneys (Fig. 2a-c).

130
Both mRNA and protein expression levels of the proximal tubule injury marker Kim1 (Havcr1) 131 were equally upregulated on day 1 after IRI in both models, confirming that the initial ischemic 132 injury was equivalent following U-IRI and IRI/CL-NX (Fig. 2a-c and Supplementary Fig. 2) 28 . Of 133 note, serum levels of both KIM-1 and NGAL were significantly higher on day 1 after IRI/CL-NX 134 than after U-IRI, likely due to clearance by the intact contralateral kidney in mice subjected to 135 U-IRI ( Supplementary Fig. 3). However, Kim1 remained substantially higher in the kidneys 136 subjected to U-IRI on days 7 and 14 at the mRNA level and day 14 at the protein level ( Fig. 2a-c).

137
As compared to Kim1, Vcam1 was not upregulated on day 1 after IRI in either model but 138 markedly increased afterwards, and was significantly higher in the U-IRI kidneys that IRI/CL-NX 139 kidneys from day 7 to day 30 (Fig. 2a), correlating with the period of progressive atrophy seen in 140 this model (Fig. 1b). Together, these data suggest that tubular cells are subjected to equivalent 141 initial ischemic injury in both models correlating with Kim1 expression, followed by delayed injury 142 and failure to redifferentiate that correlates with Vcam1 expression and is exaggerated in the 143 U-IRI model.

147
Because the difference in kidney injury marker expression between the two models became 148 apparent on day 7 (Fig. 2a), we performed single cell RNA sequencing (scRNA-seq) of the 149 injured kidneys on days 7, 14, and 30 after IRI to identify the transcriptional and cellular

161
The kidney atrophy seen on day 14 and 30 after U-IRI was found to correspond to a decrease in

176
and CD8+ cytotoxic T cells were significantly higher in the cortex of U-IRI kidneys as compared 8 to contralateral and IRI/CL-NX kidneys, and all but Ly6G were higher in the outer medulla in the 178 U-IRI model (Fig. 4b, 4c, Supplementary Fig. 6-11). Together, these data demonstrate that 179 macrophages and dendritic cells are the major cell responders in the first 7 days after IRI in both

187
To identify the pathways mediating the second wave of immune cell recruitment following IRI, we

12
To understand whether the second wave of inflammatory T cells and neutrophils promotes 282 kidney tubule atrophy, we depleted either neutrophils, T cells, or both beginning on day 5 283 following U-IRI and analyzed the phenotype of the injured kidney on day 30. We found that 284 depletion of neutrophils and T cells together in the U-IRI model of injury (Fig. 9a-c), but not either

297
Our animal model findings prompted us to investigate the relevance of T cell and neutrophil 298 accumulation for recovery of kidney function in patients with AKI. To assess this, ten kidney 299 biopsies from patients with AKI, an estimated GFR (eGFR) >60 mL/min/1.73m 2 prior to AKI  Tables 6 and 7). We quantified the number of T 305 cells and neutrophils in sections from each of these kidney biopsies, and compared to those 306 seen in kidney biopsies from healthy living kidney donors 36 . As expected, very few T cells or 13 neutrophils were found in kidneys from living donors, whereas T cells and neutrophils were easily 308 detected in the kidney interstitium of some patients at the time of biopsy for AKI (Fig. 10b).

309
Nonparametric Spearman correlation analysis of the number of T cells or neutrophils (quantified 310 as a percentage of total cells identified in the cortex) with either the absolute or relative increase 311 in eGFR at 6 months after AKI revealed a strong negative correlation between inflammatory cell 312 number and eGFR recovery following AKI (Fig. 10c,d).

323
In this study, we show that the initial macrophage, DC, neutrophil and T cell response to 324 ischemia/reperfusion injury is similar during the first 7 days after injury in the two models. total kidney area suggests that the major phenotypic change underlying kidney atrophy after 14 U-IRI is tubule loss rather than increased fibrosis (Fig. 1). In fact, the transition of macrophages 334 to a profibrotic phenotype was seen in both models of IRI ( Supplementary Fig. 5b-f), consistent 335 with evidence that even "reparative" models of kidney injury such as IRI/CL-NX or bilateral IRI 336 lead to increased kidney fibrosis 29, 41 .

338
Similar to the results of Liu and colleagues who studied the slowly progressive kidney atrophy 339 and fibrosis seen following bilateral IRI 41 , we find that the accelerated renal atrophy following

384
The question that our single cell sequencing data raised is whether or not the late influx of T cells and neutrophils, occurring after proreparative macrophage activation has waned, is responsible 386 for the late tubule dedifferentiation, sustained injury and atrophy seen following U-IRI.

387
Experiments in which we selectively depleted either T cells alone or neutrophils alone failed to 388 reduce the progressive kidney and tubule atrophy (Supplemental Figs. 23 and 24), but when we 389 depleted both T cells and neutrophils together there was a significant reduction in the degree of 390 tubule atrophy following U-IRI with preservation of proximal tubule brush border and 391 differentiation (Fig. 9). However, depletion of T cells and neutrophils did not fully prevent kidney 392 atrophy when compared to the contralateral kidneys and control kidneys. This partial effect may 393 be due to our inability to fully eliminate T cell and neutrophil infiltration (approximately 80% 394 reduction based on immunostaining), because accumulation of macrophages and DCs was not 395 prevented by the administration of the anti-Thy1.2 and anti-Ly6G antibodies (data not shown).

397
Alternatively, the partial response to cell depletion reflect that dichotomy of T cell responses to 398 kidney injury 48 . It has been shown that CCR5 was upregulated in the CD3+ infiltrating T cells, 399 and blockade of CCR5 using a neutralizing antibody in mice protected renal function after U-IRI 49 .

400
In addition, the store-operated calcium entry (SOCE) channel, Orai1, participates in the 401 activation of Th17 cells and influences renal injury 50 . Blockage of SOCE in rats attenuated Th17 402 cell activation, inflammation, and severity of AKI following IRI or intramuscular glycerol injection 50 .

594
Living donor samples were collected as previously described 36 . The banked AKI samples 595 obtained between 2015 and 2018 were acquired retrospectively after chart review. All data were 596 deidentified and maintained on a secure database. We estimated GFR (eGFR) using the 597 CKD-epidemiology equation 62 . Ten cases were identified using the following criteria: baseline 598 eGFR ≥60 mL/min/1.73m 2 , presence of AKI, exclusion of those with GN, DKD, or AIN, availability 599 of biopsy sample, and availability of 6-month follow-up eGFR (8/10 with pathologic diagnosis of 600 ATI, acute tubular injury). CD3εand CD66b-cells were detected by IF using primary monoclonal 601 antibodies against CD3ε (#NBP2-53387, Novus Biologicals) and CD66b (#305102, BioLegend), 602 respectively, and proximal tubules were visualized by IF using polyclonal antibody against LRP2 603 (#19700-1-AP, Thermo Fisher Scientific) to identify renal cortical regions on the biopsy specimen.      and 30 after injury. a. Kidney-to-body weight ratios were determined on day 1, 7, 14, and 30 after