All experiments were performed following the guidelines in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (Protocol No. 00063). Mice were maintained on a standard diet under pathogen-free housing conditions, with food and water available ad libitum.

Renal tubule-specific cadherin Cre Atg7^−/− mice (KSP1.3 Cad Cre Atg7^−/− mice) were developed by Cre recombinase technology by breeding KSP 1.3 Cad Cre mice with Atg7 floxed mice. KSP1.3 Cad Cre Atg7^−/− mice have expression of Cre recombinase in adult mice restricted to renal tubular epithelial cells with the highest expression in collecting ducts and loops of Henle and low expression in proximal tubules (22, 23). The mean age of the wild-type and Atg7^−/− mice used in the current study was 400 days old.

Renal tubule-specific cadherin Cre Atg7^−/−, Raptor^−/− mice (KSP1.3 Cad Cre Atg7^−/−, Raptor^−/− mice) were developed by Cre recombinase recombination by breeding KSP 1.3 Cad Cre mice with Atg7 floxed mice and Raptor floxed mice.

Pkd1^RC/RC mice have a hypomorphic Pkd1 gene mutation orthologous to the PKD patient disease variant, PKD1 p. R3277C (24). Pkd1^RC/RC mice in the C57BL/6 background have cysts at 3 months of age (25). Cyst expansion and size correlate with increased tubular cell proliferation (24). Wild-type C57BL/6J mice (No. 000664) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Kidneys from our previously published Torin2-treatment study (26) were examined for TLTs.

Torin2 is a potent mTOR inhibitor (27). Male and female C57BL/6 Pkd1^RC/RC mice were treated with the mTOR inhibitor Torin2 (10 mg/kg IP, daily on weekdays) or vehicle (28% DMSO in PEG300) from days 50 to 120 of age (26). There were equal numbers of males and females per group. Kidneys from our previously published study were used for the present study (26). Torin2 was purchased from ChemieTek, Indianapolis, IN, USA.

Animals received fast imaging with steady-state precession (FISP) abdominal MRI measurements by UC Denver small animal imaging core 1 month before killing, as we have described (28).

Six-month-old male wild-type C57BI/6J mice obtained from Jackson Laboratories were used. The ischemic injury was induced by clamping of the left renal pedicle for 45 min. Due to the prolonged time of the clamp, kidneys that did not reperfuse well (returned to pink color) were removed from the studies. The contralateral nonclamped right kidney was used as the control. Mice were killed 45 days after the renal pedicle clamp. The protocol for in vivo ischemic injury has previously been detailed by us (29), and the kidney phenotype of 45 min of unilateral renal ischemia in 6-month-old mice has been detailed (30). Mice were treated with the mTOR inhibitor Torin2 (10 mg/kg IP, daily on weekdays) or vehicle (28% DMSO in PEG300) for 35 days, starting from day 10 after the renal pedicle clamp until they were killed. Torin 2 was administered on day 10 after the ischemic insult because it caused mortality if administered in the first week after the ischemic insult when the mice were still recovering from the surgery. Torin2 treatment was started as early as possible after ischemia, as we wanted to determine whether mTOR inhibition could inhibit both the formation and growth of TLTs.

In a previous in vivo study, we demonstrated that Torin2 at a dose of 10 mg/kg/day IP significantly decreases p-S6 in the kidney, slows cyst growth, and improves kidney function in the Pkd1^RC/RC mouse model of ADPKD (26). Thus, the Torin2 dose of 10 mg/kg/day IP was chosen for the current study.

Blood urea nitrogen (BUN) was measured with a BioAssay Systems (Hayward, CA, USA) Urea Assay Kit according to the manufacturer’s instructions (DIUR-100). Serum creatinine was measured with high-performance liquid chromatography (HPLC) tandem mass spectrometry. [²H3] and creatinine were detected in multiple reaction-monitoring modes, examining transitions of m/z from 114 to 44.2 and from 117 to 47.2, respectively (31).

Transcutaneous glomerular filtration rate (GFR) (tGFR) was measured and performed as we have previously reported (32). The NIC-Kidney device (MediBeacon Inc, Amtsgericht, Germany) was utilized for tGFR measurements per the manufacturer’s instructions.

Histological changes due to acute tubular necrosis (ATN) score were evaluated in the outer stripe of the outer medulla on hematoxylin–eosin stained tissue and were quantified by counting the percent of tubules that displayed cell necrosis, loss of brush border, and cast formation as follows: 0 = none, 1 ≤ 10%, 2 = 10%–25%, 3 = 26%–45%, 4 = 46%–75%, and 5 ≥ 75%. At least 10 fields (× 200) were reviewed for each slide.

Protein was isolated from tissues using radioimmunoprecipitation assay (RIPA), cOmplete protease, and phoSTOP phosphatase inhibitor cocktails (Sigma, St Louis, MO). Homogenates were centrifuged, and the supernatant was taken for protein quantification by BioRad (Hercules, CA, USA) DC Protein Assay as described by the manufacturer. Samples were mixed with Laemmli sample buffer and boiled for 5 min. Samples were run on 12% fresh polyacrylamide gels. Proteins were then transferred to 0.45 µm PVDF membranes, blocked with 2% evaporated milk, and probed with antibodies listed in Supplementary Table S1 . The specificity of each of the antibodies used has been validated by the vendor (Cell Signaling Technology, Danvers, MA, USA) and has been cited in our and others’ previous publications (33–39). Blots were developed by chemiluminescence and analyzed for densitometry using ImageJ.

Tissues were fixed overnight in 10% formalin at 4°C. They were transferred to fresh 70% ethanol and left overnight at 4°C; this process was repeated twice more. Next, the tissues were processed and embedded in paraffin wax using Leica systems. Tissues were sectioned at 4 µm and baked at 60°C for 2 h. Kidneys were stained with hematoxylin–eosin and proliferating cell nuclear antigen (PCNA). TLT number, area, and index (% of the kidney) were quantified in PCNA-stained kidney sections using an NIS Element macro, as previously published (25), in tissue sections visualized by the Aperio ImageScope (Leica Biosystems, St louis, MO).

Tissue sections were deparaffinized and rehydrated, then antigen unmasking was performed in sodium citrate buffer (pH 6.0) for 25 min at 100°C. After cooling sections to room temperature in citrate buffer, endogenous peroxidase activity was blocked by immersing the sections in 3% hydrogen peroxide for 10 min, followed by a 5-min rinse in deionized water. Blocking was performed using Vectastain^® Elite^® ABC Kit blocking serum for 30 min at room temperature. Primary antibodies were diluted in tris-buffered saline with Tween20 (TBST) as indicated in Supplementary Table S1 and incubated overnight at 4°C in a dark humidified chamber. Immunoreactions were detected using the Vectastain^® standard protocol with 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) counterstained with hematoxylin. Slides were subsequently dehydrated and mounted. DAB-positive staining was analyzed using Aperio ImageScope-provided macros. Multiplexed staining for cluster of differentiation 45 (CD45), CD4, CD8, SH2 domain-containing leukocyte protein of 76 kDa (SLP76), forkhead box protein P3 (FOXP3), and programmed death ligand 1 (PDL1) was performed by the Human Immune Monitoring Shared Resource Core at the University of Colorado Anschutz using the Vectra Polaris Imaging System (Akoya Biosciences, Malborough, MA) according to manufacturer’s instructions.

Quantitation of staining was performed using the Aperio ImageScope, Leica Biosystems, by an observer blinded to the treatment modality. The TLT number was counted from the whole kidney stained with PCNA. The TLT index was calculated by dividing the total area of TLTs by the total area of the kidney. For quantitation of TLT staining of proteins, other than PCNA, the percentage of area of the TLT was calculated using the provided Aperio algorithms. As PCNA is a nuclear stain, the percentage of positive cells in the TLT was calculated using the provided Aperio algorithms. For tubular staining, the percentage of positive cells was calculated by randomly selecting 10–15 areas, each 9,000 µm², and measuring positive staining intensity using the provided Aperio algorithms.

Kidney tissues were fixed in 10% phosphate-buffered formalin (Fisher Scientific, Waltham, MA, USA) and embedded in paraffin. Sections (5 μm thick) were scanned in 15 random regions of interest. Images were acquired at × 20 magnification using a laser-scanning confocal microscope (Zeiss 780, Carl Zeiss, Jena, Germany) equipped with a titanium-sapphire laser (Chameleon Ultra II, Coherent, Santa Clara, CA, USA). The average laser power of 7% at 800 nm (tuned for a second-harmonic generation [SHG]) with 140-fs pulse duration and 80-MHz repetition rate was used. After passage through the microscope optics, the pulse duration was ~ 300 fs. SHG signal was detected on a nondescanned detector following transmission through a filter cube containing a narrow-band 390- to 410-nm emission filter (product no. hq400/20m-2p, Chroma Technology, Bellows Falls, VT, USA). Collagen was quantified using ImageJ (National Institutes of Health). The green (autofluorescence) and red (fibrillar collagens) channels were separated and a threshold was set for the collagen. The percent area was quantified using the threshold value.

Student’s t-test was used for comparisons between two independent groups. Multiple group comparisons were performed using analysis of variance (ANOVA) with posttest according to Newman–Keuls. Student’s t-test was used for comparisons between two independent groups. A p-value of < 0.05 was considered statistically significant. Values are expressed as means ± SEM.

The striking feature of 400-day-old renal tubule-specific cadherin Cre Atg7^−/− mice (KSP1.3 Cad Cre Atg7^−/− mice), a conditional Atg7 knockout mouse model, was the presence of large TLTs. On low-power magnification of hematoxylin–eosin-stained kidneys, the number and size of TLTs in kidneys were increased in Atg7^−/− kidneys vs. wild-type control kidneys ( Figures 1A, B ). As suppressed autophagy and increased mTOR play a role in kidney cyst growth in polycystic kidney disease (12, 36, 40, 41), our original hypothesis was that Atg7^−/− kidneys would be cystic. Only one out of seven Atg7^−/− kidneys ( Figure 1C ) and no wild-type kidneys were cystic. On higher power magnification of hematoxylin-eosin staining of a TLT in the kidney, the majority of cells resembled immune cells ( Figures 1D, E ). TLTs were mostly situated next to blood vessels and often engulfed blood vessels ( Figure 1F ). The number, percent of kidney area (TLT index), and average TLT size were increased in Atg7^−/− vs. wild-type kidneys ( Figure 1G ). In one Atg7^−/− kidney, the TLTs occupied 9% of the area of the kidney ( Figure 1G ). One Atg7^−/− kidney was virtually completely replaced by lymphoid tissue ( Figures 1H, I ). Due to the extreme nature of this kidney, it was not included in the analysis in Figure 1G .

There was no difference in the kidney weight ( Figure 1J ), the appearance of the kidney on fast imaging with steady-state free precession (FISP)-MRI scan ( Figure 1K ), or kidney function ( Figure 1L ) in aged Atg7^−/− vs. wild-type control (+/+) kidneys.

In aged wild-type control (+/+) kidneys there was no difference in the mean TLT number ± SEM (2.0 ± 1 vs. 3.0 ± 1.4) or mean TLT index ± SEM (0.3 ± 0.1 vs. 0.46 ± 0.2) in males versus females. In Atg7^−/− kidneys, 6/7 mice were female, so a comparison between males and females was not performed.

Tubule-specific Atg7^−/− mice were expected to have suppressed autophagy in tubular epithelium since Atg7 is an essential enzyme in the process of autophagy. The next aim of the study was to confirm that Atg7 was knocked out in the tubules in the aged renal tubule-specific cadherin Cre Atg7^−/− mice (KSP1.3 Cad Cre Atg7^−/− mice), a conditional knockout mouse model. During autophagy, the phospholipid phosphatidylethanolamine (PE) (42) anchors LC3-I (microtubule-associated protein 1A/1B-light chain 3) to the emerging phagophore membrane where it is lipidated to form LC3-II. LC3 lipidation is a multistep process, driven by the E1‐like enzymatic activity of homodimeric Atg7. Sequestosome 1/SQSTM1 (p62) is an adapter protein and a cargo receptor for autophagy (43, 44). p62 interacts with phagosomes by binding to LC3 through the LC3-interacting (LIR) domain delivering ubiquitinated cargoes to the autophagosome (45). When autophagosome/lysosome fusion is impaired, then p62 accumulates proportionally to the impairment (43). Thus, in Atg7^−/− kidneys, it was expected that there would be a block in the conversion of LC3-I to LC3-II and a build-up of LC3-I and p62 that is bound to LC3-I. On immunoblot analysis, there was a decrease in Atg7, an increase in LC3-I, and a massive increase in p62 in Atg7^−/− kidneys ( Supplementary Figures S1A, B ) but not in heart or liver ( Supplementary Figure S1C ), confirming the kidney-specific knockout of autophagy. As the KSP1.3 Cad Cre Atg7^−/− mice have an expression of Cre recombinase that is mainly present in distal tubules (medulla) with less expression in proximal tubules (cortex) (22) (23), a complete loss of Atg7 was not expected in immunoblot analysis of whole kidney extracts.

To localize p62 in the Atg7^−/− kidneys, immunohistochemistry (IHC) was performed. p62 expression was expected to correlate with the location of Cre recombinase expression in Atg7^−/− mice that are highest in collecting ducts and loops of Henle (that are known to be situated mostly in the medulla) and lowest in proximal tubules (known to be situated in the cortex). In +/+ control mice, staining for p62 was not present in the cortex or the medulla ( Supplementary Figure S1D ). As expected, in Atg7^−/− mice, staining for p62 was maximal in the medulla of the kidney with less staining in the cortex ( Supplementary Figure S1E ). In Atg7^−/− mice, staining for p62 was not seen in the TLTs, but was seen in tubules surrounding TLTs ( Supplementary Figure S1F ).

Next, IHC staining was performed in Atg7^−/− kidneys to characterize the presence of TLT markers. TLTs are characterized by PCNA, CD21, and C-X-C motif chemokine ligand 13 (CXCL13) staining and close proximity to blood vessels (1, 2). CXCL13 is a chemokine expressed by tissue fibroblasts that are thought to recruit B cells into the TLTs (2, 18). PCNA staining in tubules was increased in Atg7^−/− kidneys ( Figure 2A ). PCNA staining in tubules is a known response to tubular injury (46). There was PCNA ( Figure 2A ), CD21 (follicular dendritic cells), and CXCL13 ( Figure 2B ) staining in the TLTs ( Figure 2B ). Additional images of PCNA staining in TLTs in Atg7^−/− kidneys are demonstrated in Supplementary Figure S1G . Next, the cells present in the TLT were further characterized. There was staining in the TLTs for CD45 (a marker of all hematopoietic cells except for mature erythrocytes and platelets), CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), SLP-76 (T-cell development and maturation marker), and FOXP3 (Treg cells) ( Figure 2C ). CD3 is expressed by a high percentage of circulating peripheral T cells, forming a complex with the T-cell receptor (TCR). CD4 (cluster of differentiation 4) is a glycoprotein that serves as a co-receptor for the T-cell receptor (TCR) on CD4 T cells. Lymphocyte cytosolic protein 2, also known SLP-76, is an adaptor protein that plays a key role in signaling pathways within T cells and other immune cells (47). Studies using SLP-76-deficient T-cell lines or mice have provided strong evidence that SLP-76 plays a positive role in promoting T-cell development and activation (47).

As the proliferation of cells is a characteristic of T cells, p-S6, a downstream mTORC1 target and a potent mediator of proliferation, was studied. There was staining for p-S6 in TLTs in both +/+ and Atg7^−/− kidneys ( Figure 2D ), and p-S6 staining was increased in TLTs in Atg7^−/− compared to wild-type kidneys ( Figure 2D ). There was increased staining for p-S6 in tubules in Atg7^−/− versus wild-type mice ( Figure 2E ). There was staining for p-S6 in tubules in close proximity to the TLTs ( Figure 2F ).

Next, it was determined whether the presence of large TLTs in Atg^−/− kidneys was associated with increased fibrosis in the kidney. The percent fibrosis was low in the interstitium, and there was no difference in fibrosis between wild-type and Atg7^−/− kidneys ( Figure 2G ).

The cause of the proliferation of cells in TLTs is not well understood. mTORC1 is a well-known proliferative signaling pathway (17), and mTOR plays an important role in lymphocyte proliferation, differentiation, and survival (21). Immunoblot analysis for an extensive array of mTOR proteins in Atg7^−/− kidneys was performed ( Supplementary Figure S2 ). The increase in p-S6^S240/244 and p-Akt^T308 (markers of mTORC1) but not pAkt^S473, pSGK1^S422, p-PKCα^T638/641, p-GSK3β^S9, or p-mTOR^S2481 (markers of mTORC2) in Atg7^−/− kidneys suggested that mTORC1 but not mTORC2 was activated in the Atg7^−/− kidneys. There was no increase in p-ACC^S79 (a marker of p-AMPK), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) isoforms, or p-c-Myc (a regulator of proliferation), in Atg7^−/− kidneys. The increase in p-S6, but no increase in p-4E-BP1 isoforms, suggests that mTORC1 signals downstream to p-S6 rather than 4E-BP1 in causing the proliferation of cells within the TLTs.

To throw light on early events in the tubules that could lead to the subsequent formation of TLTs, 180-day-old Atg7^−/− kidneys were studied. Small TLTs were seen in both wild-type and 180-old Atg7^−/− mice ( Supplementary Figures S3A–C ). The most prominent tubular response in these kidneys was a massive increase in p62 in the tubules surrounding the TLTs. There was intense staining for p62 in tubules in the whole kidney sections ( Supplementary Figures S3D, E ), cortex ( Supplementary Figures S3F, G ), and medulla ( Supplementary Figures S3H, I ). p62 is a marker of cell stress or damage (43, 45). Kidneys were also stained for VCAM-1, which is a well-known chemoattractant surrounding TLTs in ischemic kidneys (16). There was little VCAM-1 staining in the Atg7^−/− kidneys, that was not localized to tubules surrounding TLTs ( Supplementary Figures S3J–L ). TNF-α is the major cytokine seen in the TLTs in ischemic kidneys and is thought to play a role in promoting inflammation, recruiting immune cells, and organizing the tissue structure in TLTs (16). There was no TNF-α staining in the small TLTs seen in wild type or Atg7^−/− kidneys ( Supplementary Figures S3M, N ). There was increased tubular injury, as indicated by kidney injury marker-1 (KIM-1) staining, in Atg7^−/− kidneys ( Supplementary Figures S3O–Q ). In summary, at an early age in Atg7^−/− kidneys, there is tubular injury and p62 in the tubules was much more abundant than VCAM-1 and TNF-α. In conclusion, intense p62 staining, a marker of cell stress and tubular injury precedes TLT formation in Atg7^−/− kidneys.

It is known that there is chronic interstitial inflammation with lymphocytes (25) and macrophages (48) in Pkd1^RC/RC mouse polycystic kidneys, a hypomorphic model of human autosomal dominant polycystic kidney disease (ADPKD). As chronic inflammation is known to play a role in the formation of TLTs (1), it was determined whether there were TLTs in the Pkd1^RC/RC mouse kidneys. TLTs were seen on low- and high-power magnification of hematoxylin–eosin-stained kidneys in 120-day-old Pkd1^RC/RC kidneys ( Figures 3A, B ; Supplementary Figures S4A, B ). There was an intense proliferation of cells in the TLTs as indicated by staining for PCNA ( Figures 3C, D ; Supplementary Figures S4C, D ).

Increased proliferation is a feature of cells lining cysts in PKD and also of the cells in the TLTs. There was also PCNA staining of the tubular cells lining the cysts ( Supplementary Figure S4D ), as we have previously described (26). Additional images of PCNA staining in TLTs in 120-day-old Pkd1^RC/RC kidneys are shown in Supplementary Figure S4D . There was increased p-S6 in the tubules in close proximity to TLTs in the TLTs in Pkd1^RC/RC kidneys ( Figure 3E ).

TLTs have been classified into three stages based on the presence of CD21-positive follicular dendritic cells (FDCs) and germinal centers (2). Stage 1 TLTs do not have CD21 FDCs. Stage 2 TLTs have CD21-positive cells but no germinal centers. TLTs with prominent CD21 FDCs and germinal centers are classified as stage 3. The TLTs in 120-day-old Pkd1^RC/RC kidneys had very slight CD21 staining and no germinal centers, suggesting that they were early-stage 1 TLTs ( Supplementary Figure S4E ). The chemokine CXCL13 is a strong factor for B-cell recruitment (2). There was very slight staining for CXCL13 in 120-day-old Pkd1^RC/RC kidneys ( Supplementary Figure S4F ).

The number of TLTs was increased in the kidney in 120-day-old and further increased in 400-day-old Pkd1^RC/RC mice compared to wild-type mice ( Figure 3F ). The size of TLTs in 400-day-old Pkd1^RC/RC mice was 50% smaller compared to the 400-day-old Atg7^−/− kidneys ( Figure 3G ). TLTs can show phenotypic heterogeneity, ranging from less organized structures composed of intermingled lymphocyte aggregates to highly organized structures made up of discrete clusters, sometimes even with germinal centers (1). The architecture of the TLTs in Pkd1^RC/RC kidneys ( Figure 3A ) was less organized, and the cells were less densely packed than the highly organized structures with densely packed cells seen in Atg7^−/− kidneys ( Figures 1B–D, F ). The less organized TLTs in Pkd1^RC/RC compared to Atg7^−/− kidneys may be due to the less advanced stage of the TLTs in Pkd1^RC/RC kidneys and the presence of large cysts, which results in the lack of interstitial space for TLTs to grow.

Increased proliferation (PCNA) ( Figures 3C, D ) and p-S6 ( Figure 3E ) in the TLTs was seen in Pkd1^RC/RC kidneys. The mTOR inhibitor Torin2 is a potent antiproliferative agent (26). Thus, the number and size of TLTs were determined in Pkd1^{RC /RC} kidneys from mice treated with Torin2 at an early age, from days 50 to 120, using the dosing strategy we have previously described (26). The number of TLTs in Pkd1 ^RC/RC kidneys was virtually eliminated by treatment with Torin2 ( Figures 3H, I ). In our published study, treatment of mice with Torin2 resulted in a 30% decrease in cyst index and improved kidney function (26).

Based on the p-S6 staining seen in tubules and TLTs in +/+, Atg7^−/−, and PKD kidneys, and studies that p-S6 is increased in tubules in ischemic kidneys in mice (49), we sought to examine whether TLTs were present in ischemic kidneys. Therefore, adult wild-type mice were subjected to 45 min of unilateral ischemia and the contralateral kidney was used as a control. Forty-five days after reperfusion, ischemic kidneys were examined for TLTs. After unilateral renal pedicle clamp in aged mice, the kidney phenotype shows an increase in kidney injury markers, inflammation fibrosis, and TLT formation and growth, which worsens with a longer period of renal pedicle clamp (30). TLTs were seen on low- and high-power magnification of hematoxylin-eosin-stained ischemic kidneys compared to no TLTs seen in the contralateral normal kidney ( Figures 4A–C ). Ischemic kidneys were smaller than the contralateral control kidney. There were TLTs with intense PCNA staining in ischemic kidneys versus no TLTs in contralateral normal kidneys ( Figures 4F–H ). Mice were treated with the mTOR inhibitor Torin2 for 35 days, starting from day 10 after the renal pedicle clamp until they were killed. Representative images of ischemic kidneys treated with Torin2 showing significantly less TLT number, size, and index are shown in Figures 4D, E, I, J . The number, TLT index, and size of TLTs were significantly reduced by treating ischemic acute kidney injury (AKI) mice with the mTOR inhibitor Torin2 ( Figures 4K–M ). The percentage of cells within the TLTs staining for PCNA was significantly reduced by treatment with Torin2 ( Figures 4N–P ), indicating that Torin2 reduces the proliferation of cells in the TLTs. TLTs in ischemic kidneys showed intense staining for p-S6 ( Figure 4Q ). Staining for p-S6 and size of TLTs was significantly reduced by treatment of mice with Torin2 ( Figure 4R ). The percent of TLT staining with p-S6 was significantly less in Torin2 versus vehicle-treated ischemic kidneys ( Supplementary Figure S4 ). Additional images of TLTs, PCNA, and p-S6 staining in vehicle versus Torin2-treated ischemic kidneys are shown in Supplementary Figures S5A–L . There was staining for p-S6 in tubules in ischemic kidneys that was reduced by Torin2 ( Figure 4T ). There was staining for p-S6 in tubules in close proximity to the TLTs ( Figure 4U ).

Torin2 resulted in a decrease in staining for CD3, a marker of T cells; CD20, a B-cell marker; and CD21, a marker of FDCs, in TLTs in ischemic kidneys ( Figures 5A–I ). There was a decrease in staining (% of TLT area) for CD3, CD20, and CD21 in Torin2-treated mice ( Figures 5C, F, I ). Torin2 resulted in a nearly complete inhibition of CD21 staining in the TLTs. T and B cells and FDCs are three of the major components of TLTs. CD21 follicular dendritic cells organize B-cell homeostasis and humeral responses in TLTs and are essential for B-cell function in TLTs (2).

There was intense p62 staining, a marker of suppressed autophagy, in immune cells in TLTs in vehicle-treated ischemic kidneys ( Figures 5J, L ). p62 staining was reduced, indicating increased autophagy in Torin2-treated ischemic kidneys ( Figures 5K, L ).

Torin2 has been described to inhibit mTORC2 (26), so p-AKT^S473, a marker of mTORC2 activation, was measured in the TLTs. The best-characterized target of mTORC2 is the AKT kinase, and mTORC2 is known to phosphorylate AKT on Serine473 (50). TLTs in aged wild-type, Atg7^−/−, Pkd1^RC/RC, and ischemic kidneys were stained for p-AKT^S473. Sections from lung cancer tumors from our previous studies (51) were used as a positive control and showed intense staining for p-AKT^S473 ( Supplementary Figure S6A ). There was minimal staining for p-AKT^S473 in a few of the TLTs in aged wild-type ( Supplementary Figure S6B ) and aged Atg7^−/− ( Supplementary Figure S6C ) kidneys. There was no staining for p-AKT^S473 in any of the TLTs in Pkd1^RC/RC ( Supplementary Figure S6D ) or ischemia ( Supplementary Figure S6E ) kidneys. This is compared to the intense staining for p-S6, a marker of mTORC1, seen in all the TLTs from aged wild type, Atg7^−/−, Pkd1^RC/RC, and ischemic kidneys. Thus, it is unlikely that the effect of Torin2 on TLTs was related to inhibition of mTORC2.

A common feature of Atg7^−/− kidneys, polycystic kidneys, and ischemic kidneys was p-S6 staining in the tubules in close proximity to TLTs. The mTOR inhibitor Torin2 resulted in a decrease of p-S6 in both the tubules and TLTs in ischemic kidneys ( Figures 4Q–T ). To determine whether p-S6 in tubules versus TLTs was driving proliferation, formation, and growth in TLTs, tubule-specific Atg7^−/−Raptor^−/− mice and Atg7^−/− controls were exposed to ischemia to induce TLT formation. Raptor, the functional unit of mTORC1, was knocked out in the tubules in Atg7^−/− mice. Renal tubule-specific cadherin Cre Raptor^−/− mice (KSP1.3 Cad Cre Raptor^−/− mice) were developed by Cre recombinase recombination. The hypothesis was that if increased p-S6 in the tubules was driving TLT formation and growth, the Atg7^−/−Raptor^−/− mice exposed to ischemia would have fewer TLTs than control Atg7^−/− mice exposed to ischemia.

In PCNA-stained sections of contralateral control nonischemic kidneys, the number of TLTs was not significantly different in Atg7^−/− ( Figure 6A ) vs. Atg7^−/−Raptor^−/− kidneys ( Figure 6B ). The TLT index was higher, rather than lower, in Atg7^−/−Raptor^−/− kidneys ( Figure 6B ). However, TLTs were very small, as indicated by a very low TLT index, as shown in the higher magnification insert of Figure 6B . The quantitation of TLT number and index is shown in Figure 6C .

In PCNA-stained sections of ischemic kidneys, the number of TLTs was the same in Atg7^−/− ( Figure 6D ) vs. Atg7^−/−Raptor^−/− kidneys ( Figure 6E ), and the TLT index was slightly lower in Atg7^−/−Raptor^−/− kidneys ( Figure 6E ). High magnification inserts of TLTs in Atg7^−/− ( Figure 6D ) and Atg7^−/−Raptor^−/− kidneys ( Figure 6E ) are shown. The quantitation of TLT number and index is shown in Figure 6F .

There was decreased p-S6 staining in tubules in the inner and outer medulla, as well as in the cortex, in Atg7^−/− and Rictor^−/− mice ( Figures 6G–I ).

In summary, there was p-S6 staining in tubules in close proximity to TLTs. However, knockout of mTORC1 in tubules reduces p-S6 staining in the cortex and medulla but does not affect the number of TLTs.

Kidneys exposed to ischemia develop tubular injury, and tubular injury can be a stimulus for TLT formation and growth (2). Therefore, the relationship between tubular injury (KIM-1 staining) and TLTs was investigated in Atg7^−/−, Pkd1 ^RC/RC, and ischemic kidneys. KIM-1 is a marker of kidney tubular injury and regeneration in mice (51) and in humans (52).

KIM-1 staining in tubules was increased in Atg7^−/− vs. age-matched wild-type mice, indicating tubular injury in Atg7^−/− tubules ( Figure 7A ). Representative images of KIM-1 staining and KIM-1 staining in tubules close to a TLT ( Figure 7A ) and quantitation of KIM-1 staining ( Figure 7B ) are shown. As tubular injury is known to produce cytokines and chemokines that play a role in TLT formation and growth (16, 53), an array of proinflammatory cytokines was measured in Atg7^−/− kidneys ( Table 1 ). There was an increase in tumor necrosis factor-α (TNF-α), IL-1β, IL-2, IL-6, and C-X-C motif chemokine ligand 1 (CXCL1) in Atg7^−/− vs. age-matched wild-type control kidneys ( Table 1 ). As TLTs form in on average less than 4% of the kidney and tubules make up the bulk of the kidney, and injured kidney tubules are known to produce TNF-α, IL-1β, IL-6, and CXCL1 (54), it is likely that the major source of such large increases in cytokines was coming from the injured tubules rather than the TLTs. Proinflammatory cytokines like TNF-α, IL-1β, IL-2, IL-6, and CXCL1 are known to play a role in TLT formation and growth (16, 53), so it is likely that these cytokines produced by the tubules surrounding the TLTs were contributing to TLT formation and growth in Atg7^−/− kidneys.

Next, the relationship between tubular injury and TLTs was investigated in Pkd1 ^RC/RC kidneys. KIM-1 staining was increased in normal tubules in Pkd1 ^RC/RC mice versus wild-type controls ( Figure 7C ). Representative images of KIM-1 staining ( Figure 7C ) and quantitation of KIM-1 staining ( Figure 7D ) are shown. In a separate analysis of IHC, Torin2 that reduced TLTs had no effect on KIM-1 staining ( Figures 7E, F ). These data suggest that Torin2 reduces TLTs in Pkd1 ^RC/RC kidneys independently of an effect on tubular injury.

The outer stripe of the outer medulla (S3 segment of proximal tubules) is considered to be the major site of tubular injury in renal ischemia (55, 56). In the renal ischemia experiments, KIM-1 was quantitated in the outer stripe of the outer medulla. In ischemic kidneys, there was large KIM-1 staining in tubules, as expected. Torin2 did not affect KIM-1 staining in ischemic kidneys. Representative images of KIM-1 staining ( Figure 7G ) and quantitation of KIM-1 staining ( Figure 7H ) are shown. As expected, serum NGAL, a marker of kidney injury (51, 52), and serum creatinine were increased in mice with kidney ischemia compared to a group of age-matched mice without kidney ischemia ( Figures 7I, J ). There was no difference in serum NGAL or serum creatinine between vehicle- or Torin2-treated mice with kidney ischemia ( Figures 7I, J ). Torin2 did not have a significant effect on kidney function as determined by transcutaneous glomerular filtration rate (GFR) measurements ( Figure 7K ). There was a significant decrease in ATN scores in ischemic kidneys treated with Torin2 ( Supplementary Figures S7A–E ). There was minimal fibrosis in contralateral nonischemic kidneys ( Supplementary Figures S7F, H, J ). Torin2 reduced TLTs but did not affect fibrosis in ischemic kidneys ( Supplementary Figures S7G, I, J ). In summary, the reduction of TLTs was associated with less tubular necrosis but no effect on KIM-1 (a marker of tubular injury and regeneration) or kidney function. Interstitial fibrosis, a known consequence of chronic tubular injury (57), was not affected by Torin2.

Next, VCAM-1, p62, and TNF-α were explored as potential tubule-derived factors surrounding TLTs that may be influenced by mTOR. VCAM-1 is the main chemokine in the tubules surrounding TLTs and is thought to play a role in TLT development (16). There was VCAM-1 staining in the tubules distant from TLTs ( Figures 8A, B ) and in tubules surrounding TLTs ( Figures 8C, D ) in both vehicles and Torin2-treated ischemic kidneys. Torin2 did not have a significant effect on VCAM-1 staining ( Figure 8E ). p62 was prominent in tubules around TLTs in Atg7^−/− mice ( Figure 1F ), so p62 in tubules in ischemic kidneys was studied. p62 plays a role in inflammatory responses, like inflammasome activation (43), expression of inflammatory genes via NF-κB (43), and production of chemokines like CXCL1 that could potentially influence TLT formation and growth (58). p62 was seen in a few tubules in both vehicle- and Torin2-treated kidneys ( Figures 8F, G ). p-62 was not seen in the tubules surrounding TLTs in ischemic kidneys. Torin2 treatment had no significant effect on p62 in tubules ( Figure 8H ). TNF-α is a major pro-inflammatory cytokine known to be expressed in injured tubules (59) and TLTs (16) in ischemic kidneys. TNF-α was seen in tubules surrounding TLTs and in TLTs ( Figures 8I, J ), and Torin2 had no effect on TNF-α in tubules or in TLTs ( Figures 8K, L ). Next, the effect of ischemia and Torin2 on VCAM-1 and TNF-α in immunoblot analysis of whole kidney homogenates was determined. Ischemia resulted in an increase in VCAM-1 and TNF-α compared to nonischemic kidneys ( Figures 8M–O ). Torin2 had no effect on VCAM-1 and TNF-α in ischemic kidneys ( Figures 8M–O ). In summary, these data suggest that mTOR-mediated tubule-TLT interactions in ischemic kidneys are independent of VCAM-1, p62, and TNF-α in the tubules.