Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish

ABSTRACT Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified 4-(phenylthio)butanoic acid (PTBA), a histone deacetylase inhibitor (HDI), as an enhancer of renal recovery, and showed that PTBA treatment increased RTEC proliferation and reduced renal fibrosis. Here, we investigated the regenerative mechanisms of PTBA in zebrafish models of larval renal injury and adult cardiac injury. With respect to renal injury, we showed that delivery of PTBA using an esterified prodrug (UPHD25) increases the reactivation of the renal progenitor gene Pax2a, enhances dedifferentiation of RTECs, reduces Kidney injury molecule-1 (Kim-1) expression, and lowers the number of infiltrating macrophages. Further, we found that the effects of PTBA on RTEC proliferation depend upon retinoic acid signaling and demonstrate that the therapeutic properties of PTBA are not restricted to the kidney but also increase cardiomyocyte proliferation and decrease fibrosis following cardiac injury in adult zebrafish. These studies provide key mechanistic insights into how PTBA enhances tissue repair in models of acute injury and lay the groundwork for translating this novel HDI into the clinic. This article has an associated First Person interview with the joint first authors of the paper.

. Dominant negative RARα expression reduces UPHD25 efficacy Uninjured (A-E) or gent-AKI (F-J) Tg(hsp70l:EGFP-HS-dnRARα) ci1008 transgenic larvae were heat shocked at 5 dpf to express a dominant-negative form of human RA receptor alpha, which inhibits RA signaling. Larvae were treated with UPHD25 or DMSO then harvested for PT cell proliferation Quantification of scar size at 20 dpa after injection of UPHD25. MeanDMSO=84.54 (N=14) vs.

Zebrafish pronephros and RNA isolation
6 dpf Tg(PT:EGFP) larvae were used to collect GFP+ pronephros, 100 pronephros were used per condition, and all experiments were repeated three times. The larvae were incubated in 10mM DTT in E3 and Tricaine for 1.5-2 hrs at room temperature. The larvae were washed in Hank's Balanced Saline Solution (HBSS, Gibco). The larvae were incubated in 5mg/ml collagenase I in HBSS (Sigma Aldrich) for 2.5-3.5 hrs at 32 ºC. The larvae were washed in Minimum Essential Media and 10% fetal calf serum (MEM, FCS, Gibco). Using p100, the larvae were titurated to further digest the fish. Under a fluorescent scope, fine forceps and p10 pipette were used to dissect GPF+ pronephros to aspirate and collect pronephric kidneys from larvae on ice. The collected tubules were used for RNA isolation and sequencing. To collect total RNA, MEM+10% FCS was aspirated from pronephric tubule mass and then immediately processed with RNeasy Micro kit.
(Qiagen). Tissue lysis and homogenization was performed in 500 μL Trizol (Thermofisher) and an electric homogenizer and pestle for 30 seconds. For phase separation, 50 μL BCP (Thermofisher) was added to the homogenate. Samples were centrifuged for 10 minutes at 12,000 rpm at 4C. 200 μL of top nucleic acid phase was collected and transferred into the column. Further downstream steps followed the manufacturer's standard protocol for the final elution of 10uL RNA. Total RNA quantification was performed using Nanodrop 1000 Spectrophotometer. Each condition was repeated with three biological replicates. sample. Four samples of the first biological replicates were pooled and sequenced on a single flow cell. Second and third replicates were pooled and ran on 2 flow cells.

In situ hybridization
The arginase-2 clone was synthesized and cloned into pEX-K248 with Sp6 promoter to drive the reverse transcription (Eurofins). The deoxygenin probe for arginase-2 targeted 500 bp of 3' end of coding region and 500 bp of 3'UTR. 6 dpf larvae were fixed in 4% paraformaldehyde Alkaline Phosphatase antibody (Roche) overnight at 4°C. The larvae were stained with BM purple (Roche) at room temperature. The reaction was stopped with 4% PFA. Larvae were then cryosectioned (see protocol for immunofluorescence) and imaged using a 20X objective on an Axiovert 40 CFL brightfield scope (Zeiss). Images were captured using Axiovision Rel v4.8 software (Zeiss). The samples were cryosectioned at 14μm to obtain serial sections of PT. The sections were washed with PBSTw (0.1% Tween-20) and processed for antigen retrieval step using sodium citrate buffer for 15 minutes at 97.5°C. Slides were washed with PBSTw and blocked using 10% normal goat serum. They were incubated with rabbit anti-Arginase-2 (Thermo Fisher, PA5-27987) at 1:100 dilution, overnight at 4°C. The slides were washed and incubated with donkey anti-rabbit Alexa568 (Thermo Fisher, A-10042) at 1:1000 dilution, 2 hours at room temperature.

Arginase-2 Antibody Staining
The slides were washed and incubated with DAPI (1μg/mL) for nuclear staining (Millipore Sigma, D9542), 10 min at room temperature. The slides were washed with PBS then mounted with Aqua Polymount (Polysciences, 18606-20). Sections were imaged as described in the main methods.

Generation of transgenic line Tg(hsp70l:EGFP-HS-dnRARa)
Tg(hsp701:GFP-dn_Hsa.rarα) transgenic fish were generated by gateway-based Tol2 transposon transgenesis. To generate the transgenic fish, gateway cloning was used with the hps70l vector as the 5' entry clone and GFP-dnRARa, a human dominant negative RARa, as the middle entry.
PolyA was used as the 3' entry clone. Following heat-shock induction, transgenic embryos expressed GFP and exhibited overt phenotypes, such as enlarged hearts and loss of forelimbs, which are consistent with loss of RA signaling. Constructs were injected into single cell embryos and screened for insertion. Progeny of P0 founder animals were used to establish the zebrafish transgenic line. In order to examine the effect of dnRAR during AKI, 2 dpi or 5dpf zebrafish larvae were heat shocked at 37°C for 1 hour. Then, larvae were immediately treated with 1μM UPHD25 or 1% DMSO for 24 hours at 28°C. After treatment, the larvae were fixed and cryosectioned as

Cardiac injury and compound treatment
Adult AB* or Tu wildtype zebrafish age 6-18 months were anesthetized with 0.168 g/L tricaine for 3-5 minutes and ventricle apex amputation was performed as previously described (Missinato et al., 2018). In adults either 3 μL of 50% DMSO vehicle in PBS or 200 μM UPHD25 (corresponding to 0.4 mg/kg) was delivered by retro-orbital injection (Pugach et al., 2009).
Injection was performed once per day, from 1-6 days post amputation (dpa), and hearts were extracted at 7 dpa to assess cardiomyocyte proliferation, and at 20 dpa to measure scar size.
Images were captured with Leica MZ 16 microscope and Q Imaging Retige 1300 camera. Clot area was measured using ImageJ (NIH). Four heart sections showing the largest clot were measured and the scar area values were measured as average of the sum of the clot area. Slides were mounted in Vectashield with DAPI (Vector Laboratories). For each experiment, at least four sections were analyzed per heart. Cardiomyocyte proliferation index was calculated as percentage of number of Mef2c + and PCNA + cells divided by the number of total Mef2c + cells.