Histone deacetylase inhibitor enhances recovery after AKI

Abstract

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Figure 1.

Zebrafish develop AKI after gentamicin treatment. Larvae injected with (A, C, E, G, and H) vehicle or (B, D, F, I, and J) gentamicin were analyzed 3 days after injury (3 dpi). (A and B) Lumen distension. Lateral view of Tg(PT:EGFP). Proximal pronephric tubules are labeled with EGFP fluorescence (green). Compared with control larvae, injured larvae display flattening of the epithelium and enlargement of the tubular lumen. con, control. (C and D) Thinning of epithelial brush border. Transverse sections of control and injured nephrons. Insets show higher magnification of the pronephric tubules. Note the thinning of the brush border in gentamicin-injected larvae (arrowheads). H&E, hematoxylin and eosin. (E and F) Loss of RTEC polarity. Immunofluorescence for Na⁺/K⁺ ATPase. Transverse sections of proximal tubules in control larvae with basolateral expression of Na⁺/K⁺ ATPase lost after gentamicin-induced injury. Insets show higher magnification of the proximal tubule. Injured tubules show flattening and sloughing of cells into the tubular lumen (asterisk). (G–J) Pax2 reactivation. Transverse cryosections of proximal tubules (outlined in white) immunostained with Pax2 antibody (red) and counterstained with DAPI (blue). No Pax2 staining was observed in the control larvae, whereas injured larvae show nuclear Pax2 staining (white arrows). Scale bars, 10 μm.

Figure 2.

m4PTB treatment increases larval survival and proliferation. (A) Survival after gentamicin-induced AKI. Gentamicin-injected larvae were treated with either 1% DMSO vehicle or 4 μM m4PTB 2 days after injury, and the survival rate was evaluated at 4, 5, and 6 dpi: 4 dpi, 87% (69/79) in vehicle-treated larvae versus 93% (74/80) in m4PTB-treated larvae; 5 dpi, 58% (46/79) in vehicle-treated larvae versus 79% (63/80) in m4PTB-treated larvae; 6 dpi, 55% (38/69) in vehicle-treated larvae versus 74% (52/70) in m4PTB-treated larvae. Data pooled from three independent survival assay experiments (Fisher exact test): *P<0.05, **P<0.005. (B–E) Proximal tubule cell proliferation. (B and D) Control and (C and E) gentamicin injured larvae were treated with (B and C) 1% DMSO or (D and E) 4 μM m4PTB at 4 dpf (2 dpi). At 5 dpf (3 dpi), larvae were incubated with the thymidine analog EdU (green), and transverse sections were counterstained with 3G8 antibody (red) to mark proximal tubular apical brush border. Scale bars, 20 μm. White lines outline the tubules. (F) Quantitative analysis of proliferating proximal RTECs. Cell counting analysis shows a significant increase in the percentage of 3G8 EdU-positive proliferating cells in m4PTB-treated larvae at 3 dpi 24 hours post-m4PTB treatment in both the absence of gentamicin (3.5%±0.46, n=14 versus 7.1%±0.68, n=14) and after gentamicin injection (7.3%±0.71, n=14 versus 10.7%±1.7, n=8). Data are expressed as mean ± SEM; t test: *P<0.05; ***P<0.001.

Figure 3.

Beneficial effect of m4PTB on functional recovery after moderate IR-AKI. Male BALB/c mice underwent right nephrectomy and left renal pedicle clamping for 26 minutes to induce moderate IR-AKI; 24 hours after injury, mice with serum creatinine values of 0.8–1.2 mg/dl were randomly allocated daily treatment with vehicle or 100 mg/kg m4PTB for 6 days. All data are expressed as mean ± SEM. (A) Functional recovery. Serum creatinine was measured by enzymatic assay in 10 mice per group for 14 days after injury. t test: *P<0.05; ***P<0.005 versus vehicle controls. (B) Tubular injury scores 3 days after injury in the cortex and OM in 8 mice per group (0–4, arbitrary units). (C) Renal Kim1 mRNA expression 3 days after injury expressed as the ratio of Kim1 to Gapdh mRNA control (five uninjured controls, seven vehicle mice, and eight m4PTB-treated mice). One-way ANOVA (P<0.005). *Versus uninjured controls, P<0.05 after Bonferroni correction for multiple between group testing.

Figure 4.

Beneficial effect of m4PTB on functional recovery, tubular injury, and fibrosis after severe IR-AKI. Male BALB/c mice underwent unilateral left renal pedicle clamping for 30 minutes to induce severe IR-AKI and were treated with vehicle or 100 mg/kg m4PTB daily for 6 days starting 24 hours after injury. (A) Functional recovery. Mice underwent right nephrectomy 8 days after the initial injury, and serum creatinine was measured after the nephrectomy to assess functional recovery in the contralateral injured kidney (six mice per group). t test: *P<0.05; **P<0.01 versus vehicle controls. (B–D) Tubular injury 28 days after injury. (B) Tubular injury scores in the cortex and OM obtained in four mice per group (0–4, arbitrary units). t test: *P<0.05 versus vehicle control. (C and D) Representative images showing renal tubular injury in OM of vehicle and m4PTB-treated mouse kidneys 28 days after severe IR-AKI. Periodic acid-Schiff–stained tissue sections. Scale bars, 50 μm. (E–G) Fibrosis scores 28 days after injury. (E) Percent fibrosis (sirius red-stained tissue) in the cortex and OM (two uninjured controls, six vehicle mice, and six m4PTB-treated mice). One-way ANOVA (P<0.001). *Versus uninjured controls; **versus vehicle-treated mice, P<0.05 after Bonferroni correction for multiple between-group testing. All data are expressed as mean ± SEM. (F and G) Representative images showing renal tubular injury in OM of vehicle and m4PTB-treated mouse kidneys 28 days after severe IR-AKI. Sirius red-stained tissue sections. Scale bars, 50 μm.

Figure 5.

Beneficial effect of m4PTB on tubular injury and fibrosis markers 28 days after severe IR-AKI. Whole-kidney RNA was extracted from five uninjured control, eight vehicle, and eight m4PTB-treated mice. (A–F) Gene expression is displayed as the ratio of gene to Gapdh mRNA control. Results are expressed as mean ± SEM. One-way ANOVA (P<0.001 for all genes). *Versus uninjured controls; **versus vehicle-treated mice, P<0.05 after Bonferroni correction for multiple between-group testing.

Figure 6.

m4PTB treatment increases expression of genes involved in cell cycle processes. Male BALB/c mice underwent moderate IR-AKI, and kidneys were harvested for RNA extraction 12 hours later. Mouse Genome 1.0 ST microarrays (Affymetrix) were hybridized with labeled RNA, and hierarchical clustering of samples and normalized gene probe set signals were performed using dChip algorithms. Genes are clustered according to the expression profiles into three main groups: group I genes involved in cellular metabolic processes, group II genes involved in cell cycle processes, and group III genes involved in microtubule-based processes. By convention, genes that are upregulated are represented in red, and genes that are downregulated are represented in blue.

Figure 7.

m4PTB treatment changes the cell cycle profile of RTECs after injury. Cellular markers of actively cycling (Ki67), S-phase (PCNA), and G2- and M-phase (pH3) RTECs in intact mouse kidneys 3 days after induction of (A–E) moderate and (F–J) severe IR-AKI treated with vehicle or m4PTB as indicated (six mice per group). (A and F) Ki67 index (Ki67+/4',6-diamidino-2-phenylindole). (B and G) PCNA index (PCNA+/4',6-diamidino-2-phenylindole). (C and H) Actively cycling (Ki67+) cells in S phase (PCNA+). (D and I) Actively cycling (Ki67+) cells in G2 (pH3 low, speckled) and M (pH3 high) phases. (E and J) Distribution of actively proliferating (Ki67+) RTECs in G1, S, G2, and M phases of the cell cycle. Data are expressed as mean ± SEM. t test: *P<0.05; **P<0.005; ***P<0.001 versus vehicle controls.