Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1

Abstract

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.

Figures

Figure 1

Oxalate metabolism pathway overview. ADH, alcohol dehydrogenase; AGT, alanine-glyoxylate aminotransferase; ALDH, aldehyde dehydrogenase; GLO, glyoxylase; GRHPR, glyoxylate reductase hydroxypyruvate reductase; GULO, gulonolactone oxidase; HAO1, hydroxyacid oxidase 1; HOG, 4-hydroxy-2-oxoglutarate; HOGA, 4-hydroxy-2-oxoglutarate aldolase; HYPDH (PRODH2), hydroxyproline dehydrogenase; LDH, lactate dehydrogenase.

Figure 2

HAO1-1 displays potent in vivo activity in mice. The lead HAO1 DsiRNA formulated in LNP (HAO1-1) was i.v. injected into wild-type mice with varying doses as indicated. Liver samples were collected for analysis of mRNA after 24 hours and for protein on day 5 after a single-dose injection. (a) Results of real-time reverse transcription PCR (mean ± SD) indicate that HAO1-1 inhibits Hao1 mRNA expression with high potency (ED50: 0.022 mg/kg). (b) Results of western analysis show that HAO1-1 displayed potent inhibition of GO protein expression 5 days after injection. (c) Hao1 mRNA was detected in liver tissues by in situ hybridization and showed that samples isolated from animals injected at 0.1 or 1 mg/kg have lower hybridization signals compared to PBS injected animals. Bar = 100 µm. (d) GO protein levels were measured by immunohistochemistry. Results showed that HAO1-1, either at 0.1 or 0.3 mg/kg, effectively and homogenously reduced liver GO protein in mouse hepatocytes, compared to PBS-injected controls. Spleen sections were used as negative controls. Bar = 30 or 60 µm. LNP, lipid nanoparticle.

Figure 3

HAO1-1 achieves rapid and durable target knockdown in mice. Wild-type mice were injected with a single i.v. dose of HAO1-1 at 0.3 or 1 mg/kg. (a) At the indicated time points, five animals were sacrificed, and liver samples were evaluated for mRNA levels by real-time reverse transcription PCR. The results (mean ± SD) demonstrated that Hao1 mRNA levels were quickly diminished and the inhibitory effect of a single dose of HAO1-1 lasted for more than a month. (b) Representative results of western analysis from each time point of mice injected with a single dose of PBS or HAO1-1 at 0.3 mg/kg. Results showed that GO protein gradually decreased starting 6 hours postdose, reaching levels below the detection limit by day 10 and reemerging between days 29 and 40.

Figure 4

HAO1-1 reduces urinary oxalate and elevates urinary glycolate levels in a mouse PH1 model. Male Agxt-/- mice were injected i.v. with HAO1-1 (N = 4) at 0.3 mg/kg or PBS (N = 5) as a control on day 0, day 33, and day 40. Urine samples were manually collected on predose day −2, as well as days 2, 5, 8, 12, 14, 16, 20, 23, 26, 28, 33, 35, 36, 41, and 48, and analyzed for oxalate, glycolate, and creatinine concentrations by LC/MS. (a) The change in urine oxalate levels in the HAO1-1 treatment group was calculated as the urine oxalate concentration normalized to creatinine (milligram of oxalate per gram of creatinine) relative to the average normalized urine oxalate concentration of the PBS control group, where the urine oxalate (milligram of oxalate per gram of creatinine) in the PBS control group was set at 1. LC/MS analysis (mean ± SD) revealed that HAO1-1 reduced oxalate levels rapidly, and the effects lasted for up to 15 days before beginning to return to baseline levels. (b) Urine glycolate levels were measured, normalized to creatinine, and expressed as a value relative to the average of the PBS control group (which was set at 1, as in Figure 3a). HAO1-1 simultaneously induced the elevation of urinary glycolate levels and the reduction of oxalate levels. Results of these experiments with values plotted without normalization to PBS treatment group and expressed as individual animal are provided in Supplementary Figure S2. (c) Twenty-four hour urine samples were also collected using metabolic cages from day 41 to 42 and from day 71 to 72. The results (mean ± SD) indicated that oxalate excretion was substantially lower after HAO1-1 treatment compared to control (PBS). (d) Oxalate levels were measured by both enzymatic assay and LC/MS methods. The normalized oxalate values (milligram per gram of creatinine) determined by both methods were compared using urine samples collected on day 0, day 2 and day 5. The data (mean ± SD) indicate a good correlation between both methods and further confirm that HAO1-1 effectively reduces oxalate production. The arrows indicate the days on which the animals were treated with HAO1-1 or PBS.

Figure 5

HAO1-1 protects against EG-induced kidney damage in a PH1 mouse model. (a) Male Agxt-/- mice were divided into four groups containing seven mice each and were supplied with either 0.7% EG in their drinking water (groups 2 to 4) or regular drinking water as a control (group 1). Animals of groups 1 and 2 were injected with three doses of PBS on days 0, 7, and 14. Animals of group 3 were injected with two doses of PBS on days 0 and 7 as well as a single dose of HAO1-1 on day 14. Group 4 received three doses of HAO1-1 on days 0, 7, and 14. Both PBS and HAO1-1 were given i.v.. Urine samples were collected weekly and analyzed for oxalate levels with enzymatic assay. All animals were sacrificed, and the kidneys were collected and evaluated for CaOx crystals on day 22. (b) Animals drinking 0.7 % of EG (EG/PBS) and treated with PBS displayed continuous elevation of urinary oxalate levels. Animals drinking EG and injected with HAO1-1 (EG/HAO1-1) exhibited urinary oxalate levels that remained at baseline concentration. Mice drinking EG and given delayed treatment of HAO1-1 (EG/PBS/HAO1-1) showed an initial elevation of urine oxalate levels (milligram per gram of creatinine) during the first 2 weeks that returned back to baseline levels following HAO1-1 treatment. Dashed lines indicate estimated range of oxalate levels of wild-type C57BL/6 mice that are not littermates and, therefore, not fully matched to the Agxt-/- mice in terms of genetic background. (c) Histological analysis of kidney tissue was performed to detect CaOx crystals using Pizzalato staining. Representative images of kidney sections show animals of the EG/PBS group developed CaOx crystals that exhibited characteristic birefringence under polarized light (upper panel) and positive staining using the Pizzalato's method (lower panel). (d) Histological analysis indicates that HAO1-1 is able to prevent the formation of CaOx crystals and protect the kidneys from further damage in the Agxt-/- mice that received EG (compare EG/PBS and EG/HAO1-1 groups). Delayed treatment of HAO1-1 also effectively reduced CaOx crystals in the kidneys, though to a lesser extent. Each picture shows a representative section from an individual animal of each group. Bar = 150 μm. (e) Whole kidney sections were imaged, scanned, and reconstituted with imaging software to further demonstrate that HAO1-1 prevents the formation of CaOx crystals in treated animals.

Figure 6

HAO1 DsiRNA displays specific and potent activity in NHPs. A selected HAO1 DsiRNA formulated in LNP, designated DCR-PH1, was i.v. injected into cynomolgus monkeys with varying doses as indicated. Liver biopsies were collected for analysis of mRNA and protein at varying time points as indicated. Urine and plasma samples were also collected at varying time points as indicated and then analyzed with LC/MS for glycolate and creatinine levels. (a) Results of real-time reverse transcription PCR indicated that HAO1 DsiRNA inhibits HAO1 mRNA expression in a dose-dependent manner. Expression of each individual animal was plotted. Each group contains four animals. (b) The results of western blot analysis indicated that HAO1 DsiRNA inhibits GO protein expression on days 7 and 14. Expression in each individual animal was analyzed. Note that only three biopsies were recovered from the 3 mg/kg dose group at day 7, and lane 5 was empty with no sample. (c) The results of real-time reverse transcription PCR indicate that HAO1 DsiRNA, when injected at 0.3 mg/kg durably inhibits HAO1 mRNA expression (mean ± SD). (d) LC/MS analysis shows that urinary glycolate levels (normalized with creatinine) increased in HAO1 DsiRNA-treated animals, in a dose-dependent manner (mean ± SD). (e) Similarly, plasma glycolate was elevated with comparable kinetics and dose dependency in treated animals. (f) Animals receiving HAO1 DsiRNA at 0.3 mg/kg once every 3 weeks were analyzed for their HAO1 mRNA expression and plasma glycolate levels at steady state. There was a notable correlation (R2 = 0.72) between the knockdown of HAO1 mRNA and elevation of plasma glycolate in individual animals. This indicates that plasma glycolate could be explored as a potential pharmacodynamic marker for target knockdown. LNP, lipid nanoparticle.