Research ArticleExperimental Studies
Open Access

Thiamet G as a Potential Treatment for Polycystic Kidney Disease

WEN-CHENG SU, CHI-FENG HUNG, YI-CHIEH WANG, HUBERT PENG, WEN-HUNG HUANG, YI-LUN LO, YUN-HWA LO, YI-CHENG CHEN, HSIN-HUI SU and YUNG-LIANG CHEN
In Vivo November 2023, 37 (6) 2524-2532; DOI: https://doi.org/10.21873/invivo.13360

Abstract

Background/Aim: Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder primarily caused by mutations in Pkd1 (PC1), which account for the majority of ADPKD cases. These mutations contribute to the formation of cysts in the kidneys and other organs, ultimately leading to renal failure. Unfortunately, there are currently no available preventive treatments for this disease. Materials and Methods: In this study, we utilized Pkd1-knockdown mice and cells to investigate the potential involvement of O-GlcNAcylation in the progression of PKD. Additionally, we examined the effects of thiamet G, an inhibitor of O-GlcNAcase (OGA), on PKD mice. Results: Our findings indicate that both O-GlcNAcylation and OGT (O-GlcNAc transferase) were downregulated in the renal tissues of Pkd1-silenced mice. Furthermore, O-GlcNAcylation was shown to regulate the stability and function of the C-terminal cytoplasmic tail (CTT) of PC1. Treatment of PKD mice with thiamet G resulted in a reduction of renal cytogenesis in these animals. Conclusion: These results highlight the unique role of O-GlcNAcylation in the development of cyst formation in PKD and propose it as a potential therapeutic target for the treatment of PKD.

Key Words:

The most common genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is caused by mutations in Pkd1 (PC1, more than 85% of cases) that lead to the formation of cysts in the kidneys and organs, ultimately resulting in renal failure (1, 2). PC1 is a large membrane protein composed of 4,302 amino acids and 11 transmembrane (TM) domains, indicating its involvement in cell-cell interactions (1).

PC1 undergoes multiple cleavages, including one in the C-terminal cytoplasmic tail (CTT), which is involved in several signaling pathways (3, 4). Recent studies have demonstrated that CTT accumulates in the nucleus and regulates transcriptional pathways in the murine kidney and cell models (1, 3). Additionally, CTT mRNA injection into Pkd1-knockdown zebrafish embryos can rescue the dorsal body curvature phenotype, indicating the CTT’s significance in kidney morphogenesis in PKD (4). However, the mechanism underlying CTT protein stability remains largely unknown.

Post-translational modifications (PTMs) of proteins play a crucial role in allowing cells to respond to diverse environmental and physiological cues by regulating protein function (5-7). O-GlcNAcylation is a reversible glycosylation process that involves the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) to serine (S) or threonine (T) residues of a protein via a single pair of enzymes that form an O-linked glycosidic bond. Specifically, O-GlcNAc transferase (OGT) catalyzes the glycosylation of the hydroxyl group of a S or T residue on the target protein using the donor substrate UDP-GlcNAc. The O-GlcNAc group is then rapidly removed from the substrate protein by the glycosidase O-GlcNAcase (OGA) (5, 8).

Many of the identified O-GlcNAc sites have high “PEST” scores; these are proline (P), glutamic acid (E), serine (S), and threonine (T)motifs that are typically found in short-lived proteins targeted for degradation via the ubiquitin pathways. This observation suggests that O-GlcNAcylation may interfere with protein degradation (8).

The primary theme in protein regulation involves the interplay and crosstalk between ubiquitination and phosphorylation (9). Consequently, O-GlcNAcylation competes with phosphorylation at the same residue of the target protein to regulate protein function in various cellular processes. O-GlcNAcylation can disrupt protein-protein interactions, regulate protein stability, act as a protein trafficking signal, and modulate cellular metabolism (8).

A deeper understanding of pathophysiological mechanisms has resulted in the development of safer and more effective drugs. In the pathogenesis of many human diseases, including diabetes, cancer, and neurodegeneration, disruption of O-GlcNAc homeostasis has been implicated (5, 8). Thiamet G (TG) is a stable inhibitor of OGA that can cross the blood-brain barrier (10). Studies have demonstrated its ability to alleviate tau phosphorylation by increasing the O-GlcNAcylation of tau in mouse brains, and it has shown potential as a therapeutic for Alzheimer’s disease (AD) in preclinical validation (10). A derivative of thiamet G (MK-8719) has been used in the first clinical trial for the treatment of tauopathies (11).

The pathogenesis of PKD is still not fully understood, making the development of effective therapies challenging. Currently, the only FDA-approved drug for adult PKD patients is tolvaptan, a vasopressin V2 receptor antagonist. However, its availability, side effects, and high cost limit its widespread use (2). In our study, we investigated the relationship between PKD and O-GlcNAcylation, as the PEST motif is present in PC1-CTT (3). Using cellular and mouse models of PKD, we examined the functional roles of O-GlcNAcylation and provided a molecular basis for the potential use of thiamet G to alleviate cyst formation in PKD. These findings may contribute to the development of new therapeutic strategies for PKD.

Materials and Methods

Cell culture and reagents. We maintained human embryonic kidney 293T cells (obtained from the Bioresource Collection and Research Center in Taiwan) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and streptomycin. We purchased PUGNAc (10 μM, 4h pretreatment, A7229), Thiamet G (10 μM, 4h pretreatment, SML0244), and Cyclohexane (50 μg/ml, 227048) from Sigma-Aldrich (Saint Louis, Missouri, MO, USA). OGT with a small molecule inhibitor (OSMI-1), ab235455, (αR)-α-[[(1,2-Dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide), (10 μM, 4h pretreatment) was purchased from Abcam (Boston, MA, USA).

Immunoprecipitation and western blot analysis. The cells were lysed in ice-cold lysis buffer, and blots were incubated with primary antibody as previously described (12). OGT (1:1,000, GTX637222) and MYC (1:1,000, GTX103436) were purchased from Genetex (Taipei city, Taiwan, ROC), while O-GlcNAc (1:1,000, sc-59623) was purchased from Santa Cruz (Dallas, TX, USA). FLAG (1:1,000, F3165) and β-actin (1:3,000, A1978) were from Sigma-Aldrich, and O-GlcNAc (1:1,000, ab2739) was from Abcam (Waltham, MA, USA). Proteins were visualized using horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:2,000; AB_10015 289, AB_2313567, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and an enhanced chemiluminescence detection kit (ECL, Millipore, Billerica, MA, USA). For all immunoprecipitation experiments, the lysis buffer contained 0.3% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS, Sigma-Aldrich, 19899) instead of 1% Triton X-100. Cell extracts were clarified by centrifugation at 12,000 rpm for 10 min at 4°C, and the supernatants were subjected to immunoprecipitation (IP) with Flag antibody (1:50). After incubation at 4°C overnight, protein A/G agarose beads (Santa Cruz, sc-2003) were added and incubated at 4°C for another 2 h. Immunocomplexes were then subjected to Western blot analysis (12, 13).

Plasmids and DNA transfection. The OGT (#29760) and PC1-CTT (#41520) plasmids were obtained from Addgene (Watertown, MA, USA). The PC1-CTT amino acid sequence corresponds to residues 4107-4303 of PC1. The Lipofectamine 2000 transfection kit was purchased from Invitrogen (Carlsbad, CA, USA). For transfection, Flag-OGT (1 μg) and Myc-CTT (1 μg) were mixed with 4 μl of Lipofectamine 2000 and added to 293T cells plated onto 6-well tissue culture plates, which were grown to about 80% confluency prior to transfection. After 24 hours, the cells were harvested and analyzed by Western blot.

shRNA lentiviruses. Plasmids encoding lentiviruses that express either scrambled sequences or shRNA against Ogt were obtained from the RNAi Core Facility of Academia Sinica, Taipei city, Taiwan, ROC. Two different sequences were specifically targeted for Ogt shRNA (clone #1, #2). Lentiviruses were prepared in 293T cells using an established protocol (13). In brief, 293T cells were transfected with either the lentiviral plasmid carrying Ogt shRNA (1 μg) or scrambled shRNA (1 μg) together with the psPAX2 (0.9 μg) packaging plasmid and pMD2.G envelope plasmid (0.1 μg) using Lipofectamine 2000. After transfection for 6 hours, the medium containing the transfection reagent was removed and replaced with fresh complete DMEM supplemented with 2% FBS. The lentiviral particles were harvested at 24, 48 and 72 h post-transfection in individual harvests; all the individual harvests were then pooled. 293T cells with about 80% confluency in 6 cm tissue culture plates were infected with appropriate amount of lentiviral particles together with 10 μg/ml polybrene (Sigma-Aldrich, TR-1003-G) for 24 h. The virus-containing medium was then removed and replaced with fresh medium with 5 μg/ml puromycin (Sigma-Aldrich, P8833) (13).

Mice and treatment. The Pkd1L3/L3 mice (C57BL/6) were a gift from Professor ST. Jiang (14). These mice carry the insertion of the loxP-flanked mc1-neomycin (mc1-neo) cassette into intron 34 in reverse orientation relative to the targeted Pkd1 gene, resulting in lower Pkd1 expression levels. Approximately 50% of Pkd1-null newborn mice died within 1 to 2 months after birth, with most survivors experiencing sterility in adulthood. For our experimental approach, we bred Pkd1L3/+ offspring and initiated intercrossing when they reached 8 weeks of age. The experimental protocol was performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the National Laboratory Animal Center. Thiamet G was used to examine the effect on cyst formation. Pkd1+/+ and Pkd1L3/L3 mice were injected intraperitoneally daily from postnatal day 7 (P7) to P14 with thiamet G (20 μM/g/day in 100 μl PBS) or vehicle DMSO and with each group comprising n=6.

Cyst index. Kidney sections from mice were stained with hematoxylin and eosin (H&E). The area of each individual cyst within the entire kidney section was calculated to determine the total cyst area. The cystic index was defined as the ratio of the total cyst area to the whole kidney area and was measured using ImageJ software.

BUN assay. The mice were sacrificed, and their urine samples were collected at P14. Blood urea nitrogen (BUN) levels were measured using a Quantichrom Urea Assay Kit (Bioassay Systems, DIUR-100, Hayward, CA, USA) following the manufacturer’s instructions. Urea concentration was detected by measuring the optical density (OD) at 570 nm.

Quantitative real-time RT-PCR. The kidneys from the mice were harvested, and total RNA was purified using the RNeasy RNA purification kit according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen), and the resulting cDNA template was mixed with SYBR Green PCR Master Mix (Applied Biosystems, 43-091-55, Carlsbad, CA, USA) in a StepOne Real-Time PCR System (Applied Biosystems). The following primers were used for RT-qPCR: mOGT forward: 5′-TTCGGGAATCACCCTACTTCA, reverse: 5′-TACCATC ATCCGGGCTCAA; mGAPDH forward: 5′-TGCA GTGGCAAAGT GGAGAT, reverse: 5′-TTTGCCGTGAGTGGAGTCATA.

Hematoxylin and eosin stain. The kidneys of the mice were fixed with 4% paraformaldehyde. Kidney sections of 4 μm thickness were prepared and counterstained with H&E for examination under a light microscope.

Statistical analysis. The results are presented as mean±standard deviation (SD) and are derived from at least three independent experiments. Statistical analysis was performed using the Student’s t-test and significance was determined at p<0.05.

Results

The level of CTT protein is positively correlated with the presence of O-GlcNAc transferase (OGT). The injection of CTT mRNA rescues the dorsal body curvature phenotype in Pkd1-knockdown zebrafish embryos, indicating that CTT of polycystin-1 (PC1) is essential for kidney function. It was hypothesized that an increase in CTT levels could compensate for the abnormal kidney function in polycystic kidney disease (PKD) in mammals. Additionally, O-linked β-N-acetylglucosamine (O-GlcNAc) addition to a protein, known as O-GlcNAcylation, has been shown to regulate protein stability, a widespread step in controlling biological processes such as cell signaling, metabolism, development, and aging (5, 8). O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), and it was tested whether OGT may enhance or stabilize CTT in an ex vivo study using kidney 293T cells. Co-transfection of Myc-CTT DNA and Flag-OGT DNA in these cells showed that overexpression of OGT enhanced CTT levels in a dose-dependent manner (Figure 1A), while knockdown of OGT significantly reduced CTT levels (Figure 1B). These results suggest that OGT may stabilize or protect CTT from degradation. To test this hypothesis, 293T cells with or without overexpressed OGT were treated with cycloheximide to inhibit newly synthesized proteins, including OGT and CTT. Figure 1C shows that overexpressed OGT prolonged CTT half-life by reducing the degradation rate of CTT, while OGT and beta-actin levels were unaffected. These experiments reveal that OGT can stabilize CTT, possibly by O-GlcNAcylation of CTT, which is often associated with protein folding and stability to protect against degradation.

Figure 1.
Figure 1.

The role of O-GlcNAc transferase (OGT) in regulating the stability of the C-terminal tail (CTT) protein. (A) Overexpression of OGT in 293T cells increased the protein levels of Myc-CTT, as evidenced by Western blot analysis using the indicated antibodies. (B) Depletion of OGT in 293T cells expressing Flag-CTT using shRNA decreased the protein levels of CTT, again confirmed by Western blot analysis. (C) OGT was found to prolong the stability of CTT protein as 293T cells expressing Flag-CTT with or without Flag-OGT were treated with cycloheximide (CHX), and Western blot analysis was performed at various time points. The band intensities were measured using ImageJ and normalized by dividing the FLAG signal to β-actin signal. p-Values were generated by Student’s t test. The results are presented as mean±SD. ***p<0.001, n=3.

Increasing CTT levels by using O-GlcNAc hydrolase inhibitors in 293T cells. O-GlcNAcylation is a prevalent post-translational modification that occurs on various proteins (5, 6, 8). It involves the combined activity of O-GlcNAc transferase (OGT) and the hydrolase OGA, as illustrated in Figure 2. This figure demonstrates that O-GlcNAcylation and de-O-GlcNAcylation can occur simultaneously but are carried out by two different enzymes (5).

Figure 2.
Figure 2.

O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) work together as an enzyme pair to integrate O-GlcNAcylation cycles. OGT catalyzes the addition of O-GlcNAc to serine or threonine residues of proteins, while OGA removes it. Thiamet G is an OGA inhibitor that prevents the removal of O-GlcNAc.

The experiments described here aimed to investigate the effect of inhibiting the O-GlcNAc hydrolase on CTT levels. We used two different hydrolase inhibitors, PUGNAc and thiamet G, and found that both resulted in an increase in CTT levels in 293T cells. The increase was more significant with PUGNAc than thiamet G (Figure 3A, B). We also investigated the interaction between O-GlcNAc transferase and CTT using co-immunoprecipitation (co-IP). We found that OGT could bind to CTT in a complex form (Figure 3C) and that newly synthesized CTT is being O-GlcNAcylated (Figure 3D). Treatment with an OGT inhibitor (OSMI-1) attenuated CTT O-GlcNAcylation (Figure 3E), suggesting that OGT is physically associated with CTT in an enzyme-substrate complex to promote CTT O-GlcNAcylation. These results suggest that inhibiting the O-GlcNAc hydrolase can elevate CTT levels, and OGT can interact with CTT to promote its O-GlcNAcylation.

Figure 3.
Figure 3.

The importance of O-GlcNAcylation in the regulation of stability of the C-terminal tail (CTT) protein. (A) Increasing the global level of O-GlcNAcylation through treatment with PUGNAc/thiamet G in 293T cells expressing Flag-CTT resulted in increased CTT protein levels, as determined by Western blot analysis using the indicated antibodies. (B) Co-immunoprecipitation (co-IP) experiments revealed that O-GlcNAc transferase (OGT) associates with CTT in 293T cells expressing Flag-OGT and/or Myc-CTT. Thirdly, O-GlcNAcylation of CTT was confirmed through IP with FLAG Ab, followed by Western blot analysis using O-GlcNAc or FLAG antibody. (C) Reduction of CTT O-GlcNAcylation was achieved by inhibiting OGT using the small molecule inhibitor OSMI-1 (10 μM, 4 h), as evidenced by IP and Western blot analysis using the indicated antibodies in cellular lysates of 293T cells expressing Flag-CTT treated with or without OSMI-1. The light chain band (LC) is indicated by an asterisk.

Low expression of OGT is associated with low levels of cellular PC1 in PKD mice. In Figure 1, we used 293T cells in an ex vivo study to demonstrate that O-GlcNAc transferase is positively correlated with the level of newly synthesized CTT. To investigate whether the low levels of PC1 could affect the expression of O-GlcNAc transferase in our knockdown mice, we used the lysate from the homogenate of the kidney cells.

Firstly, we showed that overall protein O-GlcNAcylation was substantially reduced in Pkd1-knockdown mice using a monoclonal antibody specific to O-GlcNAc on a Western blot (Figure 4A) and reached statistical significance (Figure 4B, p<0.05). Next, we demonstrated that the protein levels of O-GlcNAc transferase were also reduced (p<0.05) using an O-GlcNAc transferase-specific antibody (Figure 4C). Finally, we showed that this decrease was due to the low expression of mRNA of O-GlcNAc transferase in Pkd1-knockdown mice using qPCR (Figure 4D). Although the mechanism involved in the low expression of O-GlcNAc transferase is presently unclear, our results indicate that low levels of O-GlcNAc transferase in Pkd1-knockdown mice are associated with low levels of PC1.

Figure 4.
Figure 4.

The effect of Pkd1 knockdown on O-GlcNAcylation in mice. (A)Western blot analysis of P14 kidneys from WT and Pkd1L3/L3 mice was performed to determine the level of global protein O-GlcNAcylation and O-GlcNAc transferase (OGT) expression. (B, C) The relative global protein O-GlcNAcylation and OGT expression in the kidneys were standardized to β-actin. (D) qRT-PCR analysis was performed to determine the expression of Ogt mRNA. The data were analyzed using Student’s t-test and n=4 was used for the analysis. The results are presented as mean±SD. *p<0.05.

Rescue of polycystic formation in Pkd1L3/L3 mice using a O-GlcNAc hydrase inhibitor. We have shown inhibition of O-GlcNAc glycosidase (deglycosylation inhibition) resulted in an increase of CTT O-GlcNAcylatiom and its half-life (Figure 1, Figure 3A, B and E). A previous study also indicated that injection of the CTT mRNA can rescue the dorsal body curvature phenotype in Pkd1-knockdown zebrafish embryos (4). In addition, we have shown that the overall protein O-GlcNAcylation is drastically reduced in Pkd1 knockdown mice (Figure 4A, B). To investigate whether enhancing the O-GlcNAcylation by blocking the O-GlcNAc hydrolase with the hydrolase inhibitor thiamet G may reduce cyst formation in Pkd1L3/L3 mice, we used 24 mice divided into two groups with (n=12) and without PKD (n=12). Each wild-type or PKD group was then treated with (n=6) and without thiamet G (n=6). In the treated group, mice were administered daily injections of thiamet G into both wild-type and Pkd1L3/L3 mice from postnatal day 7 to day 14. Subsequently, the mice were analyzed on day 15. We show that inhibition of O-GlcNAc hydrolase significantly reduced renal cyst formation by a histological examination and reduced the size of kidney compared to the untreated PKD group (Figure 5A). A typical example of kidney appearance following the thiamet G treatment in the PKD group is also shown (Figure 5B). Figure 5C shows that the weight of PKD kidneys was significantly reduced upon treatment of thiamet G (n=6) relative to untreated PKD group (n=6). We also measured kidney function using a standard blood urea nitrogen (BUN) test. Figure 5D and E shows that thiamet G significantly restored the kidney function in the PKD group relative to the control untreated groups.

Figure 5.
Figure 5.

Thiamet G treatment alleviates cyst formation in Pkd1’ knockdown mice. (A) Histological examination of P14 kidneys from WT and Pkd1L3/L3 (PKD) mice injected daily with thiamet G (TG) or DMSO (C) vehicle control from 7 to 14 days post coitum (dpc). Scale bar=2 mm. (B) Gross morphology of the kidneys of Pkd1L3/L3 mice at P14, injected daily with thiamet G or DMSO. (C) Kidney weight/body weight ratios. Data represent all sections quantified for each condition. (D) Percentage of cystic area relative to the total kidney section area of P14 kidneys from WT and Pkd1L3/L3 (PKD) mice treated with DMSO (C) or thiamet G (TG). (E) Blood urea nitrogen (BUN) levels were decreased in Pkd1L3/L3 mice treated with thiamet G (TG) compared to DMSO (C) treatment (n=6 per treatment group). p-Values were generated by Student’s t-test. The results are presented as mean±SD. *p<0.05 and ***p<0.001.

Finally, the percentage of area with polycysts was determined using histological staining. As shown in Figure 5D, the progression of PKD was significantly reduced, as indicated by the cyst index. These results further support the notion that blocking O-GlcNAc hydrolase may slow the progression of PKD and suggest that O-GlcNAc hydrolase could be a potential therapeutic target for the treatment of PKD.

Discussion

Autosomal dominant polycystic kidney disease (PKD) is the most commonly inherited renal disorder, affecting 1 in 400 to 1 in 1000 live births (2, 15). This condition is characterized by the gradual development and expansion of renal cysts, ultimately leading to massive kidney enlargement and end-stage renal disease (ESRD) (15). In 85-90% of autosomal dominant PKD cases, the disease is caused by mutations in the Pkd1 gene, which encodes for polycystin-1 (PC1). PC1 is an integral membrane protein with 4,302 amino acid residues, consisting of 11 transmembrane domains, a long extracellular N-terminus containing 3,074 amino acids, and a short cytoplasmic C-terminus with 193 amino acids that interacts with multiple proteins, including PC2 (1).

The C-terminal tail (CTT) fragment of PC1 has been shown to be necessary for the physiological functions in kidney morphogenesis in polycystin-1 knockdown zebrafish, and it may explain why CTT translocates into the nuclei of kidney epithelial cells, although the exact physiological roles for this trafficking remain unclear (4). While the polycystin-1 knockdown mouse model was created in 2006 (14) to study the role of polycystin-1 in the development of PKD, the possible role of O-GlcNAc transferase in PKD has never been explored. Interestingly, we have found that the CTT contains a putative motif (PEST region) corresponding to possible O-GlcNAcylation via serine and threonine residues, and we have begun to explore the possible interaction between O-GlcNAc transferase and CTT.

There are several lines of evidence to indicate this interaction exists and plays a crucial role for the pathogenicity of PKD in the present study. Firstly, using 293T cells we show for the first time, that O-GlcNAc transferase is able to increase the levels of newly synthesized CTT in a dose-dependent manner (Figure 1A). Secondly, such an increase in CTT was hindered using two specific interfering shRNAs of O-GlcNAc transferase (Figure 1B). Thirdly, the rate of degradation of CTT was retarded in the presence of O-GlcNAc transferase (Figure 1C). This indicates the transferase can stabilize CTT via the O-GlcNAcylation on CTT. Fourth, co-immunoprecipitation containing both moieties of O-GlcNAc transferase and CTT suggests the complex formation is via enzyme-substrate binding (Figure 3C). It is of interest to note that the presence of the protein synthesis inhibitor cycloheximide, does not affect the level the transfected O-GlcNAc transferase, indicating that the levels of transferase have been established and reached a steady state, earlier than the CTT synthesis. Under these circumstances, the transferase can facilitate the O-GlcNAcylation for CTT stability (Figure 1C and D).

Our initial experiments, as mentioned above, suggest that the O-GlcNAc transferase plays a crucial role in CTT O-GlcNAcylation in 293T cells. Figure 2 shows that both O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA) work together to maintain the steady-state levels of O-GlcNAcylation on a given protein. We then tested the role of O-GlcNAc hydrolase in CTT O-GlcNAcylation using the hydrolase inhibitors (PUGNAc and thiamet-G). We confirmed that inhibition of the hydrolase resulted in an increase in the final expression of CTT. Our data further suggests that O-GlcNAc transferase may stabilize CTT expression by O-GlcNAcylation, while inhibiting the O-GlcNAc hydrolase activity substantiates this effect.

In terms of therapeutic application, there is currently no agonist for O-GlcNAc transferase. Therefore, we attempted to investigate whether inhibiting O-GlcNAc hydrolase could result in an elevation of CTT, which could have therapeutic benefits for PKD. We chose Pkd1L3/L3 mice as an in vivo model because they are well characterized and exhibit renal cystic lesions characteristic of PKD.

Thiamet G was chosen for this study due to its high selectivity and potency as an O-GlcNAc hydrolase inhibitor, with a Ki of ~20 nM. In contrast, the other inhibitor used in this study (PUGNAc) is less specific and may affect the activity of other enzymes (11). Our data show that inhibition of OGA with thiamet G is a promising therapeutic strategy. Firstly, treated Pkd1L3/L3 mice showed a reduction in kidney size and weight (Figure 5A-C). Secondly, kidney function was improved in the treated mice (Figure 5E). Thirdly, thiamet G delayed cyst formation in the kidneys of Pkd1L3/L3 mice (Figure 5D). Notably, while we observed a modest increase in CTT levels in 293T cells treated ex vivo with thiamet G, the therapeutic effect in vivo was surprisingly effective. The discrepancy between the in vitro and in vivo results may suggest the need for further experiments to screen OGA inhibitors in vitro or ex vivo.

Conclusion

Our data presents a unifying mechanism to explore the link between PKD and O-GlcNAcylation. We illustrate how O-GlcNAcylation influences the CTT of PC1, consequently impacting protein stability and function. This modulation leads to a deceleration in the advancement of PKD. Additionally, O-GlcNAcylation plays a distinct role in cyst formation during PKD development. Furthermore, the administration of thiamet G shows promising results in alleviating the progression of PKD, indicating its potential as a therapeutic agent for ADPKD in the future.

Acknowledgements

The Authors acknowledge Dr. Chao-Liang Wu from the Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, for kindly providing experimental materials. This work was supported by a research grant from the Ditmanson Medical Foundation Chia-Yi Christian Hospital Research Program (R110-023).

Footnotes

  • Authors’ Contributions

    The projects were designed by Wen-Cheng Su, Chi-Feng Hung, Hsin-Hui Su, and Yung-Liang Chen, who also provided the final approval of the version to be published. Yi-Chieh Wang, Hubert Peng, Wen-Hung Huang, and Yi-Cheng Chen performed animal experiments and analyzed data. Hubert Peng, Yi-Lun Lo, and Yun-Hwa Lo performed IHC experiments and analyzed data.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received June 1, 2023.
  • Revision received August 15, 2023.
  • Accepted August 30, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Thiamet G as a Potential Treatment for Polycystic Kidney Disease
WEN-CHENG SU, CHI-FENG HUNG, YI-CHIEH WANG, HUBERT PENG, WEN-HUNG HUANG, YI-LUN LO, YUN-HWA LO, YI-CHENG CHEN, HSIN-HUI SU, YUNG-LIANG CHEN
In Vivo Nov 2023, 37 (6) 2524-2532; DOI: 10.21873/invivo.13360
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