Effect of 2-Hydroxypropyl-γ-cyclodextrin on Renal Inflammation in Alport Mouse Model

Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides that encapsulate hydrophobic molecules such as cholesterol. Hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic heptasaccharide, has gained attention as a protective agent for chronic kidney diseases, particularly the genetic kidney disease Alport syndrome, due to its anti-inflammatory property and the ability to reduce cholesterol content in Alport kidneys. However, HP-β-CD has side effects of hearing loss and kidney injury. These concerns are important issues for Alport syndrome patients who have pre-existing hearing abnormalities and kidney dysfunction. We previously revealed that 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), a cyclic octasaccharide, is less toxic than HP-β-CD. Here, we examined the dose-dependent effects of HP-γ-CD (0.71, 1.42, 2.85 mmol/kg, once a week) on the progressive disease phenotype in the Alport mouse model (Col4a5-G5X). HP-γ-CD at 0.71 mmol/kg suppressed the renal inflammation, glomerulosclerosis, and tubular injury in Alport mice, but did not prevent the decline in kidney function. Moreover, HP-γ-CD at 2.85 mmol/kg increased proteinuria and decreased the body weight of Alport mice. Interestingly, HP-γ-CD did not reduce the unesterified cholesterol (UC) content in Alport mouse kidneys, especially in the glomeruli. These results suggested that HP-γ-CD exerted its anti-inflammatory effect, but did not improve the progressive phenotype in the Col4a5 G5X Alport mouse model. Our findings add more information on the use and dosage effects of HP-γ-CD for experimental Alport syndrome.

INTRODUCTION

Alport syndrome is an inherited glomerulonephritis caused by a mutation in one of the type IV collagen genes (COL4A3, A4, or A5) that compose the glomerular basement membrane (GBM).¹⁾ Mutations disrupt the structure and function of the GBM, resulting in proteinuria, renal dysfunction, and end-stage kidney disease (ESKD).²⁾ Pathological features of Alport syndrome include glomerular injury, renal inflammation, and fibrosis, which are similar to other chronic kidney diseases (CKD). Currently, renin angiotensin aldosterone system (RAAS) inhibitors are used for Alport therapy, but the disease eventually progresses into ESKD.³⁾ Therefore, it is important to continue the search for novel therapeutic targets that have different mechanisms from RAAS inhibitors.

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six, seven, and eight d-glucopyranose units, called α-, β-, and γ-CD, respectively. Their unique cyclic structure, featuring a hydrophilic outer surface and a hydrophobic inner cavity, enables the formation of water-soluble inclusion complexes with molecules of an appropriate size and low polarity, such as medicine or unesterified cholesterol (UC).⁴⁾ Recent studies show that renal dyslipidemia contributes to the pathological progression of kidney diseases and that reducing lipotoxicity by treatment with HP-β-CD ameliorated renal dysfunction in 129S1/SvImJ background Col4a3 knockout mice.^5,6) β-CD is also considered as a candidate treatment for atherosclerosis, a chronic inflammatory disease, not only because of its drug delivery capacity but also because of its inherent anti-inflammatory property.^7,8) Because chronic renal inflammation is a common pathology in CKD, it is expected that CDs exert renoprotection in Alport syndrome via an anti-inflammatory effect in addition to the removal of lipotoxicity. However, HP-β-CD was shown to cause lung injury, hearing loss, and kidney injury.^9–11) The latter two effects are major concerns for Alport patients having pre-existing hearing abnormalities and kidney dysfunction. To address this concern, we focused on 2-hydroxypropyl-γ-CD (HP-γ-CD), which is an octasaccharide CD with a larger cavity than HP-β-CD. We previously reported that HP-γ-CD is as effective as HP-β-CD for Niemann–Pick disease type C (NPC) treatment. Importantly, while the therapeutic dose of HP-β-CD that demonstrated efficacy in NPC model mice showed ear and pulmonary toxicity, HP-γ-CD has a wider margin of safety.^10,12,13) Based on these studies, we examined the potential effects of HP-γ-CD treatment on experimental Alport syndrome, which has a nonsense mutation at glycine 5 in Col4a5 (B6.Cg-Col4a5^tm1Yseg/J).¹⁴⁾ This Alport mouse spontaneously shows proteinuria, glomerulosclerosis, tubular injury, renal inflammation, and fibrosis. We found that HP-γ-CD had a significant anti-inflammatory effect but did not ameliorate renal dysfunction, including proteinuria and plasma creatinine in Alport mice, indicating that the anti-inflammatory effect alone is not enough to improve the progressive phenotype of Alport syndrome. Moreover, the dose of HP-γ-CD equivalent to the therapeutic dose of HP-β-CD increased proteinuria and decreased body weight in Alport mice. These findings highlight the need to explore better and safer potential therapeutic candidates and their dosing.

MATERIALS AND METHODS

Animals and in Vivo Treatment

The X-linked Alport mouse model (Col4a5^tm1Yseg G5X mutant) has been described previously.¹⁴⁾ These mice were obtained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). Age-matched wild-type (WT) C57BL/6 mice (Charles River Laboratories, Wilmington, MA, U.S.A.) were used for experiments as the controls. Male mice were used to eliminate sex differences due to sex-linked inheritance of Col4a5tm1Yseg G5X mutation.^15–22) HP-γ-CD was reported to reach the kidneys at a concentration of 2.85 mmol/kg, which is equivalent to approximately 4000 mg/kg HP-β-CD, and once-a-week subcutaneous administration of 2.85 mmol/kg HP-γ-CD improved NPC-related pathologies in a mouse model of NPC.^10,23) Based on these studies, we set the dosing condition of HP-γ-CD at 0.71, 1.42, and 2.85 mmol/kg, once a week, as low, middle, and high doses, respectively, delivered subcutaneously. We assessed the effect of HP-γ-CD on the renal function in Alport mice (Col4a5-G5X) at 6–22 weeks old (Fig. 1A) based on our previous work with the Col4a5-G5X Alport mouse model.^15–18) HP-γ-CD (degree of HP substitution: 4.6, molecular weight: 1563.92) was donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). HP-γ-CD was dissolved in distilled water, and the osmotic pressure was adjusted with sodium chloride to near physiological osmolality. The subcutaneous injection volume was 20 μL per gram of body weight for all experimental groups. All animal experiments were approved by the Animal Care and Use Committee of Kumamoto University, Kumamoto, Japan (Approval #A2025-097R1).

Fig. 1. HP-γ-CD Ameliorated the Renal Inflammation in the Col4a5 G5X Alport Mice

(A) Experimental design for the administration of HP-γ-CD in Col4a5 G5X Alport mouse model. (B) Renal sections were analyzed using F4/80 immunohistochemistry staining. Representative images are shown. Scale bar, 100 μm. (C) F4/80-positive area was evaluated based on the F4/80-stained section, using Bio-Revo imaging and analysis software. (D–J) The level of the indicated mRNA was measured and normalized to the level of Gapdh mRNA (internal control). Data are presented as mean ± S.E. (n = 8 per group). p-Values were assessed by Tukey’s test.

Measurement of Proteinuria Score

Mouse urine samples were collected for 24 h once every two weeks using a metabolic cage (AS ONE Corporation, Osaka, Japan). Urinary protein and creatinine were measured by the Bradford (Bio-Rad, Hercules, CA, U.S.A.) and Jaffe’s methods (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively, as described previously.²¹⁾ Urinary protein concentration was normalized with urinary creatinine concentration and presented as proteinuria score.

Measurement of Plasma Creatinine, Blood Urea Nitrogen (BUN), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Cholesterol

Mouse blood samples were obtained from the abdominal aorta. Fresh blood samples were centrifuged at 3000 rpm, 4°C for 15 min, and blood plasma was collected. Plasma creatinine, BUN, ALT, AST, and cholesterol were measured by DRI-CHEM (FUJIFILM, Tokyo, Japan).

Histological Analysis

Kidney tissues were fixed in 10% formalin and embedded in paraffin. Tissue blocks were sliced into 4 μm thickness using a microtome and stained with Periodic acid-Schiff (PAS) and Masson’s trichrome (MT). The protocols for PAS and MT staining are described in detail in our recent work.¹⁵⁾ The glomerulosclerosis score and fibrosis area were evaluated as previously described.^15–18) For glomerulosclerosis, tissues were imaged on a BZ-X700 microscope (KEYENCE, Osaka, Japan), and each glomerulus was visually scored by manual observation. More than 100 glomeruli selected randomly per mouse were objectively scored based on the following criteria, 0: no lesion, 1: expansion of mesangial area, 2: expansion of Bowman’s epithelial cells, adhesion of glomeruli and Bowman’s capsule and partial sclerosis, 3: sclerotic area in 50–75% of glomeruli, and 4: sclerotic area in 75–100% of glomeruli. For renal fibrosis, the MT-positive area was quantified using a BZ-X700 microscope and image analysis software (KEYENCE). Fibrosis area was normalized to tissue area at 10 different points in each mouse.

Immunostaining

A total of 4 μm paraffin sections were used for staining as described.^15,17) After deparaffinization, sections were antigen-retrieved for 10 min at room temperature with Proteinase K (S3020, Dako) or 20 min at 121°C with Dako Target Retrieval Solution (S2369, Agilent). Blocking was performed with Protein Block (X090930-2, Agilent) and Avidin/Biotin Blocking Kit (SP-2001, Vector). The tissue sections were incubated with the following primary antibodies overnight at 4°C: anti-F4/80 antibody (ab6640, Abcam, Cambridge, U.K.), biotinylated-LTL (Lotus tetragonolobus lectin; B-1325-2, Vector Laboratories, Burlingame, CA, U.S.A.), and anti-KIM-1 (Kidney injury molecule-1; AF1817, R&D Systems, Minneapolis, MN, U.S.A.). HRP or fluorescent secondary antibodies were applied for 1 h at room temperature. F4/80-, LTL-, and KIM-1-positive area of kidney tissue was quantified using a BZ-X700 microscope and image analysis software (KEYENCE).

Real-Time RT-PCR Analysis

Total RNA was isolated from the mouse whole kidney using NucleoSpin RNA (TaKaRa, Shiga, Japan) with homogenization. Reverse transcription using equal amounts of RNA and PCR amplification were performed using PrimeScript RT Reagent Kit with gDNA Eraser and SYBR Premix ExTaq II (TaKaRa), respectively, according to the manufacturer’s recommended protocol. Relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized using mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an endogenous control. The sequences of primers used for Q-PCR are listed in Table 1.

Table 1. Primers Used in Quantitative RT-PCR

Gene	Sense	Antisense
Kim-1	5′-GGAAGTAAAGGGGGTAGTGGG-3′	5′-AAGCAGAAGATGGGCATTGC-3′
Lcn2	5′-GAGAAGGCAGCTTTACGATG-3′	5′-CCTGGAGCTTGGAACAAATG-3′
Il-1β	5′-GCTGAAAGCTCTCCACCTCAATG-3′	5′-TGTCGTTGCTTGGTTCTCCTTG-3′
Il-8	5′-TGTCAGTGCCTGCAGACCAT-3′	5′-CCTCGCGACCATTCTTGAGT-3
Il-6	5′-GAGGATACCACTCCCAACAGACC-3′	5′-AAGTGCATCATCGTTGTTCATACA-3′
Mcp1	5′-GAAGCTGTAGTTTTTGTCACCAAG-3′	5′-AGGTAGTGGATGCATTAGCTTCA-3′
Tnfa	5′-CATCTTCTCAAAATTCGAGTGACAA-3′	5′-TGGGAGTAGACAAGGTACAACCC-3′
Nqo1	5′-TTCTGTGGCTTCCAGGTCTT -3′	5′-AGGCTGCTTGGAGCAAAATA -3′
Ho-1	5′-GCCACCAAGGAGGTACACAT -3′	5′-GCTTGTTGCGCTCTATCTCC-3′
Tgf-β	5′-CACCTGCAAGACCATCGACAT-3′	5′-GAGCCTTAGTTTGGACAGGATCTG-3′
Col1a1	5′-CTGGCGGTTCAGGTCCAAT-3′	5′-TTCCAGGCAATCCACAGAC-3′
Mmp9	5′-GGACCCGAAGCGGACATTG-3′	5′-CGTCGTCGAAATGGGCATCT-3′
Mmp12	5′-CATGAAGCGTGAGGATGTAGAC-3′	5′-TGGGCTAGTGTACCACCTTTG-3′
Gapdh	5′-CCTGGAGAAACCTGCCAAGTATG-3′	5′-GGTCCTCAGTGTAGCCCAAGATG-3′

Filipin III Staining

For Filipin III staining, 10 μm frozen sections were stained with Filipin III (70440, Cayman Chemical, Ann Arbor, MI, U.S.A.). Phase contrast images were taken, and the Filipin III-positive area in the kidney tissue was quantified using a BZ-X700 microscope and image analysis software (KEYENCE).

Statistical Analysis

All data are presented as mean ± standard error (S.E.). For four-group comparisons, we used ANOVA with Tukey’s test. p-Values <0.05 were considered to be statistically significant.

RESULTS

HP-γ-CD Ameliorated the Renal Inflammation in the Col4a5 G5X Alport Mice

Because Alport mice exhibit renal inflammation, we investigated the effect of HP-γ-CD on inflammation by F4/80 staining. Vehicle-treated Alport mice showed infiltrating macrophages in the interstitial area that were significantly suppressed by 0.71 mmol/kg HP-γ-CD, while the other doses tended to suppress macrophage infiltration (Figs. 1B, 1C). Consistent with the immunostaining results, the mRNA expression levels of inflammatory cytokines (Il-1β, Il-8, Il-6, and Mcp1) and oxidative stress markers (Nqo1 and Ho-1) were statistically down-regulated by treatment with 0.71 mmol/kg HP-γ-CD (Figs. 1D–1J). The doses 1.42 and 2.85 mmol/kg HP-γ-CD also decreased all inflammatory cytokines except Il-6 and the oxidative stress marker Ho-1 (Figs. 1D–1J). Collectively, these results revealed anti-inflammatory and antioxidant effects of HP-γ-CD in the kidney.

Low-Dose HP-γ-CD Slightly Ameliorated the Kidney Injury in the Col4a5 G5X Alport Mice

Alport mice exhibit typical glomerulosclerosis, renal fibrosis, and tubular injury with renal inflammation. To evaluate the effect of HP-γ-CD on kidney injury, we conducted PAS staining, Masson’s trichrome staining, and immunofluorescence staining of kidney injury molecule (Kim)-1 and lotus tetragonolobus lectin (LTL), which is a tubular marker, using renal tissue. HP-γ-CD at 0.71 mmol/kg, but not at 1.42 and 2.85 mmol/kg, ameliorated the glomerulosclerosis (Fig. 2A; PAS). Assessment of the glomerular injury score revealed that in vehicle-treated Alport mice, more than 50% of glomeruli showed severe glomerulosclerosis (score 4), which was not improved by 1.42 and 2.85 mmol/kg HP-γ-CD treatment (Fig. 2B). Notably, 0.71 mmol/kg HP-γ-CD reduced the severe glomerulosclerosis (score 4) by 10% and significantly increased the mild glomerulosclerosis (score 2) by 15% (Fig. 2B), suggesting that 0.71 mmol/kg HP-γ-CD slightly ameliorated the glomerulosclerosis. By Masson’s trichrome staining, we detected pervasive renal fibrosis in Alport mice (Fig. 2A; Masson). HP-γ-CD at 0.71 mmol/kg tended to decrease the fibrotic region in Alport kidneys, whereas 1.42 and 2.85 mmol/kg had no detectable effect (Fig. 2C). The mRNA expression levels of fibrosis-related genes (Tgf-β, Col1a1, and Mmp9) were significantly down-regulated by HP-γ-CD at 0.71 mmol/kg (Figs. 2D–2G). Next, we evaluated the effect of HP-γ-CD on tubular injury in Alport mice. Vehicle-treated Alport mice displayed increased Kim-1- and decreased LTL-stained areas, indicating tubular injury (Fig. 2A; Kim-1/LTL, H, I). HP-γ-CD reduced the Kim-1-positive area dose-dependently but did not change the LTL-positive area in Alport mice (Figs. 2H, 2I). We also measured the mRNA levels of Kim-1 and another tubular injury marker, Lipocalin-2 (Lcn2). The results showed that Kim-1 was decreased by HP-γ-CD, while Lcn2 remained unchanged compared with the vehicle-treated Alport mice. (Figs. 2J, 2K). Because HP-γ-CD did not alter the level of the tubular marker LTL and the Lcn2 mRNA level in Alport mice, we surmised that the reduction in Kim-1 by HP-γ-CD was due to the slight amelioration of the proximal tubular injury, and not by tubular loss. Together, these results suggest that low-dose HP-γ-CD slightly ameliorated kidney injury in Alport mice.

Fig. 2. Low-Dose HP-γ-CD Slightly Suppressed the Kidney Injury in the Col4a5 G5X Alport Mice

(A) Renal sections were analyzed by PAS staining, Masson-trichrome (MT) staining, or Kim-1 and LTL immunofluorescence staining. Scale bars, 50 μm (for PAS staining) and 100 μm (for other staining). (B, C) Glomerulosclerosis scores and the fibrotic region were evaluated based on the PAS-stained section or the MT-stained section, using Bio-Revo imaging and analysis software. (D–G) The level of the indicated mRNA was measured and normalized to the level of Gapdh mRNA (internal control). Data are presented as mean ± S.E. (n = 8 per group). (H, I) Kim-1- and LTL-positive area was evaluated, using Bio-Revo imaging and analysis software. Data are presented as mean ± S.E. (n = 4 per group). (J, K) The level of the indicated mRNA was normalized to the level of Gapdh mRNA (internal control). Data are presented as mean ± S.E. (n = 8 per group). p-Values were assessed by Tukey’s test.

HP-γ-CD Did Not Improve the Kidney Function in Col4a5 G5X Alport Mice

Alport syndrome is characterized by proteinuria with glomerular damage and progressive loss of renal function, so we monitored proteinuria over time in Alport mice treated with HP-γ-CD. The 0.71 mmol/kg dose did not improve proteinuria, and 2.85 mmol/kg increased proteinuria in 20- to 22-week-old Alport mice (Fig. 3A). We assessed the renal function by measuring plasma creatinine and BUN levels. HP-γ-CD did not reduce the plasma creatine at any dose and increased the BUN at 2.85 mmol/kg (Figs. 3B, 3C). Moreover, we observed a slight weight loss in the group treated with 2.85 mmol/kg HP-γ-CD (Fig. 3D), suggesting that high-dose HP-γ-CD may induce a side effect. The urine volume was unaltered in the HP-γ-CD-treated Alport mice compared with vehicle-treated Alport mice (Fig. 3E). To determine the toxicity of HP-γ-CD, we measured plasma alanine transaminase (ALT) and aspartate transaminase (AST). ALT and AST levels were unchanged in vehicle-treated Alport mice compared with wild-type mice. By contrast, HP-γ-CD reduced the ALT, but not the AST, in a dose-dependent manner (Figs. 3F, 3G).

Fig. 3. HP-γ-CD Did Not Improve the Kidney Function in Col4a5 G5X Alport Mice

(A) Urinary protein and creatinine were measured using Bradford and Jaffe’s methods, respectively. Urinary protein was normalized with urinary creatinine and presented as proteinuria. (B, C) Plasma creatinine and BUN were measured using DRI-CHEM (FUJIFILM) in 22-week-old wild-type and Alport mice. (D, E) Body weight and urine volume were measured every two weeks. (F, G) Plasma ALT and AST were measured by DRI-CHEM (FUJIFILM) in 22-week-old wild-type and Alport mice. Data are presented as mean ± S.E. (n = 8 per group). p-Values were assessed by Tukey’s test.

HP-γ-CD Did Not Decrease the Accumulation of Unesterified Cholesterol in the Kidneys of the Col4a5 G5X Alport Mice

CDs conjugate cholesterol, so we investigated the effect of HP-γ-CD on the plasma cholesterol level, which is higher in vehicle-treated Alport mice compared with WT mice. HP-γ-CD at all doses did not affect the level of cholesterol (Fig. 4A). We next assessed the accumulation of unesterified cholesterol (UC), which is the target of CDs in NPC, in the kidneys of Col4a5 G5X Alport mice by Filipin III staining. Filipin III-positive region is more pervasive in Alport kidneys, especially in glomeruli, compared with WT mice, suggesting that UC accumulated in Alport kidneys (Figs. 4B, 4C). Despite the anti-inflammatory effect and slight suppression of kidney injury by HP-γ-CD, it did not reduce the accumulation of UC (Figs. 4B, 4C). Therefore, these results suggest that HP-γ-CD worked without involving UC in the Alport kidneys, or by targeting other lipids in Alport mice.

Fig. 4. HP-γ-CD Did Not Ameliorate the Accumulation of Unesterified Cholesterol in the Kidneys of the Col4a5 G5X Alport Mouse Model

(A) Plasma cholesterol was measured by DRI-CHEM (FUJIFILM) in 22-week-old wild-type and Alport mice. (B) Renal sections were observed using phase-contrast imaging and Filipin III staining. (C) The Filipin III-positive area was evaluated based on the Filipin III-stained sections, using Bio-Revo imaging and analysis software. Data are presented as mean ± S.E. (n = 8 per group). p-Values were assessed by Tukey’s test.

DISCUSSION

In this study, we examined the effects of HP-γ-CD on the Col4a5 G5X Alport mouse model. We revealed that 0.71 mmol/kg HP-γ-CD suppressed renal inflammation, glomerulosclerosis, and tubular injury, but did not decrease proteinuria, plasma creatinine, and BUN in Alport mice, suggesting that HP-γ-CD did not improve the decline in kidney function. At a higher dose of 2.85 mmol/kg, HP-γ-CD also showed an anti-inflammatory effect, but it increased proteinuria and did not ameliorate glomerulosclerosis and renal fibrosis in Col4a5 G5X Alport mice. While HP-γ-CD showed a significant anti-inflammatory effect, our results suggested that the anti-inflammatory effect alone is not enough to ameliorate the progressive phenotype of Alport syndrome. To our knowledge, this is the first in vivo study to determine the impact of HP-γ-CD on the pathological phenotypes in Col4a5 G5X Alport mice.

Several studies have indicated that CDs exhibit anti-inflammatory effects by extracting cholesterol accumulated in organs,^24,25) but HP-γ-CD did not decrease the accumulation of UC in the kidney of Col4a5 G5X Alport mice (Figs. 4B, 4C). Interestingly, β-CD has been reported to exert its anti-inflammatory effects by working directly on macrophages.^8,26) Mechanistically, β-CD has been suggested to remove 7-ketocholesterol, a class of oxysterols regarded as pro-inflammatory factors, from macrophages.²⁷⁾ Indeed, we observed the macrophage infiltration in the kidneys of Alport mice and the reduction of the mRNA expression levels of inflammatory cytokines by HP-γ-CD (Fig. 1). Therefore, we surmised that the anti-inflammatory effect of HP-γ-CD may be due to its effect on macrophages. We also found that HP-γ-CD significantly suppressed Kim-1 expression at both protein and mRNA levels (Figs. 2A, 2H–2J). This was not due to tubular loss because the expression of the tubular marker LTL was unaffected by HP-γ-CD treatment. Because Kim-1 is upregulated by acute or chronic kidney injury, which indicates inflammation,^28,29) it is possible that HP-γ-CD decreased the renal inflammation, thereby suppressing Kim-1 expression.

A recent study showed that HP-β-CD ameliorated renal dysfunction in Col4a3 knockout Alport mice.⁵⁾ Moreover, a phase 2a clinical trial of HP-β-CD (Var-200) for diabetic kidney disease is planned (NCT06489340). However, concerns exist regarding the administration of HP-β-CD, as it has been shown to cause lung injury, hearing loss, and kidney injury.^9–11,30) On the contrary, we have previously demonstrated that HP-γ-CD (8.0 mmol/kg, s.c.) does not cause thickened alveolar septa, hepatocellular necrosis, vacuolization of the tubular epithelium, and an increase in serum BUN levels in WT mice.¹⁰⁾ However, at 2.85 mmol/kg, which is equivalent to the effective dose of HP-β-CD, HP-γ-CD reduced the body weight and increased proteinuria in Col4a5 G5X Alport mice, indicating some side effects even though HP-γ-CD has a wider margin of safety than HP-β-CD.^10,12) We also confirmed that HP-γ-CD significantly reduced ALT in Col4a5 G5X Alport mice to a level much lower than that in WT mice (Fig. 3F). Although ALT is primarily expressed in the liver, it is also expressed in the kidneys, suggesting that the HP-γ-CD-induced reduction of ALT may indicate hepatotoxicity or nephrotoxicity. The decrease in ALT may also indicate reduced vitamin B6, a complement of ALT, that has been reported to decrease in chronic glomerulonephritis as renal function declines.^31,32) Thus, the exacerbation of renal dysfunction in Col4a5 G5X Alport mice by high-dose HP-γ-CD may have contributed to the decrease in ALT. Future investigations will elucidate the detailed mechanism by which 2.85 mmol/kg (high dose) HP-γ-CD impairs the renal function of Alport mice. Our results suggest the potential nephrotoxicity of high-dose HP-β-CD or HP-γ-CD and the need to establish a safe, effective clinical dose for patients with CKD.

Our study has some limitations. First, we did not compare the efficacy and toxicity of HP-γ-CD and HP-β-CD in Col4a5 G5X Alport mice. A comparative study to determine the effect of not only HP-γ-CD but also of HP-β-CD in Col4a5 G5X Alport mice may be warranted. Second, we have not confirmed the effects at doses <0.71 mmol/kg HP-γ-CD. Considering that 0.71 mmol/kg HP-γ-CD diminished the renal pathology in Col4a5 G5X Alport mice and the further practicality of HP-γ-CD in the future, it is necessary to conduct research using low doses based on 0.71 mmol/kg of HP-γ-CD as a standard.

In conclusion, we show that low-dose (0.71 mmol/kg) HP-γ-CD partially protected Col4a5 G5X Alport mice against renal inflammation, glomerulosclerosis, and tubular injury, but it did not improve renal dysfunction in experimental Alport mice. These findings provide additional insight into the possibilities and pitfalls of using CDs for kidney diseases, including Alport syndrome.

Acknowledgments

The HP-γ-CD was generously provided by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). This work was supported by the Japan Agency for Medical Research and Development (23ek0310017h0003 to H.K.), and the Japan Society for the Promotion of Science KAKENHI (JP22H02810 to H.K., and JP23K06165 to M.A.S.).

Conflict of Interest

The authors declare no conflict of interest.

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