Citrate potassium/citrate sodium prevents the renal injury of adenine-induced nephropathy in mice

doi:10.21203/rs.3.rs-4467467/v1

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Citrate potassium/citrate sodium prevents the renal injury of adenine-induced nephropathy in mice

https://doi.org/10.21203/rs.3.rs-4467467/v1

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Background

Metabolic acidosis is a result and a risk factor of chronic kidney disease (CKD). Alkali loads can neutralize acidosis, but the reno-protective effects of the supplementation on CKD have been limited. Here we evaluated the reno-protective effect of the alkali supplement, potassium citrate/sodium citrate tablet (PCSC), on an adenine-induced-CKD animal model.

Methods

To evaluate the reno-protective effects of PCSC, mice (C57BL/6J, female, 5 weeks) orally administrated with adenine for 18 days were compared between the PCSC group and the vehicle. The weights of the body, heart, bilateral kidneys, quadriceps femoris, renal function, and histological changes were statistically analyzed

Results

Renal functions of both groups were not different, however, increments of kidney weights were significantly different (p=0.047). It was considered that PCSC suppressed adenine-induced acute injury. In the histological analysis, enlargement of the papilla area and tubular dilatation of the cortex were diminished in the PCSC group.

Conclusion

This is the first report demonstrating that PCSC prevented the development of renal tubulointerstitial injury of the murine adenine-induced CKD model.

Health sciences/Diseases/Kidney diseases

Health sciences/Diseases/Kidney diseases/Chronic kidney disease

Health sciences/Diseases/Kidney diseases/Interstitial disease

citrate

acidemia

chronic kidney disease

Metabolic acidosis is a result of renal dysfunction and the accumulation of waste and uremic toxins, observed in patients with chronic kidney disease (CKD). Metabolic acidosis is also a risk factor in the progression of CKD and cardiovascular diseases (CVD). Alkali loads can neutralize acidosis, but little is known about the reno-protective effects of the supplements on CKD probably. Because exogenic alkali loads are excreted quickly in the urine. Recently we revealed an alkalizing supplementation of citrate potassium/citrate sodium (PCSC) reduced intrarenal oxidative stress in patients with mild-stage CKD. However, whether PCSC prevents renal functions in the mild stage CKD has been controversial because the study targeted mild-stage CKD patients with low proteinuria, and almost cases were not eligible for renal biopsy for the interventional cohort study. We evaluated the effects of citrate sodium/potassium citrate in the early-stage CKD-model mice.

Animal and Protocol

This study was conducted according to the Animal Research: Reporting of In Vivo Experiments Guidelines. Female 5-week-old C57BL/6J mice (n = 16, 5 weeks-old) obtained from Japan SLC, Inc. (Japan) were fed CE-2 and drank ad libitum water. Then, four mice were housed in each cage with 12-hour light/dark cycles at an average temperature of 23˚C. The experimental protocol was approved by a licensing committee of the Animal Experimentation Committee of SMC Laboratories, Japan’s IACUC (Study Protocol No: SLMC136-2309-8, Approval ID: N015).

Following the previous report, eight adenine-induced CKD model mice were used in each group for this study. Sixteen adenine-induced CKD model mice were randomly divided into two groups: vehicle (AdV) and citrate potassium/citrate sodium (PCSC) group (AdC). After three days of orally administrating 2000 mg/kg body of PCSC (kindly provided by Nippon Chemiphar Co., Ltd.) to the AdC group, both groups were orally gavaged ten mg/kg body of adenine (FUJIFILM Wako Pure Chemical Co., Japan) once a day for 18 days (Fig. 1). On the last day of the protocol, the mice were anesthetized with isoflurane and blood samples were taken, then euthanized by exsanguination under deep sedation. Their body weights, hearts, bilateral kidneys, and quadriceps femoris were measured individually.

Histological analysis of the adenine-injured kidneys

The kidneys axial-dissected at the maximal diameter were fixed with Bouln’s solution (Sigma-Aldrich Japan LLC.) and embedded with paraffin. The samples were sectioned at 3 µm thickness and observed by periodic-acid Schiff stain (PAS, Sigma-Aldrich Japan LLC.) and Sirius-red stain (SR, FUJIFILM Wako Pure Chemical Co., Japan). The histological images captured by BZ-8000 (Keyence co., Japan) were analyzed by NIH Image J (National Institute of Health, USA). The outlines of renal capsules, the papilla areas, and SR-positive areas were measured individually.

Statistical analysis

All data were statistically analyzed by t-test with JMP pro 17 (JMP Statistical Discovery LLC.). Data was presented as mean ± SE and significant value was defined as p < 0.05.

Body sizes of the AdC and AdV groups were not significantly different at Day 21, however, both kidney weights of the AdC were significantly lower than that of the AdV [AdC 209 ± 8.06 vs. AdV 234 ± 8.06, p = 0.047] (Table). Serum creatinine and blood urea N were not significantly different between the AdC and the AdV groups, except for uric acid [p = 0.0004]. Histological findings of PAS showed an enlargement of the maximal cross-section of the whole kidney and tubular dilatation of the cortex. The changes were diminished in the PCSC group (Fig. 2A). Maximal cross-section of the kidney and the papilla area were suppressed by PCSC compared to the vehicle [maximal section; AdC 80.4 ± 2.46 vs. AdV 87.8 ± 2.30, p = 0.047 and papilla area; AdC 4.00 ± 0.46 vs. AdV 5.57 ± 0.43, p = 0.027] (Fig. 2B). One kidney of AdC group could not sliced though the center of papilla and avoided the analysis because its papilla was small different from mice of AdV group. Analysis of the Sirius-red positive-area, other than the arteries, showed no difference between the AdV and the AdC [p = 0.3760].

This is the first report that citrate supplementation prevented histological changes of tubules in adenine-induced nephropathy. The adenine-induced nephropathy presents strong kidney fibrosis and tubular dilatation with positive SR staining by increasing oxidative stress.^,^, In this study we did not recognize an increment of SR positive area in AdV and AdC groups, but PCSC suppressed the tubular dilatation recognized in AdV. It meant our modified animal model had not expressed fibrotic changes yet, because the period of adenine administration was a little shorter than in the previous study⁴ to focus on the early stage of adenine-induced nephropathy. The long-term oral administration of adenine leads to tubulointerstitial fibrosis and end-stage renal failure by inflammations, oxidative stress, and apoptosis. Another alkalizing supplementation by sodium bicarbonate prevented renal injuries in nephrosis-model mice due to the suppression of intrarenal oxidative stress. The progression of renal failure from CKD influenced by worsened lifestyle and stress takes a decade year. We consider this reno-protective effect was slight, but supplementation of PCSC can suppress the renal damage by some daily diet. We concluded that PCSC intake may eliminate the chance of the first step of renal injuries.

Acknowledgements:

We appreciate SMC Laboratories for the experimental part, following the Guidelines for the management and welfare of laboratory animals established by testing facilities, i.e. SMC Laboratories, Ltd.’s Animal Experiment Regulations. Citrate potassium/citrate sodium was kindly provided by Nippon Chemiphar Co., Ltd. Thank you to Mr. Dennis Nishimura for the English proofreading.

Author contributions statement

MA and TI designed the protocol and MA was responsible for the study. KI advised animal experiments. SW, TTo, MM, TTa, and TA helped with image analysis and prepared Figure 2. All authors reviewed the manuscript.

Ethical Statements

The experimental protocol was approved by a licensing committee of the Animal Experimentation Committee of SMC Laboratories, Japan’s IACUC (Study Protocol No: SLMC136-2309-8, Approval ID: N015).

This study was conducted in accordance with the Animal Research: Reporting of In Vivo Experiments Guidelines. C57BL/6J mice (5 weeks of age, female) obtained from Japan SLC, Inc. (Japan). All animals used in this study were cared for following guidelines: Act on Welfare and Management of Animals (Ministry of the Environment, Act No. 105 of October 1, 1973), Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notice No.88 of the Ministry of the 3 Environment, April 28, 2006) and Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials

Funds

JSPS KAKENHI grant numbers (C) 22K11849.

Conflict of interests

None.

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No competing interests reported.

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Citrate potassium/citrate sodium prevents the renal injury of adenine-induced nephropathy in mice

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Abstract

Figures

Introduction

Methods

Animal and Protocol

Histological analysis of the adenine-injured kidneys

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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