High blood sugar may blunt the protective benefits of FXR for the survival of renal medullary collecting duct cells in response to hypertonic stress

doi:10.21203/rs.3.rs-2308587/v1

Backgroud:

Diabetic nephropathy is the most common renal complication of diabetes and the leading cause of end-stage renal disease. many factors lead to the occurrence of this disease. However, little attention has been paid to the effect of water deficiency on DN. This study focused on the effect of dehydration on renal injury in diabetes mellitus.

Methods

Diabetic and non-diabetic mice were deprived of water every other day for a total of 16 weeks. The effects of water deficiency on kidney of diabetic and non-diabetic mice were analyzed by physiological indexes, biochemical tests, pathology and the expression changes of proteins. Finally, we verified the important role of farnesoid X receptor transcription factor(FXR) in renal injury caused by dehydration once again through the study of FXR knockout mice.

Results

Our animal studies confirmed that kidney injury was more obvious in diabetic mice after water deprivation.Diabetic mice had increased urine volume and serum concentrations of creatinine after dehydration, pathological injury of renal medulla was also observed. We further demonstrated that glycoprotein 91/ aquaporin 2 expression increased in renal medulla under water deficiency, especially under high blood serum glucose concentrations. At the same time, in high sugar environment, dehydration caused overexpression of FXR and tonicity-responsive enhancer-binding protein(TonEBP), which led to oxidative stress damage to the renal medulla. However, this oxidative stress damage was weakened after FXR knockout.

Conclusions

Hypertonic conditions in high glucose environments promote overexpression of FXR. It binds TonEBP in the renal medulla, thus leading to excessive oxidative stress damage and ultimately to renal medulla damage.

diabetic nephropathy

water deficiency

FXR

oxidative stress damage

According to the latest epidemiological survey, diabetes has become the most important cause of secondary kidney disease [1, 2], which raises the necessity to elucidate its underlying mechanisms. Recent results proved that factors like genetic predisposition, hyperglycemia, obesity, hypertension, smoking and high protein intake are involved in the occurrence and development of diabetic nephropathy (DN)[3, 4]. However, few authors stressed the importance of water metabolism balance on DN.

The whole-body water metabolic equilibrium is indispensable for life and maintains fluid homeostasis[5]. The adult human body produces 180L of protourine every day. About 90% of the protourine is reabsorbed in the renal tubules while the residual 10% is by the kidney medullary collecting duct cells (MCDs). When the human body is in water shortage, the plasma osmotic pressure increases and the central nervous system secretes vasopressin. Vasopressin acts on the renal collecting tubes and thus increases water reabsorption[6]. In this process, a variety of cytokines, including tonicity-responsive enhancer-binding protein(TonEBP), aquaporin 2(AQP2) and urea transporter 2 (UT2) participate in the regulation of this process[7–9]. The relationship between these factors and their expression fluctuates in a high glucose environment[10], which is one of the focuses of this study.

Previous studies show that farnesoid X receptor transcription factor(FXR) is implicated in the preservation of the medullary collecting duct under harsh conditions like dehydration and high blood serum salt and sugar concentrations[11, 12]. In addition, FXR was found to inhibit fatty acid synthesis, oxidative stress response, and the expression of pro-fibrotic growth and pro-inflammatory factors [13–17]. Data show that oxidative stress triggers diabetic nephropathy and is regulated by FXR. Therefore, we focused our investigation in this paper on the relationship between FXR and oxidative stress. We analyzed the involvement of gp91-phox(gp91) in the process. gp91 is the catalytic subunit of the nicotinamide adenine dinucleotide phosphate oxidase(NOX) and a marker of its activation and an indicator of oxidative trauma in many studies[10, 18].

This study is based on clinical and animal experiments to study the effect of water deficiency on kidneys in high sugar environments to provide a reference for the prevention and treatment of DN. Our results conclude that hypertonic conditions in high glucose environments promote overexpression of FXR, thus leading to excessive oxidative stress damage and ultimately renal medulla damage. We further prove that gp91 is also involved in oxidative stress and renal function injury under water deficiency. Thus, we provide a different knowledge of the prognosis and treatment of diabetic kidney injury.

Animal models and treatment protocols

All investigations involving animals were permitted by the Experimental Animal Ethics Committee of Nanjing Pukou District Hospital of Traditional Chinese Medicine (approval number 20210028). Moreover, all animal experimentations were consistent with the National Institutes of Health (NIH) guidelines for the laboratory handling of animals. 35 C57BL/6 mice (8–9 weeks old; weight 19–21 g) were purchased from the Zhejiang Weitonglihua Laboratory Animal Technology Inc., Zhejiang, China. 15 male C57BL/6 FXR-knockout mice (8–10 weeks old; 17–19 g in weight), were obtained from the BRL Medicine Inc, Shanghai, China. All FXR knockout mice were global knockout without tissue specificity, and the gene testing sequence is shown in the attachment. All mice were provided with nutrition and water at free and were grown under optimal conditions (regulated T^°C and 12h light/dark cycle). The mice were casually separated into seven assemblies with 5 animals each after a week for adjusted nurturing (n = 5 animals per group). The groups were: 1) blank control (BC), 2) blank control + water deprivation (BC + WD), 3) diabetes mellitus (DM), 4) diabetes mellitus + water deprivation (DM + WD), 5) FXR^−/− blank control (FXR^−/−+BC) 6) FXR^−/− diabetes mellitus (FXR^−/−+DM) and finally FXR^−/− diabetes mellitus + water deprivation (FXR^−/− + DM + WD). The four groups of diabetic mice (DM groups) were intraperitoneally injected with 50 mg/kg dissolved in 100 mM NaCi (sodium citrate buffer, pH 4.5) of streptozotocin (STZ, Merck KGaA, Darmstadt, Germany, no.V900890) for 5 successive days. The remaining three groups were injected with the same amount of NaCi buffer. In a week, mandibular vein blood biochemical analyses were performed to measure glucose. Animals from the DM groups with high fasting-blood glucose (higher than 13.4mmol/L) were categorized as diabetic. All mice were fed a Western diet(21% fat, 50% carbohydrates and 1.5% cholesterol) delivered by the Jiangsu Synergetic Medicine biology Inc., Jiangsu, China. For the WD groups, Water was restricted for 24 hours on alternate days. Serum blood glucose levels, urine volume, water drinking and body weight were measured every two weeks for 16 weeks. 16 weeks after STZ treatment, before mice killing blood samples were gathered and spinned down at 4^°C at 3000 rpm for 10 min to obtain the serum. Left renal samples were preserved in 4% paraformaldehyde for pathological and immunofluorescence investigations, whereas right renal samples were immediately frozen into liquid nitrogen for gene and protein expression analysis.

Blood And Urine Chemistry

For performing blood and urine biochemistry the animals were preserved in cages for metabolic analyses for a period of 24h, which allowed collection of urine samples. The 24h-urinary protein was then measured by protein analyzer (BIOSTEC, Spain, no.BA400). The kidney function was assessed by an automated analyzer of the blood serum (Hitachi, Tokyo, Japan, no.7180).

Reagents

The primary antibodies used were: anti-FXR (Proteintech, Wuhan, China, no. 25055-1-AP), anti-TonEBP (Affinity bioscience, Cincinnati, OH, United States, no.AF7663), anti-gp91 (BD biosciences, Santa Clara, CA, United States, no.611414), anti-AQP2 (Abclonal, Wuhan, China, no.A16209), anti-UT2 (Ablonal, Wuhan, China, no.A16209), anti-caspase3 (Cell Signaling Technology, Shanghai, China, no.9662), anti-Bcl-2 (Proteintech, Wuhan, China, no.26593-1-AP), anti-BAX (Proteintech, Wuhan, China, no.50599-2Ig) and anti-β-actin (Proteintech, Wuhan, China, no. 66009-1-Ig). The secondary antibodies included anti-mouse IgG (H + L) (Proteintech, Wuhan, China, no. SA00001-1) and anti-rabbit IgG (H + L) (Proteintech, Wuhan, China, no. SA00001-2).

Western-blot Analysis

Kidney homogenates were collected and renal cells were dissolved in lysis buffer supplemented with protease and phosphatase inhibitors (Beyotime, China). Renal lysates were gathered and exposed to ultrasonication and centrifugation to allow the gathering of the supernatant. The last was mixed with sodium dodecyl sulfate (SDS) and then exposed to 100^°C for 10 min for protein denaturation. The proteins were separated by 10% SDS-PAGE electrophoresis (Bio-Rad, China) and after that were blotted to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States) which were blocked in PBST (PBS with 1‰ Tween-20) and 5% non-fat dried milk (1 hour, At room temperature). PVDF membranes were incubated with the primary Abs for 12h at 4^°C. After washing the PVDF membranes were incubated with the secondary Abs for 1h and washed again. The bands were developed with a chemiluminescent horseradish peroxidase (HRP) substrate and the results were visualized by ChemiDoc™XRS a chemiluminescence system, equipped with Image Lab Software (Bio-Rad, Hercules, CA, USA). Results were quantified by ImageJ software (1.52a).

Histopathology

Kidney samples were preserved in 4% paraformaldehyde. Alcohol dehydration followed and samples were stained with hematoxylin and eosin (Servicebio, Wuhan, China, no.GP1031), Masson's trichrome (Servicebio, Wuhan, China, no.GP1032) and Periodic Acid Schiff (PAS) stains (Servicebio, Wuhan, China, no.GP1039). ImageJ software was applied for quantification of the Masson-stained zones.

Immunofluorescence Staining

Renal samples were cut on a microtome(Leica, Shanghai, China, no.RM2016) at slices of 40 µm thickness. Immunofluorescence was carried out on free-floating sections with anti-FXR, anti-AQP2, anti-TonEBP and anti-and gp91 antibodies. Results were visualized using microscopy (Nikon, Japan, no. eclipse ci-e). Images were taken at 10×magnification by a high-resolution slide scanning system (3DHISTECH Ltd, Hungary, no. Pannoramic MIDI). ImageJ was applied to quantify immunochemistry results and to present the antibody content as an integrated density.

Tunel Assay

Apoptotic renal tissue cells were probed with terminal deoxynucleotidyl transferase dUTP nick end labelling kit (Tunel assay) following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, USA, no.11684817910). The results were quantified by an immunofluorescence microscope (Nikon, Japan, ECLIPSE Ci Series). Five microscopic fields (×200) were randomly selected to calculate the number of apoptotic cells as a percentage of all cells.

Statistical Data Evaluation

Statistical analysis was done with GraphPad Prism 7 software. All obtained results are displayed as mean ± SD. The unpaired t-test was applied to compare between two variables. The one-way ANOVA was applied for comparisons between more than two variables. Classified data was expressed as the number of cases and were analysed by the Fisher’s exact test. P values less than 0.05 were accepted as statistically significant.

Kidney injury was more obvious in diabetic mice after water deprivation.

In seven groups of mice, BC + WD,DM + WD and FXR^−/− + DM + WD had been deprived of water every other day and observed for 16 weeks. Compared with the BC mice, the DM mice had lower body weight, they drank more water and urinated more (Fig. 1A-C). These data were consistent with the general characteristics of diabetic mice. However, compared with the DM mice, DM + WD mice showed more urine output and a higher amount of urine protein. In the BC and BC + WD mice, there were no significant differences. Similarly, there was no significant difference between FXR^−/− +DM mice and FXR^−/− + DM + WD mice(Fig. 1C,D). After FXR knockdown, TG, LDL, TCHO and TBA were significantly increased in some groups (Fig. 1E-H). In addition, the BUN and Scr blood serum levels in the DM + WD mice were higher than in the DM mice, while BC and FXR^−/−+DM mice showed no difference before and after water deprivation (Fig. 1I and J). Uric acid was higher in DM + WD mice than in FXR knockout mice(Fig. 1K). DM, DM + WD, FXR^−/− + DM and FXR^−/− + DM + WD mice had significantly increased levels of fasting blood sugar after STZ injection(Blood glucose levels were all greater than 13.4mmol/L). Compared with DM and DM + WD mice, the FXR^−/− + DM and FXR^−/− + DM + WD mice showed more pronounced glucose fluctuations(Fig. 1L).

Dehydration Aggravated Renal Pathological Damage In Diabetic Mice

To further examine the impact of water deficiency on renal damage in diabetic mice, we performed histopathological examinations of the renal cortex and medulla of these mice. In the renal cortex (Fig. 2A), HE staining showed no significant difference in the glomerular structure among the studied groups. Masson staining showed that fibrosis was more obvious in DM + WD groups than in BC and BC + WD one (Fig. 2A, black arrows), but there was no significant difference between DM and DM + WD groups. PAS staining showed a slight increase of the stroma in the glomerulus (Fig. 2A, red arrow). However, in the renal medulla region, DM + WD mice showed explicit pathological damage, which was manifested in dilatation of some of the renal inner medullary collecting ducts (Fig. 2B, blue arrows), fibrosis of the interstitium (Fig. 2B, yellow arrows) and partial renal inner medullary collecting duct blockage and peritubular inflammation (Fig. 2B, green arrows). After FXR knockout diabetic mice also showed mild renal pathological damage, tubular epithelial steatosis and mild fibrosis, there was no significant difference between the FXR^−/−+DM mice and the FXR^−/− + DM + WD ones (Fig. 2B). These results suggested that the damage of water shortage to diabetic nephropathy is mainly in renal medulla and after FXR knockout, the effect of dehydration on the detected renal damage was reduced.

Gp91/AQP2 expression increased in renal medulla under water deficiency, especially under high blood serum glucose concentrations

Gp91, known as NOX2, is an important target of oxidative stress injury[19]. It has been found that inhibition of gp91 reduced oxidative stress damage and renal fibrosis in diabetic nephropathy[18]. Our experiments showed that gp91 was significantly increased in the medulla region of mice after water deprivation, and was more significantly expressed in diabetic mice (Fig. 3A,B, pink arrows). However, in contrast to the gp91 expression in DM + WD mice, its expression in the medulla region of the FXR^−/− + DM + WD group was slightly increased after water deprivation(Fig. 3C).

AQP2 is one of the key proteins regulating water metabolism[15]. Our results with its expression levels proved that AQP2 expression was significantly enhanced in the cortex and medulla of mice in BC + WD and DM + WD groups (Fig. 3D). We also verified these results by immunofluorescence stating experiment. The results are consistent with the results of AQP2 expression analysis and are displayed in (Fig. 3E, white arrows). Data show that UT2 which are members of the solute carrier 14 (SLC14) family, drive the urinary concentrating process[20]. We proved that UT2 increased after water was cut-off, especially in a high glucose environment (Fig. 3D). We assume that AQP2 expression was significantly reduced in the FXR^−/−+BC, FXR^−/−+DM, and FXR^−/− + DM + WD groups of mice because FXR is an important nuclear receptor in the process of AQP2 activation (Fig. 3F). This result was verified by the immunofluorescence analysis (Fig. 3E). Unlike AQP2, the expression of UT2, which is also regulated by TonEBP[21], was enhanced after FXR knockout (Fig. 3F), indicating that UT2 expression was not regulated by FXR and increased its protein reactivity after dehydration. Tunel staining showed that the apoptosis of renal tissue cells in diabetic mice was significantly higher than that in non-diabetic mice, but there was no significant difference between diabetic mice before and after water deprivation (Fig. 3G).

We compared the expression positions and intensities of gp91 and AQP2 proteins in the renal cortex and medulla by immunofluorescence. These results confirmed that gp91 was significantly overexpressed in the medulla (Fig. 3H, yellow arrows), but not in the cortex. However, AQP2 was intensely expressed in the renal cortex and medulla after water deprivation. The cortex is slightly stronger than the medulla (Fig. 3I, green arrows).

Fxr And Tonebp Were Overexpressed Under Water Deficiency, Especially In Diabetic Mice

TonEBP, also known as NFAT5, is the only known gene regulator of the mammalian adaptive response to osmotic stress[22]. In the hypertonic state, the activation of TonEBP causes oxidative stress [23]. Recent data show that in the renal medulla TonEBP expression is driven by FXR and its nuclear translocation potentiates its osmoprotective role[11]. This study showed that FXR expression levels were low in BC mice kidneys. Under the condition of water deficiency, FXR expression increased, which was further promoted by a high glucose environment (Figs. 4A). Immunofluorescence also showed that FXR expression levels were significantly enhanced in BC + WD mice, and the increasing rates were more obvious in DM + WD mice (Figs. 4B, red arrows). When FXR was knocked down, its expression was significantly reduced (Figs. 4C). The western blotting showed that TonEBP was significantly higher in the BC + WD and MD + WD mice. The increase was most obvious in the DM + WD mice (Fig. 4D). This conclusion was further supported by the results from immunofluorescence analysis, which also showed that TonEBP was mainly expressed in the renal medulla (Figs. 4E, white arrows). After FXR nuclear receptor knockout, the expression levels of TonEBP were yet high in the FXR^−/−+DM group of mice (Figs. 4F). This result was further supported by immunofluorescence (Fig. 4E).

We further proved that in the renal medulla, FXR, TonEBP and gp91 were expressed in the same location and with the same intensity (Fig. 4G, yellow arrows). In the renal cortex, FXR was highly expressed (Fig. 4G, green arrows), whereas the expression of gp91 decreased when TonEBP expression lowered. These results suggest that TonEBP and FXR jointly promoted the expression of gp91, leading to oxidative stress injury of the renal medulla. W

The imbalance of body fluid is a common problem in clinics, because many diseases may destroy this balance mechanism[24]. Our studies focused on the influence of water deficiency on kidney disease. By intermittently cutting off water to experimental mice, the diabetic ones had more urine and creatinine than non-diabetic mice, which was consistent with our clinical study. The renal pathology of diabetic mice 16 weeks after intermittent hydration showed destruction of the medulla collecting ducts. We assumed this damage was a trigger for FXR and TonEBP expression after water deprivation, resulting in oxidative stress damage to the medulla. When FXR was knocked out, the expression of TonEBP remained high in diabetic mice and increased after water deprivation, while AQP2 expression significantly decreased. We did not detect any statistically relevant dissimilarities in gp91 transcription levels in the cortex and medulla after water deprivation, suggesting that FXR might have mediated TonEBP activation of gp91 and AQP2 expression under water deprivation.

In animal experiments, diabetic mice with Intermittent water deprivation showed increased urine volume and creatinine, while non-diabetic mice with intermittent water showed no such changes. This change may be related to the high expression of TonEBP in high glucose environment[25]. TonEBP is the only known gene regulator of the adaptive response to osmotic stress in mammals because of increased osmotic pressure in the renal medulla following water deprivation. TonEBP plays a protective role under normal conditions[5, 26, 27], but with the development of diabetes, the physiological function of TonEBP changes, and TonEBP may become a pathogenic factor aggravating the disease[25]. So without diabetes mice's kidneys were protected from water shortages. This hypothesis proved true as we did not see increased urine production or creatinine in these mice. Among the diabetic once, though, TonEBP was significantly increased, which could be a factor in kidney injury. This kidney damage is shown to be exacerbated by the water cut off in other authors’ results[28]. Therefore, we saw decreased urinary concentration and filtration in diabetic mice after water deprivation. Some data show that high osmotic stress and overexpression of TonEBP were linked with high oxidative stress damage in the kidney[23]. In this experiment, immunofluorescence detection showed that gp91 was highly expressed in the medulla after water withdrawal, which was consistent with the high expression of TonEBP. Therefore, we concluded that TonEBP induced oxidative stress response of renal medulla through the activation of gp91, thus causing kidney injury. This oxidative stress injury was more obvious in a high glucose environment, which might be one of the important factors in the formation of diabetic nephropathy. Tunel staining showed that the apoptosis of renal medulla cells did not increase significantly in the condition of dehydration, suggesting that apoptosis may not be the main cause of dehydrated kidney injury.

FXR regulates the metabolisms of bile and fatty acids, cholesterol and glucose homeostasis[8, 29–31]. Furthermore, it is also found to mediate water metabolism as well as oxidative stress injury[32–34]. Under hypertonic conditions, it protects the renal medulla by promoting the expression of AQP2, thus increasing urine reabsorption[15, 35]. However, in this experiment, we showed that FXR protection of the renal collecting duct cells after dehydration was weakened under high glucose conditions. According to previous studies, the lack of water caused a hypertonic environment of kidney tissue, and then oxidative stress response[23]. Our results proved that FXR triggered gp91 transcription in the renal cortex after dehydration in a non-high-glucose environment. Due to the antioxidant stress effect of FXR, the expression of gp91 in the renal cortex was decreased. In the renal medulla, FXR promoted the expression of TonEBP[11, 36], resulting in the increased expression of AQP2, which increased the reabsorption of protopuria and protected renal collecting duct cells. However, in the high glucose environment, TonEBP expression was significantly increased. Under the promotion of FXR, gp91 was overexpressed, leading to injury and occlusion of renal collecting duct cells, resulting in decreased reabsorption function and increased interstitial fibrosis. The main manifestations were increased urine and decreased renal filtration function. These results are consistent with other authors’ results proving these FXR changes in different environments, expression sites, and binding to different transcription factors[14, 37, 38]. We further demonstrated that gp91 was underexpressed in TonEBP downregulation or FXR deficiency. However, the combination of the TonEBP and FXR effectively promoted the high expression of gp91. In the renal cortex, due to the low expression of TonEBP, FXR was mainly manifested durting antioxidant stress, so gp91 was low expressed in the renal cortex under water deficiency. In the renal medulla, after a water break, TonEBP was highly expressed. The combination of TonEBP and FXR promoted the expression of gp91. This effect was more obvious in the high glucose environment, which led to the oxidative stress injury of the medulla (Fig. 5).

Although AQP2 expression was weakened in the tested mice after FXR knockout, the expression of UT2 was not significantly affected. After the water was cut off, UT2 expression increased, and protopuria reabsorption increased, thus compensating for the risk of increased urine production due to reduced AQP2 expression. Therefore, FXR^−/− mice did not see a significant increase in urine volume, which we consider another important finding.

In conclusion, the study showed that under the condition of water deficiency, In our animal models, we proved that the combination of FXR and TonEBP in the renal medulla led to the overexpression of gp91 and oxidative stress damage under the stimulation of high glucose and high osmotic conditions. We consider this as one of the important factors for the poor prognosis of diabetes patients. We also found that UT2 was normally expressed in the presence of FXR deficiency, which attenuated the risk of polyuria caused by low AQP2 expression. These results provide references and ideas for future research on the prevention of diabetic nephropathy and the influence of water imbalance on the kidney. The deficiency of this study lies in the small number of clinical samples and the lack of proteins related to water metabolism and oxidative stress detected in animal experiments. This is what we will focus on next.

DN	diabetic nephropathy
FXR	farnesoid X receptor transcription factor
AQP2	aquaporin 2
UT2	urea transporter 2
gp91	glycoprotein 91
NOX	nicotinamide adenine dinucleotide phosphate oxidase
TonEBP	tonicity-responsive enhancer-binding protein
MCDs	medullary collecting duct cells
NIH	National Institutes of Health
C57BL/6	C57 black 6
STZ	streptozotocin
SDS	sodium dodecyl sulfate
PVDF	polyvinylidene fluoride
HRP	horseradish peroxidase
PAS	Periodic Acid Schiff
PBST	phosphate buffered solution

Ethics approval

All investigations involving animals were permitted by the Experimental Animal Ethics Committee of Pukou Hospital of Traditional Chinese Medicine, Nanjing City, Jiangsu Province, China (approval number 20210026, Ethics Report provided in the attachment). We confirm that all methods are carried out in accordance with the Laboratory Animal Guidelines and Regulations. We also confirm that all methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.

Consent for publication

Not applicable

Availability of data and materials

All data can be disclosed, and we have submitted it as an attachment through the submission system.

Competing interests

No conflict of interest exist in the submission of this manuscript, and manuscript was approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. The manuscript was approved by all the authors.

Funding

This work was supported by the National Natural Science Foundation of China (81873270).

Authors' contributions

Tuo Wei carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Enchao Zhou conceived of the study, and participated in its design and coordination and helped to draft the manuscript.

Acknowledgements

We thank the Central Laboratory of Jiangsu Provincial Hospital of Traditional Chinese Medicine and the Department of Basic Pharmacology for providing us with the space, equipment and related training.

Authors' information (optional)

Not applicable

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Table 1 Characteristics of mice under different drinking water conditions

	BC	BC+WD	DM	DM+WD
Weight(mg)	48.46±1.80	42.22±2.62	28.78±1.20^ab	26.8±1.59^ab
Fasting blood sugar(mmol/L)	7.4±0.8	6.7±0.4	19.9±2.0^ab	20.4±3.2^ab
24h water intake(ml)	5.44±0.48	6.8±0.52	14±1.14^ab	16.4±1.16^ab
24h urine volume(ml)	1.38±0.21	0.54±0.24	3.84±0.67^ab	7.12±0.92^abc
24-h urine protein(mg/L)	2.51±0.2	1.6±0.33	4.05±0.63^a	8.06±0.93^abc
BUN(mmol/L)	6.07±0.41	7.55±0.76	8.71±0.60	12±1.37^abc
Scr(umol/L)	14.26±0.72	16.16±1.0	14.1±1.04	29.32±8.21^abc
UA(umol/L)	127.2±8.29	164±16.87	168±7.1	165±12.12

Characteristics of mice under different drinking water conditions
	FXR^-/-+BC	FXR^-/-+DM	FXR^-/-+DM+WD
Weight(mg)	40.52±2.03	28.5±1.7^a	31.0±0.53^a
Postprandial blood glucose(mmol/L)	6.8±0.63	20.4±15.6^a	19.1±1.1^a
24h water intake(ml)	3.5±0.22	9.3±0.37^a	8.26±0.64^a
24h urine volume(ml)	0.98±0.31	4.2±0.30^a	3.12±0.65^a
24-h urine protein(mg/L)	1.76±0.09	4.89±0.41^a	5.08±0.41^a
BUN(mmol/L)	4.48±0.31	7.65±0.62	7.72±0.20
Scr(umol/L)	15.72±0.51	20.16±1.4	15.76±0.73
UA(umol/L)	139.8±10.5	115.9±18.38	147±17.7

No competing interests reported.

originaldata1.docx

High blood sugar may blunt the protective benefits of FXR for the survival of renal medullary collecting duct cells in response to hypertonic stress

Status:

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Abstract

Backgroud:

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Animal models and treatment protocols

Blood And Urine Chemistry

Reagents

Western-blot Analysis

Histopathology

Immunofluorescence Staining

Tunel Assay

Statistical Data Evaluation

Result

Dehydration Aggravated Renal Pathological Damage In Diabetic Mice

Fxr And Tonebp Were Overexpressed Under Water Deficiency, Especially In Diabetic Mice

Discussion

Conclusion

Abbreviations

Declarations

References

Table 1

Additional Declarations

Supplementary Files

Status:

Version 1