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Article

Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats †

by
Ahmed W. Al-Humadi
1,2,
Carel W. le Roux
1,3,
Neil G. Docherty
1,
Werd Al-Najim
1,3,
Martin Tze Wah Kueh
4,
Andreas C. Lazaris
5 and
Charis Liapi
1,*
1
Diabetes Complications Research Centre, School of Medicine, Conway Institute, University College Dublin, D04 V1W8 Dublin, Ireland
2
Laboratory of Pharmacology, Medical School of Athens, National and Kapodistrian University of Athens, 75 Mikras Asias, Goudi, 11527 Athens, Greece
3
Diabetes Research Centre, Ulster University, Coleraine BT52 1SA, UK
4
Royal College of Surgeons in Ireland, University College Dublin Malaysia Campus, George Town 10450, Malaysia
5
1st Department of Pathology, Medical School, University of Athens, 75 Mikras Asias, Goudi, 11527 Athens, Greece
*
Author to whom correspondence should be addressed.
†
This article is revised and expanded version of a paper entitled Nutritional Influences on One Carbon Metabolism Exacerbate Diabetic Cardiomyopathy and Nephropathy, which was presented at Endocrine Society Annual Meeting, USA, May 2020.
Diabetology 2025, 6(1), 8; https://doi.org/10.3390/diabetology6010008
Submission received: 8 November 2024 / Revised: 20 December 2024 / Accepted: 6 January 2025 / Published: 20 January 2025
(This article belongs to the Special Issue Exclusive Papers Collection of Editorial Board Members in Diabetology)
Browse Figures
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Abstract

:
Background: Choline (Ch) deprivation causes kidney injury and dysfunction, and diabetic nephropathy is also known to be a complication of diabetes; thus, this interplay could potentially aggravate diabetic kidney disease. Aim: This study aims to examine the effect of Ch-deprivation on the severity of kidney injury in streptozotocin (STZ)-induced diabetic rats. Methods: Twenty-four adult male Wistar rats were divided into four groups: control (C), nondiabetic Ch-deprived (CD), diabetic (DM), and diabetic Ch-deprived (DM + CD). Diabetes was induced by the intraperitoneal administration of 50 mg/kg body weight STZ; Ch-deprivation was induced through a choline-deficient diet. Rats were euthanized at week 5 of the study. Biochemical tests, renal histopathology, immunohistochemistry of the kidney injury molecule-1 (KIM-1), and vascular endothelial growth factor-A (VEGF-A) expression were assessed. Results: DM + CD and DM groups demonstrated significant increases in glucose levels and in the homeostasis model of insulin resistance (HOMA IR). Creatinine and blood urea nitrogen levels significantly increased in the DM + CD group compared to the control, and homocysteine levels were higher in the CD group. Kidney histopathology revealed that renal tubular necrosis, mesangial matrix expansion, and renal fibrosis substantially increased in the DM + CD group compared to all other groups, and KIM-1 and VEGF-A expressions were most pronounced in the DM + CD and DM groups, respectively. Conclusions: Ch deprivation affected kidney function and structure in STZ-induced diabetic rats. Choline deficiency and diabetes seem to have a synergistic effect, as evidenced by kidney biochemistry, histopathology, and immunohistochemistry. These findings could highlight the important role of choline in therapeutic strategies for the treatment and, potentially, prevention of chronic diabetic kidney disease.

1. Introduction

Diabetic nephropathy (DN), a common complication of diabetes mellitus which leads to end-stage renal failure, is characterized by progressive renal dysfunction and structural changes in the kidneys. The pathogenesis of diabetic kidney injury is multifactorial, involving hyperglycemia-induced oxidative stress, inflammatory responses, and altered lipid metabolism [1,2].
Among the key dietary factors influencing kidney health, choline (Ch) is gaining more interest for its potential role in modulating kidney function, particularly in the context of metabolic diseases, such as diabetes.
Choline is an essential nutrient that plays a fundamental role in human health, serving as precursor for several biomolecules, including cell membrane components (such as phosphatidylcholine and sphingomyelin), the neurotransmitter acetylcholine, platelet-dervied growth factor, and betaine [3,4,5,6,7]. Moreover, Ch and its metabolites are involved in diverse aspects of cellular function, including methylation, lipid transport, cell signalling, and brain development [7,8,9]. Although Ch is mainly de novo synthesized in the liver, adequate dietary intake remains important [7]. Dietary recommendations for Ch intake were set by the Institute of Medicine (IOM) in 1998, and later by the European Food Safety Authority in 2016 [10,11].
Choline deficiency is a prevalent condition, and suboptimal Ch intake is an important factor of choline deficiency [7]. Only around 10% of Americans and 8% of pregnant women currently meet their gender- and life stage-specific adequate intake for choline [12,13,14].
Choline has received considerable attention, due to the adverse effects of Ch deficiency on the health of the brain, liver, heart, and kidneys, and its association with metabolic diseases, including diabetes mellitus [15]. In experimental models, Ch deficiency causes renal tubular necrosis in young rats [16,17,18], and its deficiency is commensurate with elevated homocysteine levels, a salient feature of chronic kidney disease [19,20].
Ch deprivation has also been associated with dysglycaemia and insulin resistance in adult rats [21,22], which may potentially drive the progression of diabetes complications. The impact of Ch deprivation on kidney injury in streptozotocin (STZ)-induced diabetes has not been explored.
Since choline deficiency is common and it can simultaneously occur with diabetes mellitus, this study aimed to assess the effect of these two prevalent conditions on kidney injury in rats.
Key points:
  • Choline deprivation affects kidney structure and function of diabetes rats
  • Choline deprivation aggravates the progression of kidney injury in STZ-induced diabetes.

2. Materials and Methods

2.1. Animals and Diet

Twenty-four 5-week-old male Wistar rats were obtained from the National Centre of Scientific Research, “Demokritos”, in Athens, Greece, and were used in the experiment. All procedures were approved by the Medical School of National and Kapodistrian University of Athens, after the approval of the Animal Protocols (N:1623 and N:3067) by the Department of Rural and Veterinary Policy (RVP), General Sector of Rural Economy and Veterinary, Prefecture of Attica, Hellenic Republic. Rats were cared for in accordance with the principles of laboratory animal care as previously set by the European Economic Community (EEC) Council Directive 86/609/EEC (EEC) and amended according to the recommendation 2007/526/EU for experimental animals [23]. Animals were housed under controlled environmental conditions, at a constant room temperature of 22 ± 1 °C and an artificial light/dark cycle (12/12 h), with adequate ventilation and appropriate humidity (~55%). Food and water were supplied ad libitum.
The standard diet was enriched with Ch (1.5 g/kg) at the expense of sucrose. The Ch-deficient diet (CDD) was manufactured by Mucedola (Settimo Milanese, Italy) and purchased by Analab Ltd. (Athens, Greece). The analytical composition of the CDD was the following (g/kg): sugar, 413; starch, 110; dextrin, 110; hydrogenated vegetable oil, 100; pea protein, 90; soya protein isolates, 60; corn oil, 50; mineral mix, 35; vitamin mix, 10; cellulose, 10; vitamin-free casein, 10; L-cystine, 2, in addition to protein ingredients (12%), fat (16%), fibre (2%), and ash (3.5%) [24] (Al-Humadi A et al., 2021). STZ and all other reagents used were of the highest-quality grade available and were purchased from Sigma Chemicals, Sigma-Aldrich (Steinheim, Germany).

2.2. Experimental Procedure

Animals were randomly divided into four groups (n = 6 per group), as follows: control (C), nondiabetic Ch-deprived (CD), diabetic (DM), and diabetic Ch-deprived (DM + CD). After two weeks of acclimatization, the dietary intervention was started for five weeks. Three days prior to the dietary intervention, the induction of diabetes was performed by a single intraperitoneal injection of STZ (50 mg/kg of body weight with dilution in a 0.1 mol/L citrate solution, pH 4.5); the control and CD groups were injected with the citrate buffer [25]. A plasma glucose level of ≥300 mg/dL at 72 h post-STZ injection confirmed diabetes. The five-week survival rate was 100%. Animals were euthanized 5 weeks after the dietary intervention (at 3 months of age, body weight was 298.6 ± 26.2 g).

2.3. Blood and Tissue Collection

Immediately after animals were euthanized, peripheral venous blood was sampled directly from the inferior vena cava, and subsequently used for the measurement of biochemical parameters. Both kidneys were rapidly removed, weighted, and prepared for histological and immunohistochemical assessment by submersion in neutral buffered formalin. Samples were also snap frozen in liquid nitrogen and stored at −80 °C for immunohistochemistry.

2.4. Biochemical Assessment

Fasting blood glucose levels were measured using a portable glucometer (Accu-Chek, Roche Diagnostics GmbH, Mannheim, Germany), and fasting plasma insulin levels were assessed using the Elisaruo method of the DRG. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: fasting glucose (mg/dL) × fasting insulin (µU/mL)/405. Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were measured using an automated analyzer (Hitachi, Roche Modular, Roche Diagnostics, Mannheim, Germany ), as previously assessed by Al-Humadi A et al. [24]. Serum creatinine and blood urea were measured using Agappe assay kits; serum homocysteine levels were measured by fluorescent polarization immunoassay, using a kit obtained from Abbott Laboratories, AxSYMSystem, Abbott Park, IL, USA (the reference range of the kit was 6–15 μmol/L).

2.5. Histopathological Examination

The anterior portions of left and right kidney specimens were fixed in 4% (v/v) buffered formalin solution and embedded in paraffin wax using conventional techniques for histopathological assessments. Paraffin sections were cut at 4 μm, baked for 1 h at 60 °C, dewaxed, rinsed, and stained with Haematoxylin and Eosin (H&E) to assess the necrosis, Periodic acid–Schiff (PAS) stain to evaluate the mesangial expansion of the glomeruli and Masson’s trichrome stain to evaluate renal fibrosis. The assessment was performed by the visual inspection of at least 10 glomeruli per field section using an optical microscope (DM4000B photomicroscope; Leica Microsystems, Wetzlar, Germany). Each section was divided into 4 fields, the average score was assigned for each renal sample in a semiqualitative method, and the results were expressed as the percentage of findings (injury) per field. The specimens were randomized and given a code before evaluation by two experienced pathologists, who were blinded to the treatment groups to ensure consistency and minimize bias. The classification criteria were based on well-established methods from previous studies in the field, which we referenced to ensure comparability and reliability [18,26].
a
Renal necrosis:
H&E staining was performed to assess renal necrosis. The extent of necrosis was semi-quantitatively evaluated based on the presence of structural alterations on the kidney tubular necrosis, such as tubular exfoliation, cell swelling, nuclear fragmentation, and loss of renal architecture.
The severity of necrosis was classified according to previous studies [18,27,28]. The histological scoring scale ranged from grade 0 (no necrosis) to grade 8 (massive cortical necrosis in combination with extensive tubular necrosis), as follows: score 0: no necrosis observed in the renal tissue, 0%; score 1: isolated foci of cellular necrosis in a few tubules, accounting for less than 25% of the observed tissue section; score 2: small, isolated groups of tubules exhibiting necrosis, more than 25%, but less than 50%, of the total renal tissue; score 3: affected areas showing zones of tubular necrosis, with more than 50%, but less than 75%, of the tubules involved; score 4: confluent zones of tubular necrosis present, with more than 75%, but less than 100%, of the tubules exhibiting necrotic changes; score 5: isolated foci of cortical necrosis, in addition to score 4 tubular necrosis; score 6: multiple foci of cortical necrosis, along with score 4 tubular necrosis; score 7: confluent foci of cortical necrosis, combined with score 4 tubular necrosis; score 8: massive cortical necrosis present, with score 4 tubular necrosis extending across most of the renal tissue.
b
Mesangial expansion:
PAS staining was used to evaluate mesangial expansion in glomeruli. Mesangial expansion was graded based on the percentage of glomerular area affected by increased mesangial matrix deposition in the renal cortex. For the evaluation of renal glomerular alteration, a 4-scores system was used: score 0: no glomerular alteration, no mesangial expansion; score 1: mild thickening of glomerular basement membrane with mild focal mesangial expansion (0–20% of glomeruli affected); score 2: moderate mesangial expansion (20–40% of glomeruli affected); score 3: high mesangial expansion (40–60% of glomeruli affected); and score 4: diffuse segmental expansion of mesangium (>60% of glomeruli affected).
c
Renal fibrosis:
The presence of collagen deposition in the kidney was evaluated semi-quantitatively by determining the percentage of fibrotic tissue in cortical areas. Sections were scored from 0 to 4 based on the area of blue-stained collagen, and fibrosis was classified as absent, mild, moderate, or severe, based on the extent of collagen deposition, as follows: score 0: no fibrosis; score 1: mild peritubular fibrosis; score 2: moderate fibrosis, including peritubular fibrosis, with occasional glomerulosclerosis; score 3: severe fibrosis and glomerulosclerosis, with tubular atrophy and interstitial expansion and score 4: advanced fibrosis, with large fibrotic areas, significant glomerulosclerosis, and loss of kidney architecture.

2.6. Immunohistochemical Assessment

The expression of the kidney injury molecule-1 (KIM-1) and the vascular endothelial growth factor-A (VEGF-A) in posterior renal specimens was assessed. To evaluate KIM-1 expression, 4 μm frozen sections were hydrated by incubation in alcohol solution 96° for 30 min in the dark, at room temperature (RT). Overnight incubation with the primary antibody at 1:400 dilution (TIM-1/KIM-1/ HAVER of R&D Systems, Catalogue Number: AF3689, Biotechne brand) was performed, followed by the application of the secondary antibody (DAKO anti-mouse immunoglobulins/Polymer, IgG) and staining with the streptavidin-HRP complex (Dako REAL™ EnVision™/HRP) at RT, for 30 min each. The signal was developed using diaminobenzidine as a colorimetric substrate. That was followed by rinsing with hematoxylin, dehydration, rinsing with xylene, cover slipping, and visualization under a light microscope. Between each step, the slides were washed with TBS (Tris-buffered saline). The renal injury score was evaluated according to the intensity of the KIM-1 stained granules in proximal convoluted tubules and the extent of stained tubules in the examined sections, as follows: score 0: absence of the stained tubules, 0% (no renal injury); score 1: faint stain, <25% of the tubules (mild injury); score 2: moderate stain, 25–50% of the tubules (moderate injury); score 3: strong stain, >50% of the tubules (strong injury).
For VEGF-A expression assessment, deparaffinization, rehydration, and antigen retrieval were conducted by heating the slides in PTLink (Dako, Nowy Sącz, Poland), using a low pH solution [Envision FLEX TARGET RETRIEVAL SOLUTION Low pH (50×)]. Endogenous peroxidase was blocked using H2O2 3% solution for 15 min at RT, and non-specific staining was incubated with Dako REAL™ peroxidase-blocking solution, code S2023 for 30 min at RT. Slides were incubated overnight with the VEGF-A165 primary antibody [vascular endothelial growth factor antibody (VG1), Invitrogen by Thermo Scientific, Waltham, MA, USA, catalogue number: PA1-21796], at a dilution of 1:500 overnight, and the secondary antibody (Dako anti-mouse immunoglobulins/polymer, IgG) for 30 min at RT, followed by chemiluminescent detection [24].
The expression of VEGF-A in the kidney was graded according the localization and the intensity of the immunostaining in the endothelial cells in renal tubules and/or glomeruli, as follows: score 0: no stain, 0% of stained expressed endothelial cells; score 1: faint stain, <25% isolated stained endothelial cells; 2: moderate staining, 25–50% of clusters of endothelial cells, proximal or distal tubular cells; score 3: strong stain, >50% widespread stained cells throughout the renal sections. The results were expressed as a percentage of areal density.

2.7. Statistical Analysis

Data expressed as mean ± SD were analyzed using one-way analysis of variance (ANOVA) followed by multiple comparisons with Bonferroni’s and Tukey’s honest significant difference methods. The significance level for all analyses was set at p ≤ 0.05, and all analyses were performed by GraphPad Prism 10.0.2 (171) for Mac (GraphPad Software, version 10.0.2 (171), San Diego, CA, USA).

3. Results

3.1. Body Weight and Kidney Weight

Although all rats gained weight by the end of the experiment, the lowest weight gain was observed in the diabetic Ch-deprived group (14.4% increase in the DM + CD group, 28.2% in the CD group, 22.3% in the DM group, and 33.6% in the control group). The kidney weight of the diabetic Ch-deprived rats significantly increased (p < 0.05) compared to Ch-deprived rats, while the kidney weight to body weight ratio was significantly higher in the diabetic Ch-deprived group (p < 0.01) compared to the control and CD groups (Table 1).

3.2. Markers of Renal Function

Serum creatinine levels showed a significant increase (p ≤ 0.01) in the diabetic Ch-deprived DM + CD rats compared to the control; blood urea nitrogen levels in the DM + CD group were also significantly higher in the DM + CD group compared to the control, CD and DM groups (p ≤ 0.001, p ≤ 0.01 and p ≤ 0.05, respectively) (Table 1).

3.3. Glycaemic Profile

After five weeks, fasting blood glucose levels were significantly higher (p ≤ 0.001) in both the DM and DM + CD groups compared to the control and CD groups. The DM + CD group exhibited higher fasting blood glucose levels than the DM group (p ≤ 0.05). Plasma insulin measurements indicated relative hypoinsulinemia in the DM + CD and DM groups compared to the CD and control groups (p ≤ 0.01). Additionally, hypoinsulinemia was observed in the DM group (p ≤ 0.05) compared to the control and CD groups (Figure 1a,b).
The HOMA–IR was significantly higher in both the DM and DM + CD groups compared to the CD and control groups (p ≤ 0.001) (Figure 1c), and in the DM + CD group compared to the DM group (p ≤ 0.05) (Figure 1c). No significant increase was observed in the CD group compared to the control.

3.4. Lipid Profile

Table 2 presents the lipid profile and homocysteine of non-diabetic and diabetic adult rats exposed to CDD. A substantial decrease in serum HDL levels was shown in the DM and CD groups (p ≤ 0.05), and a significant increase in serum triglyceride levels (p ≤ 0.05) was observed in the DM group compared to the control. Serum homocysteine levels were significantly lower in the DM group (p ≤ 0.05) compared to the CD and control groups (Table 2).

3.5. Renal Histopathology

Histopathological findings of the kidneys in diabetic Ch-deprived rats revealed macroscopic renal damage, characterized by bluish–red discoloration and an increase in renal weight. Microscopically, extensive progressive renal disease was observed, characterized by glomerulotubular and interstitial injury, with more pronounced tubular necrosis, pyknosis, and karyolysis. These changes were associated with increased eosinophilia (primarily in the proximal renal tubules), the exfoliation of tubular endothelial cells, and vascular congestion (H&E stain) (Figure 2a,e). A relative decrease in Bowman’s capsule space, the significant thickening of the basement membrane, and the diffuse expansion of the glomerular mesangial matrix were also noted (Figure 3a,e). Additionally, there was a degree of renal tubule atrophy and significant renal interstitial fibrosis (Masson stain) (Figure 4a,e).
Exposure to a Ch-deficient diet for five weeks provoked a low grade of proximal tubular necrosis (Figure 2a,c), with mild but non-significant glomerular matrix expansion (PAS stain) (Figure 3a,c) and significant interstitial fibrosis (Figure 4a,c). Similarly, the DM group showed non-significant tubular necrosis (Figure 2a,d), significant expansion of the glomerular mesangium along with basement membrane thickening (Figure 3a,d), and a significant increase in interstitial fibrosis (Figure 4a,d).

3.6. Immunohistochemical Findings

3.6.1. KIM-1 Expression

KIM-1 immunohistochemical staining was predominantly expressed in the cytoplasm of renal tubular endothelial cells. KIM-1 expression was significantly increased in the CD group (p ≤ 0.01) compared to the control, and in the DM + CD group compared to the control, DM (p ≤ 0.001), and CD (p ≤ 0.05) groups. No significant change in KIM-1 expression was observed in the DM group (Figure 5).

3.6.2. Renal VEGF-A Stain Expression

VEGF-A immunohistochemical staining was predominantly observed in the cytoplasm of renal tubular endothelial cells and glomerular endothelial cells. The VEGF-A expression in the DM group was significantly higher compared to the CD, DM + CD, and control groups (p ≤ 0.001, p ≤ 0.001, and p ≤ 0.01, respectively) (Figure 6). Although VEGF-A expression decreased in the choline-deprived groups (CD and DM + CD), the change was not statistically significant.

4. Discussion

Given the critical role of choline in metabolism and the complications that arise from its deficiency, this study investigated the effects of Ch deprivation on the renal pathology of STZ-induced diabetic rats. The findings of the study indicate that dietary Ch deprivation exacerbates diabetic renal injury, as evidenced by the worsened renal function and structure in adult rats under a diabetes mellitus simulation. The diabetic Ch-deprived rats exhibited a significant deterioration in glycemia and renal function, as evidenced by hyperglycaemia, increased blood urea nitrogen, and elevated serum creatinine. Ch deprivation also altered renal architecture, as seen in increased kidney weight, renal tubular necrosis, mesangial matrix expansion, and tubule-interstitial fibrosis. Additionally, there was an upregulation in the expression of the tubular injury marker KIM-1, along with a downregulation in the expression of VEGF.
The pronounced hyperglycaemia observed in diabetic rats fed with CDD is in accordance with the hypothesis that choline deficiency contributes to insulin resistance supported by our findings, which show an amplified effect when co-stimulated with diabetes [22]. Homocysteine, a highly atherogenic amino acid, was significantly increased in the choline-deprived group. This could be attributed to the dysregulation of methionine metabolism, leading to a reduction in S-adenosylmethionine (SAM) availability. In contrast, homocysteine levels were lower in the DM group, which could be explained by several mechanisms: (i) the increased activities of enzymes such as betaine-homocysteine methyltransferase and cystathionine beta-synthase, which convert homocysteine to methionine or cysteine; (ii) elevated SAM levels; and (iii) enhanced betaine metabolism, promoting homocysteine clearance [29].
Our findings further corroborate previous studies, showing a deterioration of renal function (i.e., creatinine and blood urea nitrogen) in STZ-induced diabetic rats, indicating a closely intertwined impairment of renal functionality. The decreased body weight is indicative of (i) the aggravation of protein and nucleic acids catabolism; (ii) increased impairment of energy utilization under a diabetic state due to an exacerbation in hyperglycaemia and hypoinsulinaemia, and subsequent aggravated tissue wasting, in diabetic Ch-deprived rats [30,31]. In addition, the significant increase in kidney hypertrophy index in diabetic rats exposed to CDD could be attributed to an aggravated overexpression of growth factors in renal tubules, and/or as a consequence of renal necrosis [32,33]. These data are in line with a study by Denninghoff et al., who reported an increase in kidney weight index due to renal necrosis caused by a Ch-deficient diet in young rats [33].
Renal histopathology revealed an exacerbation of renal tubular tissue necrosis in the Ch-deprived state. The mechanism of Ch deprivation-related kidney injury primarily involves free radical-mediated processes, resembling hepatic necrosis following carbon tetrachloride or paracetamol intoxication [9,17,34,35]. Reported mechanisms indicate that Ch deprivation induces mitochondrial complex syndrome I-mediated oxidative stress, akin to the pathogenic mechanism in ischemic reperfusion-induced renal injury, resulting in the generation of reactive oxygen species (ROS), amplified in a time- and dose-dependent manner; these ROS are highly reactive, and can damage lipids, proteins, and DNA in the mitochondria, contributing to cellular damage [17,18,34,36,37]. On a quantitative level, according to a previous study, dietary Ch deprivation triggered a fourfold surge in plasma thiobarbituric acid-reactive substances levels, such as in the index of oxidative injury, coupled with inflammatory responses and apoptosis [38]. Oxidized lipids and lipid peroxidation products represent an important role in renal tubular necrosis progression, while antioxidant administration (e.g., butylated hydroxyanisole, butylated hydroxytoluene) can prevent renal injury induced by Ch deficiency [39]. Future studies are needed in order to elucidate the oxidative effects of both over- and underconsumption of Ch, in a time- and dose-dependent manner, on renal tubular necrosis.
Our findings revealed that the combination of Ch deprivation and diabetes was associated with a more pronounced kidney fibrosis progression. Choline deficiency results in phosphatidylcholine depletion and subsequent membrane damage and cellular dysfunction, which initiate the release of pro-inflammatory cytokines, the activation of oxidative stress, and an increase in free radicals, leading to renal necrosis and fibrosis. Other mechanisms involving the exacerbation of the fibrotic state may be due to stimulation from non-hypoxia-related pathways, including a proximal tubule-based renin–angiotensin system, the toxic effects of albumin-bound fatty acids, and the activation of epidermal growth factor receptor signalling pathways [40,41,42]. Moreover, progressive renal interstitial fibrosis in the coexisting metabolic disorder results in increased tissue rigidity, which is perceptible through mechanotransduction at the cellular level. Rather than dampening the fibrogenic process, a stiff matrix initiates a positive feedback loop, triggering further fibrotic-related transcription cofactors [43]. Another key contributor to the pathogenesis of kidney disease is mesangial expansion. The increased mesangial matrix deposition in diabetic groups suggests the effective hyperglycemic simulation that leads to the disruption of glomerular architecture and filtration impairment [44]. This feature is driven by mediators, including the transforming growth factor-beta (TGF-β) and angiotensin II, which modulate matrix metalloproteinases to enhance extracellular matrix synthesis and inhibit matrix degradation [44]. While the understanding of the direct impacts of Ch on the regulation of mesangial cell proliferation is limited, studies have shown that Ch can alleviate angiotensin II-induced cardiac hypertrophy via calcium- and p38 MAPK-signalling pathways within the cells [45,46]. Similarly, TGF-β, in a Ch-deficient state, has been implicated in worsening apoptosis and fibrosis in the brain, liver, and heart [47,48,49]. Future studies on the effects of a Ch-deprived state on extracellular matrix deposition are warranted.
In light of these immunohistochemical findings, the combined metabolic conditions of diabetes and choline deprivation acted synergistically to impair renal function and exacerbate structural damage, leading to the strongest expression of KIM, which correlated positively with blood urea and creatinine levels. Primarily expressed in renal tubules, KIM-1 serves as a hallmark biomarker for acute and chronic kidney injury [50,51]. It has been reported that KIM-1 expression may contribute to the development of interstitial fibrosis following injury and the progression of chronic nephropathy [50,51]. Dysregulated angiogenesis is also implicated in the pathogenesis of chronic kidney disease (CKD). In choline deficiency, mitochondrial dysfunction and oxidative stress can lead to the stabilization of the hypoxia-inducible factor (HIF-1α) that is upregulated in renal injury, leading to the enhanced expression of pro-fibrotic genes, including the transforming growth factor β1(TGF-β1) and VEGF, both of which contribute to fibrosis and the formation of fibrotic scars in the kidney, resulting in a fibrotic response. Activation of TGF-β1 leads to the activation of Smad-dependent and Smad-independent pathways in response to oxidation induced by choline deficiency, resulting in collagen production and increased ECM deposition and fibrosis [17,18,33,34,52].
Furthermore, the decrease in VEGF-A levels in the DM + CD group suggests a loss of cytoprotective effects, and reduced cell counts in glomerular podocytes, mesangial cells, and endothelial tubules [53,54,55]. Several mechanisms have been postulated to explain this phenomenon, including capillary rarefaction leading to the activation of the hypoxia-inducible factor, chronic inflammation, and decreased epithelial cell survival due to the inhibition of the anti-apoptotic kinase Akt/PI3K pathway [56,57].
The main limitation of this study is the relatively short study period (5 weeks), which may not be sufficient to observe a full course of kidney pathology and the evaluation of kidney injury. Future studies should also consider the evaluation of kidney injury. Future studies should also consider investigating both the short- and long-term effects of choline supplementation to assess the possibly protective impact of choline and conduct an in-depth evaluation of renal function to more accurately track renal injuries, including a quantitative analysis of KIM-1 and VEGFA.

5. Conclusions

Dietary choline deprivation accelerates the induction and progression of diabetic kidney injury, as evidenced by the renal functional, histopathological, and immunohistochemical deterioration. These findings highlight the importance of Ch supplementation in the management and prevention of kidney disease. To ensure holistic assessment, future comorbidity simulation studies should consider stratifying the degree of Ch depletion as a gradual experimental model to elucidate the mechanistic role of chronic cellular injury and renal remodelling.

Author Contributions

A.W.A.-H. and C.L.: conceived and designed the presented idea of the study. A.W.A.-H.: ran the experiment protocol, acquisition, interpretation of data, and achieved the original draft of the manuscript. C.W.l.R., N.G.D., W.A.-N. and M.T.W.K.: participated in reviewing the manuscript. A.C.L.: performed the histopathological procedure and assessment. A.W.A.-H.: performed the statistical analysis. C.L.: supervised the article and performed the critical revision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

All procedures were approved by the Medical School of National and Kapodistrian University of Athens after the approval of the Animal Protocols (N:1623 and N:3067) by the Department of Rural and Veterinary Policy (RVP), General Sector of Rural Economy and Veterinary, Prefecture of Attica, Hellenic Republic. Rats were cared for in accordance with the principles of laboratory animal care as previously set by the European Economic Community (EEC) Council Directive 86/609/EEC (EEC) and amended according to the recommendation 2007/526/EU for experimental animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the corresponding author only upon reasonable request.

Acknowledgments

We would like to express our gratitude to Eugene Danas for his valuable assistance in various laboratory techniques. We would like to acknowledge that the data presented in this manuscript are part of the PhD dissertation of Ahmed W. Al-Humadi, the first author.

Conflicts of Interest

There are no conflicts of interest to declare.

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Figure 1. (a) Fasting blood glucose levels (mg/dL) in diabetic and non-diabetic rats exposed to CDD for five weeks. The number of symbols (*, #, †) corresponds to the level of the statistical significance value, ***: p ≤ 0.001 versus control; †††: p ≤ 0.001 versus CD; #: p ≤ 0.05 versus DM. (b) Fasting plasma insulin levels (μU/mL) in diabetic and non-diabetic rats exposed to CDD for five weeks. **: p ≤ 0.01 versus control; ††: p ≤ 0. 01 versus CD; *: p ≤ 0.05 versus control; † versus CD. (c) Insulin resistance index (HOMA-IR) values in diabetic and non-diabetic rats exposed to CDD for a five-week period. The statistical significance value ***: p ≤ 0.001 versus control; †††: p ≤ 0.001 versus CD; #: p ≤ 0.05 versus DM. All values refer to mean ± SD. Ch: choline; control: rats receiving a standard diet; CDD: choline-deprived diet, DM: diabetic group; DM + CD: choline-deprived diabetic group. HOMA-IR: homeostasis model assessment index of insulin resistance.
Figure 1. (a) Fasting blood glucose levels (mg/dL) in diabetic and non-diabetic rats exposed to CDD for five weeks. The number of symbols (*, #, †) corresponds to the level of the statistical significance value, ***: p ≤ 0.001 versus control; †††: p ≤ 0.001 versus CD; #: p ≤ 0.05 versus DM. (b) Fasting plasma insulin levels (μU/mL) in diabetic and non-diabetic rats exposed to CDD for five weeks. **: p ≤ 0.01 versus control; ††: p ≤ 0. 01 versus CD; *: p ≤ 0.05 versus control; † versus CD. (c) Insulin resistance index (HOMA-IR) values in diabetic and non-diabetic rats exposed to CDD for a five-week period. The statistical significance value ***: p ≤ 0.001 versus control; †††: p ≤ 0.001 versus CD; #: p ≤ 0.05 versus DM. All values refer to mean ± SD. Ch: choline; control: rats receiving a standard diet; CDD: choline-deprived diet, DM: diabetic group; DM + CD: choline-deprived diabetic group. HOMA-IR: homeostasis model assessment index of insulin resistance.
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Figure 2. Histopathological findings of renal necrosis in non-diabetic and diabetic adult rats exposed to CDD. (a) Histopathological scoring and representative light microscopy (H&E; ×200) images (b–e) of the necrosis occurring in the kidneys of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: Choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant.
Figure 2. Histopathological findings of renal necrosis in non-diabetic and diabetic adult rats exposed to CDD. (a) Histopathological scoring and representative light microscopy (H&E; ×200) images (b–e) of the necrosis occurring in the kidneys of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: Choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant.
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Figure 3. PAS-stain histopathological findings for glomerular mesangial matrix expansion of non-diabetic and diabetic adult rats exposed to CDD. Histopathological scoring (a) and representative light microscopy (PAS; ×200) images (b–e) showing mesangial matrix deposition in the renal glomeruli of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet and water (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant. Data refer to mean ± SD.
Figure 3. PAS-stain histopathological findings for glomerular mesangial matrix expansion of non-diabetic and diabetic adult rats exposed to CDD. Histopathological scoring (a) and representative light microscopy (PAS; ×200) images (b–e) showing mesangial matrix deposition in the renal glomeruli of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet and water (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant. Data refer to mean ± SD.
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Figure 4. Histopathological findings of Masson’s stain for renal fibrosis in non-diabetic and diabetic adult rats exposed to CDD. Histopathological scoring (a) and representative light microscopy (Masson’s stain; ×200) images (b–e) showing renal fibrosis occurring in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant. Data refer to mean ± SD.
Figure 4. Histopathological findings of Masson’s stain for renal fibrosis in non-diabetic and diabetic adult rats exposed to CDD. Histopathological scoring (a) and representative light microscopy (Masson’s stain; ×200) images (b–e) showing renal fibrosis occurring in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; ns: non-significant. Data refer to mean ± SD.
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Figure 5. IHC KIM-1 stain expression (percentage of areal density) in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Immunohistochemical scoring (a) and representative light microscopy images (KIM-1 stain; ×400) (b–e) showing renal KIM-1 protein expression in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; IHC: immunohistochemistry; ns: non-significant.
Figure 5. IHC KIM-1 stain expression (percentage of areal density) in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Immunohistochemical scoring (a) and representative light microscopy images (KIM-1 stain; ×400) (b–e) showing renal KIM-1 protein expression in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; IHC: immunohistochemistry; ns: non-significant.
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Figure 6. IHC VEGF-A stain expression (percentage of areal density) in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Immunohistochemical scoring (a) and representative light microscopy (VEGF-A165 stain; ×400) images (b–e) showing renal VEGF-A protein expression in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet and water (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; IHC: immunohistochemistry; ns: non-significant. Data refer to mean ± SD.
Figure 6. IHC VEGF-A stain expression (percentage of areal density) in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Immunohistochemical scoring (a) and representative light microscopy (VEGF-A165 stain; ×400) images (b–e) showing renal VEGF-A protein expression in the renal tissue of non-diabetic and diabetic adult rats exposed to CDD. Control: rats receiving standard diet and water (b); CD: choline-deprived group (c); DM: diabetic group (d); DM + CD: diabetic rats exposed to CDD (e). **: p ≤ 0.01, ***: p ≤ 0.001 compared to other groups. CDD: choline-deprived diet; IHC: immunohistochemistry; ns: non-significant. Data refer to mean ± SD.
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Table 1. Biochemical profile and renal weight of non-diabetic and diabetic adult rats exposed to CDD.
Table 1. Biochemical profile and renal weight of non-diabetic and diabetic adult rats exposed to CDD.
GroupsSCr (mg/dL)BUN (mg/dL)Kidneys wt. (g)Kidneys/Body wt. Ratio
Control0.42 ± 0.0732.33 ± 4.633.6 ± 0.170.007 ± 0.0004
CD0.46 ± 0.0936.33 ± 6.182.74 ± 0.180.007 ± 0.001
DM0.50 ± 0.0340.00 ± 5.173.01 ± 0.370.008 ± 0.001
DM + CD0.58 ± 0.06 **53.66 ± 10.91 ***#††3.23 ± 0.28 †0.010 ± 0.001 **††
Data refer to mean ± SD of serum levels of blood urea, creatinine, and kidney weight, kidneys/body weight ratio after the five-week exposure of non-diabetic and diabetic adult rats to CDD. Statistical significance: **: p ≤ 0.01, ***: p ≤ 0.001 compared to control; #: p ≤ 0.05 compared to DM; †: p ≤ 0.05, ††: p ≤ 0.01 compared to CD. Where no sign, no significant difference among the groups. CDD: choline-deprived diet; CD: choline-deprived group; DM: diabetic group; DM + CD: diabetic Ch-deprived group; SCr: serum creatinine; BUN: blood urea nitrogen; wt.: weight.
Table 2. Lipidemic profile and homocysteine levels of non-diabetic and diabetic adult rats exposed to CDD.
Table 2. Lipidemic profile and homocysteine levels of non-diabetic and diabetic adult rats exposed to CDD.
GroupsTC (mg/dL)TG (mg/dL)HDL (mg/dL)LDL (mg/dL)Hcy (μmol/L)
Control75.17 ± 8.8461.17 ± 36.5552.83 ± 7.9410.10 ± 15.1824.02 ± 1.85
CD59.50 ± 12.3472.00 ± 22.6738.33 ± 11.48 *6.77 ± 4.5730.08 ± 16.02
DM72.20 ± 12.38137.00 ± 47.93 *35.80 ± 5.22 *9.00 ± 8.2810.90 ± 1.65 *†
DM + CD69.33 ± 12.0486.00 ± 64.5046.50 ± 9.815.63 ± 6.1323.25 ± 16.03
Data refer to mean ± SD of serum levels after the 5-week exposure of non-diabetic and diabetic adult rats to CDD. Statistical significance *: p ≤ 0.05 compared to control; †: p ≤ 0.05 compared to CD. Where there is no sign, there is no significant difference among the groups. CD: choline-deprived group; DM: diabetic group; DM + CD: diabetic Ch-deprived group; Hcy: homocysteine; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TC: total cholesterol; TG: triglycerides.
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MDPI and ACS Style

Al-Humadi, A.W.; le Roux, C.W.; Docherty, N.G.; Al-Najim, W.; Kueh, M.T.W.; Lazaris, A.C.; Liapi, C. Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats. Diabetology 2025, 6, 8. https://doi.org/10.3390/diabetology6010008

AMA Style

Al-Humadi AW, le Roux CW, Docherty NG, Al-Najim W, Kueh MTW, Lazaris AC, Liapi C. Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats. Diabetology. 2025; 6(1):8. https://doi.org/10.3390/diabetology6010008

Chicago/Turabian Style

Al-Humadi, Ahmed W., Carel W. le Roux, Neil G. Docherty, Werd Al-Najim, Martin Tze Wah Kueh, Andreas C. Lazaris, and Charis Liapi. 2025. "Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats" Diabetology 6, no. 1: 8. https://doi.org/10.3390/diabetology6010008

APA Style

Al-Humadi, A. W., le Roux, C. W., Docherty, N. G., Al-Najim, W., Kueh, M. T. W., Lazaris, A. C., & Liapi, C. (2025). Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats. Diabetology, 6(1), 8. https://doi.org/10.3390/diabetology6010008

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