Maternal dexamethasone exposure does not affect glucose tolerance but alters renal haemodynamics in F1 rats in a sex‐dependent manner

Abstract Introduction Prenatal programming with dexamethasone increases the risk of the development of hyperglycaemia and insulin resistance, leading to diabetes in adulthood. Dexamethasone also causes a decline in renal glomerular filtration in the adult offspring. Sodium‐glucose cotransporter‐2 (SGLT2) plays a significant role in regulating blood glucose and renal haemodynamics in diabetic patients. However, the role of SGLT2 in dexamethasone‐induced programming and the putative sex‐dependent effects on the changes named earlier is unknown. Therefore, this study aimed to investigate the impact of maternal dexamethasone treatment on glucose tolerance, insulin sensitivity, renal perfusion and renal function in adult male and female offspring and the possible contribution of SGLT2 to these changes. Methods and Results Pregnant Sprague Dawley rats (F0) were treated with either vehicle or dexamethasone (0.2 mg/kg ip) from gestation Day 15 to 20. F1 males and F1 females were randomly selected from each mother at 4 months of age. There was no change in serum Na+, Na+ excretion rate, glucose tolerance or insulin sensitivity in F1 male or female rats. However, dexamethasone caused significant glomerular hypertrophy and decreases in CSinistrin and CPAH indicating decreased glomerular filtration rate and renal plasma flow, respectively, in dexamethasone‐treated F1 male but not female rats. Dexamethasone did not affect SGLT2 mRNA or protein expression in F1 males or females. Conclusion We conclude that dexamethasone‐mediated prenatal programming of glomerular volume, renal function and haemodynamics is sex‐dependent, occurring only in adult male offspring.


| INTRODUC TI ON
Pregnant women at risk of premature labour are treated with synthetic glucocorticoids essential for developing many foetal organs. 1 Synthetic glucocorticoids are not readily metabolized by the placental enzyme-11β-hydroxysteroid dehydrogenase-2 2 ; therefore, they may play a role in prenatal programming and have detrimental effects on the foetus, such as intrauterine growth restriction 3 (IUGR) and the development of diseases later in life such as hypertension and diabetes.Maternal treatment with dexamethasone (Dex) causes an increase in fasting blood glucose level and hyperinsulinaemia in the F 1 generation, upregulation of hepatic gluconeogenesis enzymes 4 and a decrease in insulin content in pancreatic β cells. 5,6e kidneys could further exacerbate dexamethasone-induced glucose homeostasis impairment as exposure of cultured cortical tubular cells to dexamethasone causes an increase in glucose synthesis and upregulation of enzymes involved in gluconeogenesis. 7Similarly, renal gluconeogenesis increased in diabetic animals and patients (reviewed in Sharma and Tiwari 2021 8 ).The kidneys can further contribute to hyperglycaemia by altering renal glucose reabsorption.Type 1 diabetic patients showed a higher renal capacity to reabsorb glucose 9 with increased proximal tubular expression of sodium-glucose transporter-2 (SGLT2), responsible for 90% of renal glucose reabsorption, in diabetic animals 10 and humans. 11In addition to altering blood glucose levels, SGLT2 contributes to the hyperfiltration that occurs early in diabetes through tubulogmlomerular feedback. 12The increase in proximal Na + -glucose cotransport causes afferent arteriolar dilation leading to an increase in renal blood flow (RBF) and glomerular filtration rate (GFR). 12Blocking glucose reabsorption with specific SGLT2 blockers improved glucose tolerance, reduced hyperglycaemia in diabetic rats 13 and reduced glomerular filtration rate (GFR), albuminuria and renal and glomerular hypertrophy in diabetic Akita mice. 14Moreover, with the use of specific SGLT2 blockers, there was a significant decrease in HbA1C and blood glucose levels in Type 1 15 and Type 2 diabetic patients, 16,17 with improvement in renal function. 15,18e studies mentioned earlier suggest the significance of renal SGLT2 in glucose homeostasis and renal haemodynamics; however, sex differences in the susceptibility to these changes have not been addressed despite the evidence that kidneys of female rats have higher levels of SGLT2 protein expression. 19A recent review 20 concluded that females have higher insulin sensitivity and less susceptibility to diabetes than males.However, the role of sex in the possible development of hyperglycaemia, insulin resistance and diabetes due to prenatal exposure to dexamethasone and the potential contribution of SGLT2 to the above-mentioned changes are unknown.
Therefore, this study aimed to investigate the effect of prenatal dexamethasone treatment on glucose homeostasis, insulin sensitivity, renal haemodynamics and renal expression of SGLT2 in F 1 adult male and female rats.

| Animals
Sprague-Dawley rats were used in this study (200-300 g body weight).The rats were placed in a room with a 12:12 light:dark cycle, kept at 22.3 ± 0.3°C and 31.2 ± 0.8% humidity.The rats had free access to water and standard chow (801151, Special Diets Services).
All rats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals and all experimental protocols used in this study were approved by the Animal Ethics Committee at Kuwait University, Faculty of Medicine.
For mating, male and female rats were placed in cages at a ratio of 1:3, respectively.The next day, mating was confirmed by the presence of sperm in the vaginal smear and considered Day 0 of pregnancy.The pregnant rats (F 0 ) were housed individually in cages until the beginning of the experiment.

| Experimental groups
Pregnant dams were divided into two groups-a control group (C, n = 12) treated with vehicle (saline) and another group treated with dexamethasone (Dex, n = 12).Dexamethasone 21-phosphate disodium salt (Cat # D 1159, Sigma-Aldrich) was administered daily at a dose of 0.2 mg/kg ip 21 from Day 15 of gestation (dg) to 20dg.The offspring (F 1 rats) were maintained for four months and randomly divided into two groups-Group 1 contained 12 dams which were further divided into two subgroups-a control group and a Dex-treated group (n = 6/group) to study changes in glucose homeostasis, insulin sensitivity, renal function and renal plasma flow (one F 1 male and one F 1 female were randomly chosen from each mother).The Group 2 also contained 12 dams which were further subdivided into two groups as described above (a control and a Dex-treated group; n = 6/ group), but their offspring were used to study glomerular morphometric changes, and gene and protein expressions of SGLT2.The experimental design is shown in Figure 1.

| Body weights, kidney weights and sample collection
Body weights of 4-month-old F 1 males and females (n = 12-15) were measured.The F 1 rats were then euthanised; the right kidney was removed, weighed, and stored at −70°C.The midsection of the left kidney was cut and fixed in 4% paraformaldehyde and processed for paraffin embedding for morphometric measurements and immunohistochemistry. Twenty-four hours before sample collection, the rats were kept in metabolic cages and 24-h urine sample was collected.

| Fasting blood glucose and insulin levels
Fasting blood glucose (FBG, G 0 ) concentration was measured in 4-month-old F 1 rats in samples taken from the tail vein using a glucometer (GLUCOTREND 2, Roche, Germany).Fasting insulin levels (I 0 ) were measured in control and dexamethasone-treated rats using a rat insulin ELISA Kit (ERINS, Invitrogen).Insulin sensitivity was derived from (G 0 ) and (I 0 ) levels using quantitative insulin sensitivity check index (QUICKI) = 1/[log(I 0 ) + log(G 0 ), which is highly correlated with glucose clamp technique. 22

| Oral glucose tolerance test (OGTT)
After 12-14 h of fasting, the blood glucose level was measured (G 0 ).
Then, a 20% glucose solution at a dose of 2 g/kg body weight was administered to the rats via a gastric tube.Blood glucose level was measured at 15, 30, 60 and 120 min and blood samples were collected, centrifuged and the serum samples were stored at −20°C.
The glucose values were plotted against time and the area under the curve (AUC) was calculated as a measure of glucose tolerance as

| Protein excretion rate
The rats were placed in metabolic cages and 24-hour urine was collected.Urinary protein concentration was measured using a Bradford Coomassie blue assay 24 using a commercially available assay kit (Cat # 23236, Thermo Scientific).Briefly, 300 μL of the Coomassie Plus reagent was added to 10 μL of standard or sample onto the microplate wells.Standards and samples were prepared in duplicates, and after 10 minutes incubation at room temperature the absorbance was measured at 595 nm with Versa Max microplate reader (Molecular Devices, USA).Protein excretion rate (PER) was calculated as: PER = U p •V, where U p is urinary protein concentration (mg/ ml) and V is urine flow rate (mL/24 h).

| Assessment of Na + concentration in serum and urine
Concentration of Na + in serum and 24 h urine samples was measured using an electrolyte measuring system SPOTCHEM ™ EL (SE-1520, Arkray, Inc).The principle of the method is to convert an electrical potential into an ion concentration using an ion-selective membrane F I G U R E 1 Experimental design.CF, control female; CM, control male; DF, dexamethasone-treated female; DM, dexamethasone-treated male.
electrode (SPOTCHEM-E plate).Serum samples were undiluted while urine samples were diluted 1:1 in distilled water as recommended by the manufacturer.

| Sodium excretion rate
Na + excretion rate (E Na + ) was calculated as E Na + = U Na + •V, where U Na + is urinary Na + concentration (mM) and V is the urine flow rate (mL/24 h).

| Estimation of glomerular filtration rate (GFR) and effective renal plasma flow (RPF)
The GFR and RPF were estimated in 4-month-old F 1 male and F 1 female of control and dexamethasone-treated mothers (F 0 ) by measuring the clearances of sinistrin (Inutest®; Fresenius-Kabi, Linz, Austria) and para-aminohippuric acid (PAH, A1422, Sigma-Aldrich, Germany), respectively.Each rat was anaesthetized with inactin (Inactin hydrate, 24899957, Sigma-Aldrich, Germany) at a dose of 100 mg/kg ip.The left carotid artery was catheterized for continuous monitoring of arterial blood pressure.The jugular vein was catheterized and an infusion with sterile Ringer's solution containing 6% albumin at a rate of 60 μL/min was performed for 45-60 min.The urinary bladder was cannulated for urine collection after which infusion of Ringer's solution containing (36 mg/mL) sinistrin and (9 mg/ mL) PAH in 1% BSA was started.Priming doses (160 mg/kg) of sinistrin and (8 mg/Kg) PAH were given intravenously.After a 30-min equilibration period, four samples of urine were collected at 20-min intervals.At the mid-point of each period, an arterial blood sample was collected in dry heparinized tubes and centrifuged at 2000 g for 10 min.Concentrations of sinistrin and PAH in plasma and urine samples were measured using the anthrone method (319899, Sigma-Aldrich, Germany method 25 ) and Bratton and Marshall's protocol, 26 respectively.

| Renal SGLT2 mRNA expression
The SGLT2 mRNA expression was studied in renal tissue by realtime quantitative polymerase chain reaction (ReT-PCR).Total RNA was extracted using TRIzol method.The RNA was treated with DNase and reverse transcribed.Real-time PCR reaction was carried out on a ReT-PCR system (Applied Biosystems, model 7500) using the following primer-Slc5a2, (Rn00574917_ m1).The reaction was performed using Solis BioDyne PCR master mix and cycled as follows-1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 60 cycles of alternate 15 secs at 95°C and 1 min at 60°C.Eukaryotic 18S was used as the housekeeping gene (Cat # 4319413E, Applied Biosystems).

| Immunohistochemistry for renal expression of SGLT2
Four-micrometre thick paraffin sections of kidneys were cut and deparaffinized in xylene and dehydrated in descending alcohol grades.
The kidney slides were incubated overnight at 4°C with the SGLT2 primary antibody (A gift from Prof. Hermann Koepsell, University of Würzburg (1:250 dilution) in a humidification chamber.The slides were incubated for 30 min with a biotinylated secondary antibody (anti-rabbit IgG, 1:200 dilution; Vector Laboratories) for 30 min and washed twice with PBS for 5 min each.After incubation for 30 min with the tertiary antibody (Vectastain Elite ABC kit, PK-6100, Vector Laboratories) and a PBS wash, diaminobenzidine peroxidase substrate (SK-4100, Vector Laboratories) was applied, and the slides were observed under a light microscope for optimal staining.The slides were washed in distilled water and immersed in haematoxylin for 3 min, followed by a dip in 1% eosin.The sections were dehydrated through ascending grades of alcohol, xylene and mounted with DPX.

| Western blotting for SGLT2 protein expression in renal membrane fractions
Renal protein expression of SGLT2 in membrane fractions was studied by western blotting.The renal tissue was homogenized in buffer (10 mM Tris, 1.5 mM EDTA, 10% v/v Glycerol) with 100 μL of 100 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitors (Pierce protease inhibitor tablets, Cat # SA 2286912, Ther-moFisher Scientific).The tissue was centrifuged for 10 min at 4°C at 6000 g.The supernatant was centrifuged at 4°C at 150,000Xg for an hour and the pellet was resuspended in the homogenisation buffer and used for western blotting. 19Forty microgram of protein for each sample was loaded on stain-free gel and electrophoresis was run at 200 V for 30-40 min (Mini-protein tgx stain-free gels (Cat # 4568096, Biochemicals and Radiochemicals (BioRad)).The proteins were then transferred to nitrocellulose membranes (Trans-blot turbo transfer pack cat no: 1704158, BioRad) at 25 V and 1.0 Amp for 30 min, visualized on BioRad ChemiDoc MP Imaging System to measure total protein loaded.After washing the membranes, rabbit polyclonal anti-SGLT2 antibody was added (Aviva Systems Biology, #ARP43832_ P050) at 1:500 dilution after which the membranes were blocked using 1x Tris-buffered saline (TBS) (1% casein blocker cat no: 161078, BioRad).Donkey anti-rabbit IgG secondary antibody (Horseradish Peroxidase-Linked, Cytiva Amersham ECL, NA934) was added at a dilution of 1:1000.Negative controls with elimination of the primary antibody were run for every gel.The bands were visualized using chemiluminescence on the Chemidoc Imager, then analysed using imaging using Image Lab software.β-Actin is not a reliable internal control 27 especially for membrane fractions as it is variable within the same group (data not shown).Therefore, the expression of each protein was taken as a ratio of SGLT2 band density to the cumulative densities of all proteins loaded per sample.glomeruli from the renal cortical zone of each rat were measured as previously described 29 and were assessed by a blind observer.

| Statistical analysis
Data were expressed as mean ± SEM for each group.For body weights, kidney weights and physiological data (FBG, PER, glucose and insulin), comparison between groups was performed using one-way ANOVA followed by Bonferroni or Games-Howell post hoc tests dependant on the homogeneity of variances.For GFR, RPF, glomerular morphometry and SGLT protein expression, two-way ANOVA was performed to assess if sex and/or treatment influenced the parameters tested, followed by Bonferroni or Games-Howell post hoc test as stated in the figures.For the ReT-PCR protocol, the expression of SGLT2 was normalised to 18S using the 2 −∆∆Ct method. 30Two-way ANOVA was performed to assess the effect of sex and/or treatment on SGLT2 mRNA expression.In all experiments, the statistical significance was considered when the p value was less than 0.05.1).
Dexamethasone had no effect on protein excretion rate in either males or females.

| The effect of maternal treatment with dexamethasone on glucose homeostasis in the 4-month-old F 1 males and females
Fasting blood glucose and fasting insulin levels were not affected by maternal treatment with dexamethasone in either F 1 males or F 1 females.In addition, GTT and quantitative insulin sensitivity check index showed no change in insulin sensitivity in any of the groups (Table 2).

| The effect of maternal dexamethasone exposure on serum and urine sodium levels in 4-month-old F 1 males and females
Maternal dexamethasone treatment did not affect Na + concentration in serum or urine nor sodium excretion rate (Table 3).

TA B L E 1
Effects of maternal dexamethasone exposure on body and kidney weights and protein excretion rate in 4-month-old F 1 rats.

| The effect of maternal treatment with dexamethasone on renal function and effective renal plasma flow in 4-month-old F 1 males and females
Two-way ANOVA showed that sex does not affect C Sinistrin or C PAH,

| The effect of maternal treatment with dexamethasone on glomerular morphometry in 4-month-old F 1 males and females.
Maternal treatment with dexamethasone caused significant glomerular hypertrophy in F 1 adult males with substantial increases Abbreviations: CF, control female; CM, control male; DF, dexamethasone-treated female; DM, dexamethasone-treated maleNote: Data are expressed as mean ± SE. n = 6 in each group.Two-way-ANOVA showed no effect of treatment and/or sex on any of the parameters measured.There was no significant difference between the groups in any of the parameters studied when compared using Bonferroni post hoc test.

TA B L E 3
The effect of maternal dexamethasone treatment on serum and urine sodium in 4-month-old F 1 males and females.

F I G U R E 2
The effect of maternal treatment with dexamethasone on renal plasma clearance of sinistrin (A) and PAH (B) in 4-month-old F 1 males and females.CM, control male; DM, dexamethasone-treated male; CF, control female; DF, dexamethasone-treated female.Results are expressed as mean ± SE. n = 6 in each group.Data were compared with two-way ANOVA followed by Bonferroni post hoc test and showed that dexamethasone had a significant effect on clearances of sinistrin and PAH while the combination of treatment and sex affects only clearance of sinistrin (GFR).Sex alone has no effect on either parameter.Clearances of sinistrin and PAH significantly decreased only in F 1 males.**p < 0.01; ***p < 0.001 when compared to controls.
in the glomerular area (p < 0.01) and volume (p < 0.01) (Figure 3; Table 4).The treatment did not cause glomerular hypertrophy in F 1 females, and the mesangial matrix area was not affected by dexamethasone treatment in any group.Two-way ANOVA showed that the glomerular area and volume are not affected by sex or treatment but rather by the interaction between sex and treatment.

| The effect of maternal treatment with dexamethasone on SGLT2 gene expression in 4-month-old F 1 males and females
Analysis of the ReT-PCR data showed no significant change in SGLT2 mRNA expression in F 1 males or females exposed to maternal dexamethasone and two-way ANOVA analysis showed no effect of sex and/or treatment on SGLT2 mRNA expression (Figure 4).

| Localisation of SGLT2 protein in F1 males and females
Immunohistochemistry showed that the SGLT2 protein is expressed in S1 segments of proximal convoluted tubules and not expressed in glomeruli (Gs) or distal convoluted tubules (Figure 5).

| SGLT2 protein expression in renal membrane fractions in 4-month-old F1 males and
Immunohistochemistry for SGLT2 showed localized expression of SGLT2 in the brush border of the proximal convoluted tubules (Fig- ure 5).Western blotting data indicated no effect of sex and/or treatment on the expression of SGLT2 in renal membrane fractions of 4-month-old rats (Figure 6).

| DISCUSS ION
This study examined the effect of prenatal dexamethasone treatment on glucose homeostasis and renal function in 4-month-old F 1 males and females.Prenatal programming with glucocorticoids during pregnancy causes IUGR and diseases in the offspring, such as diabetes, that develop later in life. 31In our study, at 21 days gestation foetuses of dexamethasone-exposed mothers were smaller than that of controls, but females and males were studied simultaneously; however, other studies have shown growth retardation in both males and females in the same animal model as ours. 32The weights of F 1 males remained less than that of controls at 4 months of age, similar to earlier reports in which only males had smaller body weights than controls at 6 months. 33ternal treatment with dexamethasone did not affect glucose tolerance or insulin sensitivity either in 4-month-old F 1 males or F 1 F I G U R E 3 Glomerular morphometric changes in four-month-old control males (CM), dexamethasone-treated male (DM), control female (CF) and dexamethasonetreated female (DF).There was significant increase in glomerular tuft area (GTA), but no change in and mesangial matrix area (MMA, arrows) or MMA/GTA in dexametbasone-treated males, No significant changes in glomerular morphometry was observed in 4-monthold female rats.(4Km paraffin sections, Periodic-acid Schiff stain, X 40).
females.In consensus with our results, a previous study showed increased insulin resistance only when dexamethasone treatment was combined with a high-fat diet in 4-month-old male rats. 34Conversely, other studies showed increased glucose intolerance in other IUGR models induced either by a low-protein diet 35 or intrauterine artery ligation 36 at 4-months postnatally; however, these models are incomparable to ours.On the other hand, the same dose of dexamethasone caused hyperglycaemia at six months of age; therefore, this difference may be due to the difference in sample time. 4spite not detecting hyperglycaemia or insulin resistance in our model; the role of prenatal dexamethasone treatment as a risk factor for the development of diabetes is strongly suggested by a random clinical trial on human subjects that showed impaired insulin response to glucose in adults exposed to maternal glucocorticoids. 37wever, the role of sex in dexamethasone-induced impaired insulin response was not addressed by that study.Prenatal treatment with dexamethasone did not affect renal handling of proteins as no increase in protein excretion rate was detected at 4 months in either F 1 males or females.It was noted that PER was significantly higher in males than that in females and is believed to be because of the effect of androgens on the kidneys in males. 38 the same model as ours, proteinuria develops only at 6 months of age in male rats. 33Similarly, dexamethasone had no effect on sodium levels in serum or sodium excretion rate, a finding that agrees with what was reported earlier. 33Maternal dexamethasone treatment did not affect sodium excretion in F 1 rats.Despite the significant role of SGLT2 in glucose reabsorption, it is responsible for a small portion of proximal tubular sodium reabsorption, therefore any change in SGLT2 activity or expression would have minimal effect on sodium excretion.
Prenatal treatment with dexamethasone caused a significant decrease in GFR in F 1 males but not in F 1 females.The studies on the effect of dexamethasone treatment on renal function are contradictory, with some showing a decline in GFR 39,40 and others not detecting any change in GFR. 21,41The differences between these studies and ours are the dose and duration of the dexamethasone treatment.
Few studies investigated the role of sex in renal function response to prenatal glucocorticoid treatment programming.Betamethasone caused a reduction in GFR only in adult male sheep. 42,43However, in another study, dexamethasone did not affect GFR either in male or female rat offspring, even when combined with a high-protein diet, 41 probably due to the lower dose of dexamethasone used (0.1 mg/kg/ day) and the shorter duration of the experimental model of 70 days.
Recently, in the same model as the one used in this study, dexamethasone caused a decrease in GFR, proteinuria and glomerulosclerosis in male but not female offspring at 1 year postnatally. 40ny factors can alter glomerular filtration, such as glomerular surface area, nephron number, renal plasma flow and tubuloglomerular feedback. 12The decrease in GFR in dexamethasone-treated males was attributed to decreased nephron number. 40Prenatal dexamethasone treatment caused a decline in nephron number at 70 days 44 and at 6 months of age in rats, 21 but in these studies males and females were analysed as one group.Martins et al (2003)   showed a decrease in nephron number in 70-day-old male but not Abbreviations: CF, control female; CM, control male; DF, dexamethasone-treated female; DM, dexamethasone-treated male; GTA, glomerular tuft area; GV, glomerular volume; MMA, mesangial matrix area.
Note: Two-way ANOVA showed that the interaction between sex and treatment has a significant effect on glomerular area and volume.Results are expressed as mean ± SE and compared using Games-Howell post hoc test.*p < 0.05, **p < 0.01 when compared to control.#p < 0.05 when compared to males in the same treatment group.

TA B L E 4
The effect of maternal treatment on glomerular morphometry in 4-month-old F 1 males and females.

F I G U R E 4
The effect of maternal treatment with dexamethasone on gene expression of SGLT2 in 4-month-old F 1 males and females.CF, control female; CM, control male; DF, dexamethasone-treated female; DM, dexamethasone-treated male.N = 6 in each group.The results using 2 −ΔΔCt method normalised to 18S were compared using 2-way-ANOVA analysis of data and showed that there was no effect of sex and/or treatment an SGLT2 mRNA expression.
female offspring when treated with dexamethasone combined with a postnatally high-protein diet.Similarly, IUGR caused by protein restriction 45 or intrauterine artery ligation 44 caused a reduction in nephron number in male but not female offspring, with compensatory glomerular hypertrophy only in male rats. 46The effect of dexamethasone on nephron number cannot be verified in our model as it was not measured; however, it may be inferred from the glomerular hypertrophy that occurred only in dexamethasone-treated males.The decline in renal function cannot be explained solely by the reduction in nephron number as some models reported that GFR remained constant despite lower nephron number. 42 for tubular factors, SGLT2 is believed to play a significant role in glucose regulation and renal haemodynamics.Renal SGLT2 ex- F I G U R E 6 SGLT2 protein expression in renal membrane fractions of control males (CM), dexamethasone-treated males (DM), control females (CF) and dexamethasone-treated females (DF).The SGLT2 protein band was at the right size around 66-73 kDa (A).Data (n = 6) are expressed as mean ± SEM.SGLT2 expression, taken as the ratio of SGLT2 band density to the cumulative densities of all proteins loaded, was not statistically different in dexamethasone-treated Fl males or Fl females when compared to their controls (B).Negative controls showed no band at the expected size of SGLT2.Data were compared using two-way ANOVA and analysis showed that there was no effect of sex and/or treatment on SGLT2 membrane protein expression.
glucose as it was within normal values in the sera of dexamethasonetreated F 1 rats; and the lack of diuresis suggesting normal glucose excretion rate.Therefore, data suggest that SGLT2 does not play a role in the altered renal function seen in male rats.
Our study is the first to show that maternal dexamethasone causes a decrease in effective renal plamsa flow in F1 males, which would be a major cause for the decreased filtration rate in those rats.The decrease in renal plasma flow suggests increased renal vascular resistance although in our study we did not assess vascular resistance.Studies have shown that the direct effect of glucocorticoids on the kidney is a decrease in renal vascular resistance and an increase in renal blood flow, 47 which is believed to be due to a reduction in the sensitivity to angiotensin II 47 or increased expression of eNOS. 48The only plausible explanation for a decline in renal plasma flow in our study, where dexamethasone caused IUGR, is an increase in preglomerular resistance due to other factors, such as an increase in renal levels of angiotensin II 49,50 and increased expression of angiotensinogen and angiotensin II type 1 receptor (AT1R), 51 decrease in renal nitric oxide production, 51 reduction of vascular compliance, 52 which is associated with low birth weight 53 or altered sympathetic activity. 54e role of sex in the vascular response to dexamethasone treatment was not addressed by the above-mentioned studies.Oestrogen and its receptors, which are known to be important regulators of body weight and insulin sensitivity, 55 were proven to be protective in other models of prenatal programming 56 and could be the reason behind the sex-specific renal haemodynamic response to prenatal dexamethasone treatment.Oestradiol activates renal eNOS, which is expressed more in kidneys of female rats 57 and protects against renal injury. 58Few studies explore the possible involvement of RAS in this male-specific decline in renal function.Ovariectomy increased renal angiotensin converting enzyme (ACE) activity and AT 1 R binding densities and these effects were reversed with oestradiol. 59With dexamethasone treatment, female offspring showed lower expression of Agt than males 60 and higher basal expression of angiotensin II type 2 receptor (AT2R) and angiotensin converting enzyme 2, which is the vasodilatory arm of the renin angiotensin system (RAS), 61 therefore females may respond differently to the activation of RAS. 61The role of RAS in the sex specific renal response to maternal dexamethasone treatment should be further investigated.
In conclusion, maternal dexamethasone treatment during late gestation causes glomerular hypertrophy and a decline in renal plasma flow and glomerular filtration rate only in adult male offspring.

2. 8 |
Glomerular morphological changes: The effect of maternal dexamethasone treatment on glomerular area, volume and mesangial matrix area Rats were anaesthetized by urethane at a dose of 1.2 g/kg i.p.The left kidney was flushed with normal saline, removed and 3 mm-thick transverse sections were cut and placed in 10% formalin.Three to four days later, the sections were embedded in wax, cut into 4 μm-thick sections, and mounted on APES (3-aminopropyltriethoxysilane )-coated slides for morphological studies.Periodic Acid-Schiff (PAS) stain was used and the total glomerular tuft area and mesangial matrix areas in 15-20 however both are affected by Dex treatment.The combination of sex and treatment had an effect on C Sinistrin but not C PAH .Maternal dexamethasone treatment caused a significant decrease in C Sinistrin (p < 0.001) and C PAH (p < 0.05) in 4-month-old F 1 males but not in females, indicating a decline in GFR (Figure 2A) and effective RPF (Figure 2B), respectively.

3 | RE SULTS 3.1 | The effect of maternal dexamethasone exposure on body weights, kidney weights and protein excretion rates in 4-month-old F 1 male and female rats
The effect of maternal treatment on glucose homeostasis in 4-month-old F 1 males and females.
TA B L E 2