Knockout of Na⁺-glucose cotransporter SGLT1 mitigates diabetes-induced upregulation of nitric oxide synthase NOS1 in the macula densa and glomerular hyperfiltration

Abstract

Na⁺-glucose cotransporter (SGLT)1 mediates glucose reabsorption in late proximal tubules. SGLT1 also mediates macula densa (MD) sensing of an increase in luminal glucose, which increases nitric oxide (NO) synthase 1 (MD-NOS1)-mediated NO formation and potentially glomerular filtratrion rate (GFR). Here, the contribution of SGLT1 was tested by gene knockout (−/−) in type 1 diabetic Akita mice. A low-glucose diet was used to prevent intestinal malabsorption in Sglt1^−/− mice and minimize the contribution of intestinal SGLT1. Hyperglycemia was modestly reduced in Sglt1^−/− versus littermate wild-type Akita mice (480 vs. 550 mg/dl), associated with reduced diabetes-induced increases in GFR, kidney weight, glomerular size, and albuminuria. Blunted hyperfiltration was confirmed in streptozotocin-induced diabetic Sglt1^−/− mice, associated with similar hyperglycemia versus wild-type mice (350 vs. 385 mg/dl). Absence of SGLT1 attenuated upregulation of MD-NOS1 protein expression in diabetic Akita mice and in response to SGLT2 inhibition in nondiabetic mice. During SGLT2 inhibition in Akita mice, Sglt1^−/− mice had likewise reduced blood glucose (200 vs. 300 mg/dl), associated with lesser MD-NOS1 expression, GFR, kidney weight, glomerular size, and albuminuria. Absence of Sglt1 in Akita mice increased systolic blood pressure, associated with suppressed renal renin mRNA expression. This may reflect fluid retention due to blunted hyperfiltration. SGLT2 inhibition prevented the blood pressure increase in Sglt1^−/− Akita mice, possibly due to additive glucosuric/diuretic effects. The data indicate that SGLT1 contributes to diabetic hyperfiltration and limits diabetic hypertension. Potential mechanisms include its role in glucose-driven upregulation of MD-NOS1 expression. This pathway may increase GFR to maintain volume balance when enhanced MD glucose delivery indicates upstream saturation of SGLTs and thus hyperreabsorption.

INTRODUCTION

Diabetes mellitus is a leading cause of chronic kidney disease. The early responses to hyperglycemia include kidney growth, tubular hyperreabsorption, and glomerular hyperfiltration. Early kidney growth and hyperfiltration have been considered risk factors for the later development of diabetic nephropathy (36), but their pathophysiology is still incompletely understood, including how the kidney “senses” hyperglycemia.

Most renal glucose reabsorption occurs in the early proximal tubule (S1/S2 segments) and is mediated by high-capacity Na⁺-glucose cotransporter (SGLT)2 in the apical brush border (32). As a consequence, individuals with loss-of-function gene mutations in SGLT2 (SLC5A2) have persistent renal glucosuria (20). In comparison, SGLT1 (SLC5A1) is expressed in the apical membrane of further downstream segments of the nephron, including the late proximal tubule (S2/S3 segments) and thick ascending limb (13, 40). In normoglycemic conditions and with intact SGLT2, SGLT1 reabsorbs the glucose that has not been reabsorbed by upstream SGLT2 and is delivered to the late proximal tubule, which equals ~3% of filtered glucose (4, 19). In accordance, individuals with gene mutations in SGLT1 (SLC5A1) have little or no glucosuria (20). Overall, SGLT1 has a three to five times lower glucose transport capacity in the kidney than SGLT2 (3, 37).

Driven by hyperglycemia-induced tubular growth and increased glomerular filtration of glucose, SGLT2 contributes to the enhanced proximal tubular Na⁺ and fluid reabsorption in the early diabetic kidney (29, 36, 37). The increase in proximal reabsorption lowers Na⁺-Cl⁻-K⁺ concentrations at the macula densa (MD) and reduces tubular backpressure (hydrostatic pressure in the Bowman space), which, through the physiology of tubuloglomerular feedback (TGF) and an increase in glomerular hydrostatic filtration pressure, contributes to diabetic glomerular hyperfiltration (27, 33, 35). Evidence for primary hyperreabsorption upstream of the MD and a potential role of SGLT2 in glomerular hyperfiltration has also been proposed in patients with diabetes (2, 8, 39, 42). Pharmacological inhibition of SGLT2 is a new antihyperglycemic approach that increases glucosuria, thereby lowering blood glucose levels (37). SGLT2 inhibition induces additional pleiotropic effects that may contribute to the renal and cardioprotective effects observed in patients with diabetes (15, 37, 43). Based on the role of SGLT2 in tubular hyperreabsorption and hyperfiltration in the diabetic kidney, one early effect of SGLT2 inhibition is a modest reduction in glomerular filtration rate (GFR), which, by lowering the transport burden on the kidney, may help to preserve kidney function in the long term (17). In addition, the natriuretic and diuretic effects of SGLT2 inhibition lower blood pressure (37).

The quantitative contribution of SGLT1 to renal glucose reabsorption has been well established in euglycemic conditions; however, little is known about the consequences of its inhibition on renal glucose transport, blood glucose control, or early changes in the diabetic kidney (22). Previous studies have indicated that SGLT2 and SGLT1 together can explain all renal glucose reabsorption in euglycemic nondiabetic mice (19), but whether the same is true in the diabetic setting has not been specifically tested. Moreover, glucose uptake via SGLT1 may be a critical stimulus for the diabetic kidney to grow. Furthermore, SGLT1 expression has been localized to the apical membrane of the MD in mice (13), rats (1), and humans (46), where it has recently been proposed to contribute to the sensing of luminal glucose and expression and activation of neuronal nitric oxide (NO) synthase (NOS1) (46).

NOS1 is the predominant NOS isoform expressed in MD cells (14, 45). NO generated by the MD attenuates the TGF-mediated reduction in glomerular capillary pressure and GFR (10, 38, 45) and has been implicated in TGF resetting (24, 25, 44). Previous studies have proposed a role for enhanced renal NO activity (28) and increased expression of NOS1 in the MD (MD-NOS1) in diabetes-induced glomerular hyperfiltration (9, 26). A role for MD-NOS1 has recently been demonstrated in the acute increase in GFR in response to glucose infusion in nondiabetic mice (46). Moreover, the MD NO generation and TGF inhibition induced by luminal glucose were blocked by a SGLT1 inhibitor (46). To assess the contribution of SGLT1 in a diabetic setting, Sglt1 gene-targeted diabetic and nondiabetic mice were studied with regard to renal glucose transport, blood glucose control, glomerular hyperfiltration, and MD-NOS1 expression. Moreover, the present study assessed the functional contribution of SGLT1 when SGLT2 was pharmacologically inhibited.

METHODS

Animals.

All animal experimentation was conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and was approved by the local Institutional Animal Care and Use Committee. The generation of gene-targeted mice lacking Sglt1 on the C57BL/6J background has been previously described (4). Since the DBA/2J genetic background may enhance renal susceptibility to injury in Akita diabetic mice, a genetic model of type 1 diabetes (5), Sglt1^−/+ mice were first backcrossed to DBA/2J mice (stock no. 000671, Jackson Laboratories) for four generations. Subsequently, mice were crossed to DBA/2J Ins2(C96Y/+) Akita mice (Akita/+, stock no. 007562, Jackson Laboratories) to generate the final breeders (Sglt1^+/− and Akita/+ Sglt1^+/− mice). Littermate experimental male mice were generated [control wild-type (WT), control Sglt1^−/−, Akita/+ WT, and Akita/+ Sglt1^−/− mice) and housed in the same animal room with a 12:12-h light-dark cycle. Mice had free access to tap water and a diet with minimal content of glucose and galactose and high content of fructose (40% fructose, 20% casein, and 20% cellulose; by weight: 17.7% protein, 40.4% carbohydrate, and 15.2% fat; TD.150497, Harlan Teklad, Madison, WI). The low-glucose/galactose diet prevented diarrhea in Sglt1^−/− mice due to the role of SGLT1 in intestinal glucose uptake (4) and minimized the contribution of intestinal SGLT1 for the observed outcome. The high dietary fructose content served to induce robust hyperglycemia in WT Akita/+ mice in the absence of dietary glucose.

Renal clearance experiments to test whether SGLT2 and SGLT1 together can explain all glucose reabsorption in the diabetic kidney.

In series 1, control WT, control Sglt1^−/−, Akita/+ WT, and Akita/+ Sglt1^−/− mice were prepared for renal clearance experiments under terminal inactin/ketamine anesthesia with measurement of GFR by [³H]inulin clearance to determine fractional renal glucose excretion as previously described (19). In all mice, the SGLT2 inhibitor dapagliflozin (dapa; 10 mg/kg ip) was applied 1 h before functional experiments to eliminate SGLT2-mediated glucose reabsorption. The same clearance study approach was used in type 2 diabetic db/db mice (stock no. 000697, Jackson Laboratories) in which Sglt1 had been deleted by cross-breeding.

To confirm the selectivity of dapa for SGLT1 versus SGLT2, preliminary experiments were performed using metabolic cages with awake C57BL/6J Sglt1^−/− and C57BL/6J Sglt2^−/− mice and their WT littermates (32). After the bladder had been emptied, dapa (0.1, 1, or 10 mg/kg) or vehicle (25 µl H₂O/g body wt by oral gavage) was applied followed by quantitative urine collection over 3 h to measure glucosuria.

Experiments to determine the role of SGLT1 in Akita diabetic mice.

In series 2, experiments were performed in control WT, control Sglt1^−/−, Akita/+ WT and Akita/+ Sglt1^−/− mice. To determine potential additive effects of SGLT1 and SGLT2 inhibition, the experiments included four additional groups of mice (same genotypes as described above) that were chronically treated with dapa (7.5 mg/kg of repelleted diet; diet described above) starting at 5 wk of age. Non-dapa, vehicle-treated mice received the repelleted diet.

Systolic blood pressure and heart rate in awake mice.

At 15 wk of age, systolic blood pressure and heart rate were determined using an automatic tail-cuff system (Visitech-Systems, Apex, NC) after appropriate training as previously described (30, 32).

GFR in awake mice.

GFR measurements were performed in conscious mice at 16 wk of age using the plasma elimination kinetics of FITC-sinistrin (Fresenius-Kabi, Linz, Austria) following a single dose intravenous injection of this GFR marker (30). Briefly, FITC-sinistrin (2% in 0.85% NaCl, which also served to establish the standard curve) was injected into the retroorbital plexus (2 µl/g body wt) during brief isoflurane anesthesia. At 3, 5, 7, 10, 15, 35, 56, and 75 min after injection, blood was collected from the end of the tail into a Na⁺-heparinized 10-µl microcap (Hirschmann Laborgeräte, Eberstadt, Germany). After centrifugation, plasma was diluted 1:10 in 0.5 mol/l HEPES (pH 7.4), and fluorescence was determined using a Nanodrop ND-3300 fluorospectrometer (Nanodrop Technologies, Wilmington, DE) by pipetting 2 µl of sample onto the pedestal. GFR was calculated using a two-compartment model of two-phase exponential decay (GraphPad Prism, San Diego, CA).

Kidney harvest and analysis.

At 17 wk of age, mice were anesthetized by isoflurane, kidneys were harvested and decapsulated, and kidney wet weight was determined. One-half of the kidney was fixed in 4% paraformaldehyde for 24 h at 4°C. After transfer to 70% alcohol for 24 h at 4°C, kidneys were embedded in paraffin and sectioned (5 μm), and glomerular size was determined. For the latter, images were taken using a ×10 objective on an Olympus IX81 Microscope (Shinjuku, Tokyo, Japan).

Immunostaining for NOS1.

Tissue sections were hydrated, and heat-induced epitope retrieval was applied in 10 mM citrate buffer (pH 6.0) using a microwave. Sections were incubated in blocking solution (2.5% normal goat serum in PBS) for 30 min at room temperature followed by an overnight incubation with primary antibody (anti-NOS1 antibody, 1:500, catalog no. BML-SA227-0100, Enzo Life Sciences) at 4°C. After being washed three times in Tris-buffered saline with Tween 20, sections were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG antibody for 1 h at room temperature. Immunostained slides were then scanned using a Axio Z1 slide scanner (Carl Zeiss). Mean signal intensity was determined for individual MD segments of scanned images via ImageJ software.

Reverse transcription and real-time PCR.

Frozen kidneys were homogenized using a mechanical tissue homogenizer in lysis buffer provided by the RNeasy Plus Mini Kit (catalog no. 74136, Qiagen). RNA was purified using the RNeasy Plus Mini Kit per the manufacturer’s instructions. cDNA was prepared with 5 μg of purified RNA using the SuperScript IV First-Strand Synthesis System (catalog no. 18091050, ThermoFisher) per the manufacturer’s instructions. cDNA reactions were diluted fivefold, and 1 μl was used for quantitative PCR. Real-time PCRs were performed using the Taqman assay (catalog no. 4331182, ThermoFisher) in an ABI7300 Real Time PCR System (Applied Biosystems, Foster City, CA). See Table 1 for primer details. Amplification efficiencies were normalized against the housekeeping genes β-actin and 18S rRNA, and relative fold increases were calculated using the Pfaffl technique of relative quantification, which accounts for real-time efficiencies (18). Each experiment was performed in triplicate.

Table 1.

Real-time PCR primers used

Target	Assay
Sglt1	Mm00451203_m1 (Applied Biosystems)
Sglt2	Mm00453831_m1 (Applied Biosystems)
Renin	Mm02342889_g1 (Applied Biosystems)
Actb	Mm00607939_s1 (Applied Biosystems)
18S rRNA	Mm03928990_g1 (Applied Biosystems)

Sglt, Na⁺-glucose cotransporter; Actb, β-actin.

Experiments to assess the GFR effect of Sglt1 knockout in a second experimental diabetes model.

In series 3, diabetes was induced in C57BL/6J mice lacking Sglt1 and their WT littermates by streptozotocin (STZ) following the recommendations of the Animal Model of Diabetic Complications Consortium (https://www.diacomp.org/shared/document.aspx?id=19&docType=Protocol) and as previously described (35). Briefly, mice were injected with STZ (50 mg/kg ip) on 5 consecutive days. Nondiabetic control mice received vehicle injections (Na⁺-citrate buffer with pH adjusted to 4.5). At 14 wk after STZ treatment to induce diabetes, food and fluid intake was measured in regular cages over 3 days. At 15 wk, blood glucose was measured by tail snip in awake mice, and GFR was determined by plasma elimination kinetics of FITC-sinistrin as described above. All mice in this model were fed a low-glucose/galactose diet containing high protein content, i.e., the diet previously used in studies using Sglt1^−/− mice (by weight: 52.4% protein, 1.5% carbohydrate, and 11.9% fat, TD.08212, Harlan Teklad) (4, 19).

Blood and urine collection and analysis.

Urine was collected by a bladder catheter in renal clearance experiments (series 1). In awake mice, urine was obtained by picking up the mice to elicit reflex urination and holding them over a petri dish (series 2 and 3) or using a metabolic cage for sample collection (series 1). Blood was collected from a carotid artery catheter in renal clearance experiments (series 1). Blood was collected by tail snip (for glucose measurement) immediately before urine collection in awake mice (series 2 and 3).

Blood glucose from tail snips was determined using an Ascensia Elite XL glucometer (Bayer, Mishawaka, IN). Blood glucose in clearance experiments and urine glucose was determined by the hexokinase/glucose-6-phosphate dehydrogenase method (Infinity, Thermo Electron, Louisville, CO). Concentrations of urinary albumin (Exocell, Philadelphia, PA) and creatinine (kinetic assay approach, ThermoFisher) were measured using commercial assays.

Statistical analysis.

Data are presented as means ± SE. Data were log transformed when so doing resulted in a distribution that was more normal. To analyze for statistical differences between groups, two-way ANOVA was performed to probe for a significant effect of the two specific factors or for the interaction between the two factors. If the interaction was statistically significant, a pair-wise multiple comparison procedure (Holm-Sidak method) was then used to identify the significant effects (23). One-way ANOVA followed by the Holm-Sidak method was used for comparison of individual doses with vehicle in metabolic cages. An unpaired Student’s t-test was performed when only two groups were compared. P values of <0.05 were considered statistically significant.

RESULTS

Dapa is a selective inhibitor of SGLT2 versus SGLT1 in mice.

In metabolic cage experiments, glucosuria in response to vehicle application was slightly higher in Sglt1^−/− versus WT mice (Fig. 1A, left) and strongly elevated in Sglt2^−/− versus WT mice (Fig. 1A, right). Dapa induced a similar dose-dependent glucosuric response in the two WT groups. Dapa application did not enhance glucosuria at any of the tested doses in Sglt2^−/− mice (Fig. 1A, right). In contrast, the glucosuric response to dapa was enhanced in Sglt1^−/− versus WT mice, and the difference persisted at the highest dapa dose of 10 mg/kg (Fig. 1A, left). The data were consistent with selective inhibition of SGLT2 in response to the tested dapa doses.

Fig. 1.

A: dapagliflozin (dapa) is a selective inhibitior of Na⁺-glucose cotransporter (SGLT)2 versus SGLT1 in the mouse kidney. In metabolic cage experiments, dapa induced a similar dose-dependent glucosuric response in the two groups of wild-type (WT) mice. The glucosuric effect of dapa was absent in Sglt2^−/− mice and enhanced in Sglt1^−/− mice with the differences between Sglt1^−/− and their WT mice persisting up to the highest dose of 10 mg/kg. Left, Sglt1^−/− mice (*P < 0.05 vs. WT mice by Student’s t-test). n = 5−8 mice/group. Right, Sglt2^−/− mice [#P < 0.05 vs vehicle (Veh) by one-way ANOVA followed by the Holm-Sidak method for comparison of individual doses with Veh]. n = 3–6 mice/group. B: combined inhibition of SGLT2 and SGLT1 eliminated net renal glucose reabsorption in diabetic mice. Renal clearance experiments showed that under conditions of dapa (10 mg/kg ip) pretreatment and similar filtration rates of glucose in Sglt1^−/− versus WT mice, the absence of SGLT1 significantly reduced fractional renal glucose reabsorption versus WT mice. Moreover, the data indicated that dual SGLT2/SGLT1 inhibition can eliminate net glucose reabsorption in the nondiabetic and diabetic kidney. * P < 0.05 vs. WT mice by Student’s t-test. n = 7–10 clearance periods in 4–5 mice/group. bw, body weight.

Combined inhibition of SGLT2 and SGLT1 eliminated net renal glucose reabsorption in diabetic mice.

Fractional renal glucose reabsorption in dapa-pretreated mice was 36%, 24%, and 17% in control WT, Akita/+ WT, and db/db WT mice, respectively (Fig. 1B, right). Under conditions of dapa pretreatment and similar filtration rates of glucose (Fig. 1B, left) in Sglt1^−/− versus WT mice, the absence of Sglt1 significantly reduced fractional renal reabsorption of glucose versus WT mice, such that fractional renal glucose reabsorption was not different from zero in any of the three groups with dual SGLT2/SGLT1 inhibition (Fig. 1B, right). These data document a significant contribution of SGLT1 to renal glucose reabsorption in diabetic mice and indicate that dual SGLT2/SGLT1 inhibition can eliminate net glucose reabsorption in nondiabetic and diabetic kidneys.

In Akita diabetic mice, absence of SGLT1 resulted in lesser blood glucose, MD-NOS1 expression, GFR, kidney weight, and albuminuria and greater systolic blood pressure.

Absence of renal Sglt1 mRNA expression was confirmed in nondiabetic and Akita/+ Sglt1^−/− mice (Fig. 2A). In nondiabetic mice, urinary glucose-to-creatinine ratios were higher in Sglt1^−/− compared with WT mice (Fig. 2B, left), consistent with a small contribution of SGLT1 to renal glucose reabsorption (4, 19), whereas renal Sglt2 mRNA expression was not different (Fig. 2C, left). This was associated with similar values in nondiabetic Sglt1^−/− versus WT mice for blood glucose (Fig. 2D, left), GFR (Fig. 2, E and F, left), body weight (Fig. 3A, left), kidney weight (Fig. 3B, left), and outer cortex glomerular size (Fig. 3C, left). MD-NOS1 protein expression was higher in nondiabetic Sglt1^−/− versus WT mice (Fig. 4, left). This was accompanied by a small increase in urinary albumin-to-creatinine ratios (Fig. 3D, left) and a small reduction in systolic blood pressure (Fig. 3E, left) in nondiabetic Sglt1^−/− versus WT mice, whereas renal renin mRNA expression (Fig. 3F, left) was not different.

Fig. 2.

Absence of Na⁺-glucose cotransporter (SGLT)1 and pharmacological SGLT2 inhibition independently lowered blood glucose and glomerular filtration rate (GFR) in Akita diabetic mice. A−F: data for renal mRNA expression of Sglt1 (A) and Sglt2 (C), urinary glucose-to-creatinine ratios (B), blood glucose levels (D), and GFR (E and F). See text for details. Two-way ANOVA was performed to probe for a significant effect of Sglt1^−/− (P_Sglt1), dapagliflozin (P_dapa), or the interaction between the two factors (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm-Sidak method) identified the significant effects. *P < 0.05 vs. wild-type (WT); #P < 0.05 vs. vehicle. n = 9–10 mice/group for gene expression, n = 10–15 mice/group for glucosuria, n = 11–21 mice/group for blood glucose, and n = 10–19 mice/group for GFR.

Fig. 3.

Absence of Na⁺-glucose cotransporter (SGLT)1 lowered kidney weight, glomerular size, and albuminuria independent of SGLT2 inhibition in Akita diabetic mice. A−F: data for body weight (A), kidney weight (B), outer cortex glomerular size (C), urine albumin-to-creatinine ratios (D), systolic blood pressure (BP; E), and renal renin mRNA expression (F). See text for details. Two-way ANOVA was performed to probe for a significant effect of Sglt1^−/− (P_Sglt1), dapagliflozin (P_dapa), or the interaction between the two factors (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm-Sidak method) identified the significant effects. *P < 0.05 vs. wild-type (WT) mice; #P < 0.05 vs. vehicle. n = 12–21 mice/group for body weight, n = 11–19 mice/group for kidney weight, n = 160–284 glomeruli/group from 4−6 mice/group, n = 9–14 mice/group for albuminuria, n = 10–17 mice/group for systolic BP, and n = 9–10 mice/group for renin expression.

Fig. 4.

Absence of Na⁺-glucose cotransporter (SGLT)1 attenuated the increase in macula densa (MD)-nitric oxide synthase 1 (NOS1) expression in response to Akita diabetes as well as SGLT2 inhibition in nondiabetic mice. A: the mean intensity of NOS1 in single nephron MD sections was determined using ImageJ. Scale bars = 10 μm. See text for details. B: protein expression of NOS1 in the MD. Two-way ANOVA was performed to probe for a significant effect of Sglt1^−/− (P_Sglt1), dapagliflozin (P_dapa), or the interaction between the two factors (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm-Sidak method) identified the significant effects. *P < 0.05 vs. wild-type (WT) mice; #P < 0.05 vs. vehicle. n = 68–115 single nephron MD sections in 3–4 animals/group.

In Akita/+ mice, absence of SGLT1 lowered GFR (Fig. 2, E and F, right). This was associated with modestly reduced blood glucose levels (from ~550 to 480 mg/dl; Fig. 2D, right) and increased renal Sglt2 mRNA expression (Fig. 2C, right). These changes indicated a reduction in filtered glucose and a potential increase in SGLT2-mediated glucose reabsorption, which would explain the lower urinary glucose-to-creatinine ratios in Sglt1^−/− versus WT Akita/+ mice (Fig. 2B, right). Improved metabolic control was also suggested by a modestly greater body weight in diabetic mice lacking SGLT1 (Fig. 3A, right). In contrast to the response in nondiabetic mice (see above), absence of SGLT1 in Akita/+ mice reduced MD-NOS1 expression (Fig. 4, right). This was associated with lower GFR (see above; Fig. 2, E and F, right), kidney weight (Fig. 3B, right), outer cortex glomerular size (Fig. 3C, right), and urinary albumin-to-creatinine ratios (Fig. 3D, right). In contrast to the response in nondiabetic mice (see above), absence of SGLT1 in Akita/+ mice increased systolic blood pressure (Fig. 3E, right), associated with lower renal renin mRNA expression (Fig. 3F, right). This potentially indicated some level of fluid retention in Sglt1^−/− versus WT Akita/+ mice that was in part compensated by downregulation of renin expression.

In nondiabetic mice, absence of SGLT1 enhanced the glucosuric effect of pharmacological SGLT2 inhibition and prevented the increase in MD-NOS1 expression.

Dapa treatment in nondiabetic WT mice induced a modest increase in glucosuria (Fig. 2B, left), associated with a small increase in MD-NOS1 (Fig. 4, left). This occurred without a significant effect on blood glucose (Fig. 2D, left), GFR (Fig. 2, E and F, left), outer cortex glomerular size (Fig. 3C, left), systolic blood pressure (Fig. 3E, left), or renal renin mRNA expression (Fig. 3E, left). Similar to the effect of absence of SGLT1, dapa induced a small increase in urinary albumin-to-creatinine ratios in nondiabetic mice (Fig. 3D, left).

When SGLT2 was inhibited with dapa in nondiabetic mice, urinary glucose-to-creatinine ratios were higher in the absence of SGLT1 (Fig. 2B, left). This effect of SGLT1 knockout was greater than in vehicle-treated mice, consistent with enhanced delivery of glucose to SGLT1-expressing segments during SGLT2 inhibition (19). Despite greater glucosuria and likely glucose delivery to the MD, absence of SGLT1 prevented the increase in MD-NOS1 expression in response to dapa in nondiabetic WT mice (Fig. 4, left). Dapa treatment did not significantly affect the small reduction in systolic blood pressure observed in nondiabetic Sglt1^−/− versus WT mice (Fig. 3E, left).

During pharmacological SGLT2 inhibition in Akita diabetic mice, absence of SGLT1 resulted in lesser hyperglycemia, MD-NOS1 expression, GFR, kidney weight, and albuminuria but did not enhance blood pressure.

Dapa treatment in Akita/+ mice reduced blood glucose (Fig. 2D, right), MD-NOS1 expression (Fig. 4, right), GFR (Fig. 2, E and F, right), kidney weight (Fig. 3B, right), and outer cortex glomerular size (Fig. 3C, right) and increased body weight (Fig. 3A, right). This was associated with similar glucosuria (Fig. 2B, right), possibly due to balanced reductions in filtered and reabsorbed glucose. Dapa increased renal renin mRNA expression in Akita/+ mice (Fig. 3F, right), whereas systolic blood pressure was unchanged (Fig. 3E, right).

During SGLT2 inhibition in Akita/+ mice, absence of SGLT1 reduced blood glucose levels (from ~300 to 200 mg/dl; Fig. 2D, right), MD-NOS1 expression (Fig. 4, right), GFR (Fig. 2, E and F, right), kidney weight (Fig. 3B, right), outer cortex glomerular size (Fig. 3C, right), and urinary albumin-to-creatinine ratios (Fig. 3D, right). Dapa treatment did not prevent the decrease in renal renin mRNA expression (Fig. 3F, right) but prevented the increase in systolic blood pressure (Fig. 3E, right) observed in vehicle-treated diabetic Sglt1^−/− versus WT mice.

Confirmation of blunted hyperfiltration in another model of diabetic mice lacking Sglt1.

STZ-induced diabetes increased blood glucose (~385 mg/dl) and GFR; this was associated with enhanced glucosuria, food and fluid intake, and reduced body weight (Fig. 5A). Absence of SGLT1 did not significantly affect the STZ-induced increase in hyperglycemia and food and fluid intake or the reduction in body weight but prevented glomerular hyperfiltration without significantly changing glucosuria (Fig. 5A). When we plotted urinary glucose-to-creatinine ratios versus filtered glucose, there was a leftward shift in STZ-induced diabetic Sglt1^−/− versus WT mice (Fig. 5B). In other words, STZ-induced diabetic Sglt1^−/− mice excreted similar amounts of glucose versus STZ-induced diabetic WT mice despite lower glucose filtration, consistent with lesser glucose reabsorption in the absence of SGLT1.

Fig. 5.

Absence of Na⁺-glucose cotransporter (SGLT)1 attenuated streptozotocin (STZ)-induced diabetes glomerular hyperfiltration without significantly altering blood glucose and shifted glucosuria to lower values of glucose filtration. A: absence of SGLT1 did not significantly affect STZ-induced hyperglycemia or glucosuria but blunted the STZ-induced diabetes increase in glomerular filtration rate (GFR). The latter reduced filtered glucose but glucosuria was not affected. B: thus, STZ-induced diabetic Sglt1^−/− mice excreted similar amounts of glucose versus STZ-induced diabetic wild-type (WT) mice despite lower glucose filtration, consistent with lesser glucose reabsorption in the absence of SGLT1. Two-way ANOVA was performed to probe for a significant effect of Sglt1^−/− (P_Sglt1), STZ (P_STZ), or the interaction between the two factors (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm-Sidak method) identified the significant effects. *P < 0.05 vs. WT mice; #P < 0.05 vs. nondiabetic vehicle-treated control mice. n = 5–6 mice/group in the nondiabetic groups and n = 7 mice/group for the diabetic groups.

DISCUSSION

The main findings of the present study are that the absence of SGLT1 lowers glomerular hyperfiltration, kidney weight, glomerular size, and albuminuria in Akita mice, a genetic model of type 1 diabetes mellitus. This was associated with modestly improved blood glucose control. Similar effects of SGLT1 knockout were observed during pharmacological SGLT2 inhibition, indicating independent and additive effects of SGLT2 and SGLT1 inhibition in Akita mice. The effect of SGLT1 knockout on glomerular hyperfiltration was confirmed in STZ-induced diabetes, a chemically induced type 1 diabetes model. In this case, lower GFR was observed without a significant effect on blood glucose levels. These results indicate that SGLT1 contributes to diabetic glomerular hyperfiltration independent of its glucose-reabsorbing and hyperglycemia-maintaining effects. A likely mechanism begins with the sensing of increased glucose delivery by SGLT1 in the luminal membrane of MD cells, which respond by increasing NOS1-dependent NO formation (46). This further reduces the vasoconstrictor tone set by TGF, which is already low in diabetes due to increased NaCl and fluid reabsorption driven by tubular growth and SGLT2 in the early proximal tubule (37). The present study provides several pieces of evidence that support this theory. First, SGLT1 is necessary for MD-NOS1 expression to increase when glucose delivery to the MD is promoted by giving SGLT2 blocker to nondiabetic animals. Second, MD-NOS1 expression is blunted in Akita mice lacking SGLT1. Third, SGLT1 is necessary for full expression of diabetic hyperfiltration, and this is unlikely due to further decline in MD Cl⁻ delivery.

A GFR effect that is unique to SGLT1 inhibition is proposed to be via inhibition of diabetes-induced MD-NOS1 expression and NO formation. SGLT1 has been previously determined to be localized to the luminal membrane of the MD in mice (13) and rats (1), and a recent study confirmed this expression pattern in the human kidney (46). Studies in rats and mice have implicated MD-NOS1 in the GFR increase in response to acute hyperglycemia and STZ-induced diabetes (9, 26, 46). Moreover, an increase in luminal glucose in the MD perfusate enhanced MD NO formation and attenuated TGF-mediated vasoconstriction of the afferent arteriole, and these effects were prevented by luminal addition of an SGLT1 inhibitor (46), indicating a role of SGLT1 in glucose-induced MD NO formation and reduced afferent arteriolar vasoconstriction. The present study provides the first evidence for a proposed MD-SGLT1-NOS1-GFR pathway in a diabetic mouse model, inasmuch as the absence of SGLT1 reduced MD-NOS1 expression and GFR in diabetic Akita mice.

The diabetic mice in the present study were glucosuric, indicating that high amounts of filtered glucose (due to hyperglycemia and hyperfiltration) saturated the renal glucose reabsorption capacity. Renal clearance experiments in Sglt1^−/− and WT mice unmasked a contribution of SGLT1 to renal glucose reabsorption in diabetic mice. Knockout of SGLT1, however, only modestly reduced blood glucose levels in diabetic Akita mice and left blood glucose unchanged in STZ-induced diabetic mice. These findings indicated an overall modest contribution of SGLT1 to total glucose reabsorption in the tested diabetes models. Moreover, the experiments in Akita mice showed that the contribution of SGLT1 to blood glucose control was significantly smaller than the contribution of SGLT2. The glucosuric effect of eliminating SGLT1 is further attenuated by its GFR-lowering effect. In fact, glucosuria was less in Akita mice lacking SGLT1 than in WT Akita mice. The lower GFR and blood glucose levels reduced filtered glucose in Sglt1^−/− mice, which is expected to oppose the lack of SGLT1-mediated renal glucose reabsorption. This is reminiscent of the unchanged glucosuria previously observed in response to SGLT2 inhibition in STZ-induced diabetic and Akita mice, in which the reduction in filtered glucose through lowering of GFR and blood glucose levels balanced the inhibitory effect on tubular glucose reabsorption (30, 34).

Moreover, the absence of SGLT1 in Akita mice was associated with increased renal Sglt2 mRNA expression, which may have enhanced SGLT2-mediated glucose reabsorption, thereby further reducing glucosuria. A previous study (4) in nondiabetic Sglt1^−/− C57BL/6J mice on a low-glucose, high-protein diet did not find a change in renal mRNA or protein expression of SGLT2, similar to the findings in nondiabetic Sglt1^−/− DBA2 mice on low-glucose, high-fructose diet in the present study. Therefore, Akita diabetes may have affected the effect of the absence of SGLT1 on SGLT2 expression. Moreover, absence of SGLT1 in C57BL/6J mice on a low-glucose, high-protein diet was recently found to improve kidney recovery after acute kidney injury induced by ischemia-reperfusion; this included improved recovery in SGLT2 expression (16). SGLT2 is localized to the early proximal tubule, where little SGLT1 is expressed or functionally active (32). In other words, SGLT1-mediated transport in the late proximal tubule, thick ascending limb, and MD may have implications for renal integrity beyond these tubular segments. Further studies are needed to follow up on this hypothesis, which may also relate to the observed effects of SGLT1 elimination on albuminuria, glomerular size, and kidney weight.

The present study indicates that inhibition of SGLT1 and SGLT2 can independently improve early changes in the diabetic kidney. This includes effects on blood glucose control, GFR, kidney weight, and glomerular size. The observed additive effects on blood glucose control are consistent with complementary roles of SGLT2 and SGLT1 in renal glucose reabsorption in the nondiabetic and diabetic kidney. This is consistent with the observed elimination of net glucose reabsorption in nondiabetic and diabetic mice by dual SGLT2/SGLT1 inhibition in renal clearance experiments.

The GFR-lowering effect of SGLT2 inhibition has been proposed to involve the TGF mechanism and an increase in tubular backpressure, secondary to inhibiting the enhanced Na⁺-glucose reabsorption in the early proximal tubule (37). By lowering blood glucose levels, SGLT2 inhibition also attenuates the diabetes-induced kidney weight increase and tubular growth, thereby also reducing tubular hyperreabsorption (37). To the extent that eliminating SGLT1 reduces tubular Na⁺ and fluid reabsorption upstream of the MD in the diabetic kidney, it is expected to lower GFR via TGF and tubular backpressure. SGLT1 knockout also reduced kidney weight in Akita mice, potentially due to the modest effect on blood glucose, which, through lesser tubular growth and overall transport machinery, may also have limited tubular hyperreabsorption and thereby GFR. Finally, lowering blood glucose may have limited glomerular growth and induced direct vascular effects that lessened hyperfiltration (31). Thus, effects on GFR by lowering blood glucose and tubular hyperreabsorption would be shared by SGLT2 and SGLT1 inhibition, with the magnitude of these effects expected to be much smaller in response to SGLT1 inhibition (Fig. 6).

Fig. 6.

Proposed role of Na⁺-glucose cotransporter (SGLT)1 in the early diabetic kidney. Blue arrows indicate positive interactions. Hyperglycemia induces tubular growth and enhances filtered glucose. This increases Na⁺-glucose cotransport, thereby maintaining hyperglycemia and reducing urinary Na⁺ and fluid excretion, with a larger contribution of SGLT2 versus SGLT1. Lower urinary Na⁺ and fluid excretion tends to increase effective circulating volume (ECV) and blood pressure (BP). The tubular hyperreabsorption, however, reduces tubular backpressure in the Bowman space (PBow) and lowers NaCl concentration at the macula densa (MD), both increasing glomerular filtration rate (GFR) to restore urinary Na⁺ and fluid excretion. An increase in glucose delivery to the MD indicates that upstream Na⁺-glucose cotransport has been saturated. This is sensed by SGLT1 in the MD and, by enhancing MD nitric oxide synthase 1 (NOS1) expression, further increases GFR as an additional mechanism to compensate for maximized Na⁺-glucose cotransport. It is proposed that inhibition of SGLT1 has a relatively small effect on diabetic tubular hyperreabsorption and thus induces little natriuresis and diuresis. SGLT1 inhibition, however, induces a relatively larger antinatriuretic and antidiuretic effect through inhibition of MD-NOS1 upregulation and lowering of glomerular hyperfiltration. As a consequence, ECV increases with the resulting suppression in renin and increase in BP aiming to restore renal Na⁺ and fluid excretion and ECV.

Why should an increase in MD glucose trigger an increase in GFR? Insights may have been provided by the observation that the blunted diabetes-induced increase in GFR in SGLT1 knockout mice was associated with suppressed renal renin mRNA expression and an increase in systolic blood pressure. The increase in GFR in the diabetic kidney serves, at least in part, to stabilize body volume when diabetes-induced tubular growth and hyperglycemia-induced Na⁺-glucose cotransport cause a primary increase in tubular Na⁺, glucose, and fluid reabsorption (7, 36). Blunting this compensatory increase in GFR in the absence of a robust effect on hyperreabsorption is expected to increase blood pressure, which is a first-order mechanism for Na⁺ homeostasis (6). In the diabetic kidney, the occurrence of glucose delivery to the MD indicates saturation of upstream SGLTs and thus hyperreabsorption of Na⁺, glucose, and fluid. The MD senses the increased luminal glucose via SGLT1, and the SGLT1-NOS1-GFR pathway enhances GFR to maintain urinary Na⁺ and fluid excretion and volume balance (Fig. 6). Such a mechanism would complement the classical TGF and tubular backpressure mechanisms that link tubular hyperreabsorption to an increase in GFR via lower MD Na⁺-Cl⁻-K⁺ concentrations and lesser hydrostatic pressure in the Bowman space, respectively (Fig. 6), with the latter two mechanisms also operating when tubular glucose reabsorption is below the transport maximum of glucose (36). Several lines of evidence indicate a decisive role for MD-NOS1 in lessening the swings in blood pressure otherwise required to maintain long-term Na⁺ balance on a varying diet (11, 12, 41), and mice with deletion of MD-NOS1 exhibit salt-sensitive hypertension (12). Moreover, and in accordance with a proposed MD-SGLT1-NOS1-GFR pathway and secondary effects on blood pressure, the response to SGLT1 knockout in diabetic mice resembled those of a previous study (9) that used high doses of a selective NOS1 inhibitor in diabetic rats; in both cases, GFR was reduced and accompanied by mild elevations in blood pressure.

An increase in blood pressure can have detrimental consequences on the kidney and cardiovascular system. Cotreatment with SGLT2 inhibitor prevented the increase in blood pressure in response to SGLT1 knockout in Akita mice. SGLT1 knockout reduced renal renin mRNA expression also under these conditions, but the SGLT2 inhibitor had lifted the renin levels, likely as a consequence of the more robust diuretic effect of inhibiting SGLT2 versus SGLT1. Furthermore, coinhibition of SGLT2 and SGLT1 had further lowered blood glucose and kidney weight and thereby potentially tubular hyperreabsorption. Thus, cotreatment with SGLT2 inhibitor may improve the effects of SGLT1 inhibition in the diabetic setting. On the other hand, use of dual SGLT2/SGLT1 inhibition may increase the risk of hypoglycemia and volume depletion. Further studies are needed to better understand these issues.

Through the proposed MD-SGLT1-NOS1-GFR pathway, the increase in glucose delivery to the MD in response to SGLT2 inhibition may in part offset the GFR-lowering effect induced by enhancing Na⁺-Cl⁻-K⁺ delivery to the MD and increasing tubular backpressure. An increase in MD glucose delivery is expected when SGLT2 inhibition enhances glucosuria, which typically is observed in the clinical setting with no or only moderate hyperglycemia. Under these conditions, the MD-SGLT1-NOS1-GFR pathway could lessen the GFR reduction in response to SGLT2 inhibition. In the present study in Akita mice, SGLT2 inhibition, in steady state, did not increase glucosuria and, therefore potentially, did not enhance glucose delivery to the MD, due to the concomitant reduction in filtered glucose (as a consequence of lowering GFR and blood glucose) that matched the inhibition of glucose reabsorption (30, 34). Despite a potentially unchanged glucose delivery to the MD, SGLT2 inhibition reduced MD-NOS1 expression in Akita mice. This may reflect the volume loss induced by SGLT2 inhibition (reflected by renal renin mRNA expression increase) and an inhibitory influence of volume loss on MD-NOS1 activity (Fig. 6). Clearly, multiple inputs determine MD-NOS1 expression and activity, and the net effect of SGLT2 inhibition seems more complex. Complex influences were also indicated by the upregulation of MD-NOS1 expression in the absence of SGLT1 in nondiabetic mice; while this upregulation of MD-NOS1 expression was unexpected and requires further investigation, it was associated with lower systolic blood pressure.

In summary, the absence of SGLT1 lowers glomerular hyperfiltration, kidney weight, glomerular size, and albuminuria in a genetic model of type 1 diabetes mellitus. This was associated with modestly improved blood glucose control. The present study further indicates independent and additive effects of SGLT2 and SGLT1 inhibition on blood glucose, glomerular hyperfiltration, kidney weight, and glomerular size in this diabetes model. Combined SGLT2 and SGLT1 inhibition acutely eliminated net renal glucose reabsorption in nondiabetic and diabetic mice. The hyperfiltration-lowering effect of SGLT1 knockout was confirmed in STZ-induced diabetic mice despite similar hyperglycemia. Proposed GFR-lowering mechanisms of SGLT1 knockout included lower blood glucose levels, potential inhibition of tubular growth and hyperreabsorption, and inhibiting a role of SGLT1 in sensing increased glucose delivery to the MD, which otherwise increases MD-NOS1 expression and contributes to diabetic hyperfiltration, potentially to maintain urinary Na⁺ and fluid excretion and, thus, volume balance (Fig. 6). SGLT2 inhibition also increased MD-NOS1 expression in nondiabetic mice, an effect prevented in SGLT1 knockout mice. The net response in MD-NOS1 expression to SGLT2 inhibition is more complex, as indicated by studies in diabetic mice, potentially reflecting the integration of multiple factors like MD glucose delivery and volume status.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant R01-DK-112042, University of Alabama at Birmingham/University of California-San Diego O’Brien Center of Acute Kidney Injury NIH Grants P30-DK-079337 (to V. Vallon and S. Thomson), R01-DK-106102 (to V. Vallon), R01-HL-142814 (to R. Liu and V. Vallon), DK-099276, HL-142814, and HL-137987 (to R. Liu), National Natural Science Foundation of China Grant 81800649 (to P. Song), Natural Science Foundation of Hunan Province Grant 2018JJ3727 (to P. Song), Astra-Zeneca (to V. Vallon), and the Department of Veterans Affairs.

DISCLOSURES

Over the past 36 mo, V. Vallon has served as a consultant and received honoraria from Bayer, Boehringer Ingelheim, Eli Lilly, Janssen Pharmaceutical, and Merck and received grant support for investigator-initiated research from Astra-Zeneca, Bayer, Boehringer Ingelheim, Fresenius, and Janssen Pharmaceutical. Over the past 36 mo, S. Thomson received grant support for investigator-initiated research from Merck.

AUTHOR CONTRIBUTIONS

P.S., Y.C.K., H.K., and V.V. conceived and designed research; P.S., W.H., A.O., R.P., Y.C.K., C.v.G., Y.F., B.F., and V.V. performed experiments; P.S., W.H., R.P., Y.C.K., and V.V. analyzed data; P.S., Y.C.K., and V.V. interpreted results of experiments; Y.C.K. and V.V. prepared figures; V.V. drafted manuscript; P.S., W.H., A.O., R.P., Y.C.K., C.v.G., Y.F., B.F., H.K., S. T., R.L., and V.V. edited and revised manuscript; P.S., W.H., A.O., R.P., Y.C.K., C.v.G., Y.F., B.F., H.K., S. T., R.L., and V.V. approved final version of manuscript.