Early Proximal Tubule Research Articles

Gene knockout of the Na+-glucose cotransporter SGLT2 in a murine model of acute kidney injury induced by ischemia-reperfusion.

In the early proximal tubule, Na+-glucose cotransporter 2 (SGLT2) mediates the bulk of renal glucose reabsorption. Gene deletion in mice (Sglt2-/-) was used to determine the role of SGLT2 in acute kidney injury induced by bilateral ischemia-reperfusion (IR). In Sglt2-/- and littermate wild-type mice, plasma creatinine increased similarly on day 1 after IR. This was associated with an equal increase in both genotypes in the urinary kidney injury molecule-1-to-creatinine ratio, a tubular injury marker, and similarly reduced urine osmolality and increased plasma osmolality, indicating impaired urine concentration. In both IR groups, FITC-sinistrin glomerular filtration rate was equally reduced on day 14, and plasma creatinine was similarly and incompletely restored on day 23. In Sglt2-/- mice subjected to IR, fractional urinary glucose excretion was increased on day 1 but reduced and associated with normal renal Na+-glucose cotransporter 1 (Sglt1) mRNA expression on day 23, suggesting temporary SGLT1 suppression. In wild-type mice subjected to IR, renal Sglt1 mRNA was likewise normal on day 23, whereas Sglt2 mRNA was reduced by 57%. In both genotypes, IR equally reduced urine osmolality and renal mRNA expression of the Na+-K+-2Cl- cotransporter and renin on day 23, suggesting thick ascending limb dysfunction, and similarly increased renal mRNA expression of markers of injury, inflammation, oxidative stress, and fibrosis (kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, monocyte chemoattractant protein-1, transforming growth factor-β1, NADPH oxidase-2, and collagen type 1). This was associated with equal increases in kidney histological damage scores and similar degree of capillary loss in both genotypes. The data indicate that genetic deletion of SGLT2 did not protect the kidneys in the initial injury phase or the subsequent recovery phase in a mouse model of IR-induced acute kidney injury.

Osmotic diuresis by SGLT2 inhibition stimulates vasopressin-induced water reabsorption to maintain body fluid volume.

Most of the filtered glucose is reabsorbed in the early proximal tubule by the sodium‐glucose cotransporter SGLT2. The glycosuric effect of the SGLT2 inhibitor ipragliflozin is linked to a diuretic and natriuretic effect that activates compensatory increases in fluid and food intake to stabilize body fluid volume (BFV). However, the compensatory mechanisms that are activated on the level of renal tubules remain unclear. Type 2 diabetic Goto‐Kakizaki (GK) rats were treated with vehicle or 0.01% (in diet) ipragliflozin with free access to fluid and food. After 8 weeks, GK rats were placed in metabolic cages for 24‐hr. Ipragliflozin decreased body weight, serum glucose and systolic blood pressure, and increased fluid and food intake, urinary glucose and Na+ excretion, urine volume, and renal osmolar clearance, as well as urine vasopressin and solute‐free water reabsorption (TcH2O). BFV, measured by bioimpedance spectroscopy, and fluid balance were similar among the two groups. Urine vasopressin in ipragliflozin‐treated rats was negatively and positively associated with fluid balance and TcH2O, respectively. Ipragliflozin increased the renal membrane protein expression of SGLT2, aquaporin (AQP) 2 phosphorylated at Ser269 and vasopressin V2 receptor. The expression of SGLT1, GLUT2, AQP1, and AQP2 was similar between the groups. In conclusion, the SGLT2 inhibitor ipragliflozin induced a sustained glucosuria, diuresis, and natriuresis, with compensatory increases in fluid intake and vasopressin‐induced TcH2O in proportion to the reduced fluid balance to maintain BFV. These results indicate that the osmotic diuresis induced by SGLT2 inhibition stimulates compensatory fluid intake and renal water reabsorption to maintain BFV.

Mechanisms of Protective Effects of SGLT2 Inhibitors in Cardiovascular Disease and Renal Dysfunction.

Type 2 diabetes mellitus is one of the most common forms of the disease worldwide. Hyperglycemia and insulin resistance play key roles in type 2 diabetes mellitus. Renal glucose reabsorption is an essential feature in glycaemic control. Kidneys filter 160 g of glucose daily in healthy subjects under euglycaemic conditions. The expanding epidemic of diabetes leads to a prevalence of diabetes-related cardiovascular disorders, in particular, heart failure and renal dysfunction. Cellular glucose uptake is a fundamental process for homeostasis, growth, and metabolism. In humans, three families of glucose transporters have been identified, including the glucose facilitators GLUTs, the sodium-glucose cotransporter SGLTs, and the recently identified SWEETs. Structures of the major isoforms of all three families were studied. Sodium-glucose cotransporter (SGLT2) provides most of the capacity for renal glucose reabsorption in the early proximal tubule. A number of cardiovascular outcome trials in patients with type 2 diabetes have been studied with SGLT2 inhibitors reducing cardiovascular morbidity and mortality. The current review article summarises these aspects and discusses possible mechanisms with SGLT2 inhibitors in protecting heart failure and renal dysfunction in diabetic patients. Through glucosuria, SGLT2 inhibitors reduce body weight and body fat, and shift substrate utilisation from carbohydrates to lipids and, possibly, ketone bodies. These pleiotropic effects of SGLT2 inhibitors are likely to have contributed to the results of the EMPA-REG OUTCOME trial in which the SGLT2 inhibitor, empagliflozin, slowed down the progression of chronic kidney disease and reduced major adverse cardiovascular events in high-risk individuals with type 2 diabetes. This review discusses the role of SGLT2 in the physiology and pathophysiology of renal glucose reabsorption and outlines the unexpected logic of inhibiting SGLT2 in the diabetic kidney.

Effect of renal tubule-specific knockdown of the Na+/H+ exchanger NHE3 in Akita diabetic mice.

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

The Potential Role of SGLT2 Inhibitors in the Treatment of Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is a difficult disease to treat due to the relative paucity of therapeutic options other than injectable insulin. The latter, however, can induce hypoglycemia, which has been linked to enhanced cardiovascular risk. Sodium glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral anti-hyperglycemic medications that do not increase the hypoglycemia risk and are US Food and Drug Administration (FDA) approved in type 2 diabetes mellitus. SGLT2 inhibitors may also be of benefit in type 1 diabetic patients, in addition to insulin, although they have not yet been approved for this indication. By blocking SGLT2 in the early proximal tubules of the kidney, these drugs decrease renal glucose retention, which is enhanced in hyperglycemia, thereby improving blood glucose control, in type 1 and type 2 diabetic patents. Their low hypoglycemia risk is due to the compensating reabsorption capacity of another glucose transporter, SGLT1, in the downstream late proximal tubule and the body's metabolic counter-regulation, which remains intact during SGLT2 inhibition. When insulin dosage is lowered too much, SGLT2 inhibitors can enhance ketogenesis to the extent that the risk of diabetic ketoacidosis increases, particularly in type 1 diabetic patients. SGLT2 inhibitors improve the renal and cardiovascular outcome in type 2 diabetic patients. The mechanisms likely include a reduction in glomerular hyperfiltration, blood pressure, volume overload, and body weight, as well as lowering blood glucose without increasing the hypoglycemia risk. The same mechanistic effects are induced in type 1 diabetic patients. More studies are needed with SGLT2 inhibitors in type 1 diabetic patients, including renal and cardiovascular clinical outcome trials, to fully evaluate their therapeutic potential in this specific population.

Tubular NHE3 is a determinant of the acute natriuretic and chronic blood pressure lowering effect of the SGLT2 inhibitor empagliflozin

The sodium glucose cotransporter 2 (SGLT2) inhibitor, empagliflozin, is a new antihyperglycemic drug that also lowers blood pressure and has beneficial effects in patients with heart failure (EMPA‐REG OUTCOME). These effects may relate at least in part to its natriuretic effect. In the brush border of the early proximal tubule, SGLT2 may interact with the Na+‐H+‐exchanger NHE3, which reabsorbs about 30% of Na+ in the proximal convoluted tubule. Here we determine whether the acute or chronic natriuretic effect of empagliflozin depends on intact tubular NHE3.MethodsWe generated mice with NHE3 knock‐down in the tubular system (NHE3‐KO; using Pax8‐Cre) and compared them with littermate controls (WT). In a first series, we determined acute effects of empagliflozin (0.3 mg/kg, given by oral gavage together with a water load to facilitate quantitative urine collection over 3 hours) or vehicle on urinary pH and excretion of fluid, Na, and bicarbonate in metabolic cage studies (n=9–11/group). In a second series, we treated mice for 4 months with empagliflozin (150 mg/kg of diet) or vehicle and measured systolic blood pressure (SBP) by automated tail cuff in trained awake mice and renal renin mRNA expression by RT‐qPCR (n=10/group for renin mRNA and 15–26/group for SBP).ResultsSeries 1In WT, empagliflozin significantly enhanced urinary pH and the excretion of glucose, fluid, Na and bicarbonate compared with vehicle control. In NHE3‐KO, urine pH and bicarbonate excretion were significantly greater than in WT in response to vehicle; however, these two parameters were not significantly changed in response to empagliflozin. Moreover, empagliflozin actually reduced urine Na excretion in NHE3‐KO.Series 2In WT, empagliflozin lowered SBP and enhanced renal renin mRNA expression, consistent with a sustained natriuretic effect. In vehicle‐treated NHE3‐KO, SBP was lower and renal renin mRNA higher compared with WT; however, these two parameters were not significantly changed in response to empagliflozin.ConclusionThe data are consistent with the notion that the acute natriuretic and the chronic blood pressure lowering effect of the SGLT2 inhibitor empagliflozin involve a functional interaction with tubular NHE3, and can occur in the absence of hyperglycemia. The chosen dose of empagliflozin only induced a mild glucosuria, i.e. glucose reabsorption through downstream SGLT1 had just been fully saturated. Since SGLT1 transports Na and glucose in a ratio of 2:1 versus 1:1 for SGLT2, shifting glucose reabsorption from SGLT2 to downstream SGLT1 can explain an anti‐natriuretic effect of empagliflozin that has been unmasked by the absence of tubular NHE3.Support or Funding InformationNIH R01DK112042, R01DK106102, P30DK079337, the Department of Veterans Affairs, Boehringer Ingelheim Pharma GmbH & Co.KGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Absence of the Na‐Glucose Cotransporter SGLT1 Ameliorates Kidney Recovery in a Murine Model of Acute Kidney Injury

The renal Na‐glucose cotransporter SGLT1 reabsorbs ~3% of the filtered glucose in the late proximal tubule, a hypoxia‐sensitive tubular segment that enters the outer medulla. Pharmacological blockade of SGLT2, which in the early proximal tubule reabsorbs most of the filtered glucose, was found protective in a mouse model of acute kidney injury (AKI) induced by ischemia‐reperfusion (IR). Since little is known about the role of SGLT1 in this context, we determined whether lack of SGLT1 affects kidney injury or recovery following IR.SGLT1‐knockout (KO) and WT mice (C57BL/6J males) underwent IR or sham surgery. IR was induced by 20 minutes of bilateral renal artery clamping, while body temperature was maintained at 36–37□. Sham operation was similar to IR surgery without artery clamping. All groups (WT Sham, WT IR, SGLT1‐KO Sham and SGLT1‐KO IR, n=7–8/group) were fed a low‐glucose high‐protein diet to prevent diarrhea in SGLT1‐KO. Urine and blood were collected at several time points during recovery. GFR was measured 14 days after IR by plasma elimination kinetics of FITC‐sinistrin in conscious mice. Kidneys were harvested on day 16.On day 1 after IR, plasma creatinine (by LC‐MS‐MS) increased to similar levels in SGLT1‐KO and WT (0.554±0.08 vs. 0.470±0.1 mg/dL, p=0.351), indicating similar initial kidney impairment. In accordance, IR reduced urine osmolality and increased plasma osmolality to similar levels in SGLT1‐KO and WT on day 1, possibly as a consequence of impaired urine concentration or renal retention of waste products. This was associated with a greater increase in fractional glucose excretion in SGLT1‐KO (1 to 19%) vs WT (0.1 to 7%) (p=0.003) indicating a prominent role of SGLT1 in glucose reabsorption at this time point. On day 14, GFR was significantly lower in WT IR vs SGLT1‐KO IR (6.56±0.6 vs. 9.13±0.8 μL/min/g BW, p<0.05), associated with higher plasma creatinine levels on harvest day (0.130±0.016 vs. 0.093±0.009 mg/dL, p<0.05). Moreover, plasma osmolality remained elevated in WT IR but was normalized in SGLT1‐KO IR on day 16 (345±11 vs. 316±4 mosm/kg, p=0.006). On day 16, plasma urea levels in sham groups were higher in SGLT1 KO vs WT (18.0±0.3 vs. 13.2±0.9 mmol/L, p=0.028), possibly reflecting a proposed role of SGLT1 in renal urea secretion, unmasked by high protein diet. Plasma urea was strongly increased in WT IR vs sham (24.6±2.2 vs. 13.2±0.9 mmol/L, p<0.001), whereas levels were preserved in SGLT1‐KO IR vs sham. RT‐qPCR analyses on whole kidney tissue revealed that Na‐2Cl‐K cotransporter NKCC2 mRNA expression was decreased in WT IR but preserved in SGLT1‐KO IR, whereas renal mRNA expression of markers of injury, fibrosis and inflammation (KIM‐1, type 1 collagen, MCP‐1) was lower in SGLT1‐KO IR vs WT IR.Lack of SGLT1 improved kidney recovery in a mouse model of IR‐induced AKI. The results may reflect a deleterious role of SGLT1 activity in the late proximal tubule for outer medullary integrity in the aftermath of IR‐induced AKI. This may impair tubular function (reduced NKCC2) with consequences on GFR, kidney integrity, and body homeostasis, and have therapeutical implications.Support or Funding InformationNIH R01DK106102, R01DK112042, P30DK079337, and the Department of Veterans AffairsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organic anion transporter OAT3 enhances the glucosuric effect of the SGLT2 inhibitor empagliflozin

The sodium-glucose cotransporter SGLT2 inhibitor empagliflozin (plasma protein binding ~88%) may reach its target in the brush border of the early proximal tubule by glomerular filtration and tubular secretion. Here we determined whether empagliflozin is secreted by renal tubules in mice and whether genetic knockout of the basolateral organic anion transporter 3 ( Oat3-/-) affects its tubular secretion or glucosuric effect. Renal clearance studies in wild-type (WT) mice showed that tubular secretion accounted for 50-70% of empagliflozin urinary excretion. Immunostaining indicated that SGLT2 and OAT3 localization partially overlapped in proximal tubule S1 and S2 segments. Glucosuria in metabolic cage studies was reduced in Oat3-/- vs. WT mice for acute empagliflozin doses of 1, 3, and 10 mg/kg, whereas 30 mg/kg induced similar maximal glucosuria in both genotypes. Chronic application of empagliflozin (~25 mg·kg-1 ·day-1) in Oat3-/- mice was associated with lower urinary glucose-to-creatinine ratios despite maintaining slightly higher blood glucose levels than WT. On a whole kidney level, renal secretion of empagliflozin was largely unchanged in Oat3-/- mice. However, the absence of OAT3 attenuated the influence of empagliflozin on fractional glucose excretion; higher levels of plasma or filtered empagliflozin were needed to induce similar increases in fractional renal glucose excretion. We conclude that empagliflozin is excreted into the urine to similar extent by glomerular filtration and tubular secretion. The latter can occur largely independent of OAT3. However, OAT3 increases the glucosuric effect of empagliflozin, which may relate to the partial overlap of its localization with SGLT2 and thus OAT3-mediated tubular secretion of empagliflozin in the early proximal tubule.

Abstract P464: Sodium Glucose Cotransporter 2 Inhibition by Canagliflozin Attenuates Intrarenal Angiotensinogen Augmentation in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a complex disease where hyperglycemia occurs as a result of the development of insulin resistance. T2DM leads to complications, including renal and cardiovascular injury. Renal proximal tubular angiotensinogen (AGT) is stimulated by hyperglycemia via elevated oxidative stress, and is associated with activation of the intrarenal renin-angiotensin system which contributes to development of high blood pressure and diabetic nephropathy in diabetes mellitus. Sodium glucose co-transporter 2 (SGLT2) is mainly expressed in early renal proximal tubules and is responsible for most of the glucose reabsorption by the tubules. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on intrarenal AGT regulation in T2DM mice. Male New Zealand Obese mice were fed a regular fat diet (RFD, 4% fat) or a high fat diet (HFD, 40% fat) to induce diabetes. When the mice fed with HFD exhibited >350 mg/dl blood glucose levels (week 0), both RFD and HFD fed mice were treated with 10 mg/kg/day CANA or vehicle for 6 weeks by daily oral gavage. Systolic blood pressure (SBP) levels were measured by a tail cuff system and 24-hour urine samples were collected using metabolic cages. Intrarenal AGT mRNA and protein levels were determined by digital PCR and western blot analysis. CANA treatment decreased blood glucose levels in HFD mice, which remained suppressed for duration of the study. SBP in HFD groups were higher than in the RFD and RFD+CANA groups (134.7±3.6, 118.7±10.8 and 108.3±7.6 mmHg) at week 6. The elevated SBP was normalized by CANA (110.0±6.0 mmHg). The HFD group exhibited greater renal cortical AGT mRNA levels than the RFD group (3.7±0.4 vs. 7.4±1.0 copies/reaction). Likewise, intrarenal AGT mRNA expression was lower in CANA treated group (4.5±0.8 copies/reaction). Western blot analysis also showed lower AGT protein levels in CANA treated group (1.02±0.08-fold compared with RFD) than in HFD group (1.49±0.08-fold). In the HFD group, CANA treatment also suppressed elevated urinary 8-isoprostane levels, a marker of renal oxidative stress. These results demonstrate that CANA attenuates intrarenal AGT augmentation that occurs in T2DM, which may mitigate the development of diabetic nephropathy and progression of high blood pressure.

Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging.

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman’s capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

Temporo-spacial microanatomical distribution of the murine sodium-dependent ascorbic acid transporters Slc23a1 and Slc23a2 in the kidney throughout development.

The two membrane transporters Slc23a1 and Slc23a2 mediate ascorbic acid uptake into cells. We recently determined the key role of Slc23a1 in renal re-absorption of ascorbic acid in a knockout mouse model. However, the renal spatial and temporal expression patterns of murine Slc23a1 and Slc23a2 are not defined. This study utilizes database evidence combined with experimental confirmation via in-situ hybridization to define the spatial and temporal expression of Slc23a1 in the murine kidney. Slc23a1 is expressed in the early proximal tubule, but not in its precursors during embryonic development, and exclusive proximal tubular expression persists throughout the animal's lifetime. In contrast, Slc23a2 is uniformly expressed in metabolic cell types such as stromal cells. The expression patterns appear to be conserved from rodent lineages to humans.

Targeting renal glucose reabsorption to treat hyperglycaemia: the pleiotropic effects of SGLT2 inhibition.

Healthy kidneys filter ∼160g/day of glucose (∼30% of daily energy intake) under euglycaemic conditions. To prevent valuable energy from being lost in the urine, the proximal tubule avidly reabsorbs filtered glucose up to a limit of ∼450g/day. When blood glucose levels increase to the point that the filtered load exceeds this limit, the surplus is excreted in the urine. Thus, the kidney provides a safety valve that can prevent extreme hyperglycaemia as long as glomerular filtration is maintained. Most of the capacity for renal glucose reabsorption is provided by sodium glucose cotransporter (SGLT) 2 in the early proximal tubule. In the absence or with inhibition of SGLT2, the renal reabsorptive capacity for glucose declines to ∼80g/day (the residual capacity of SGLT1), i.e. the safety valve opens at a lower threshold, which makes it relevant to glucose homeostasis from day-to-day. Several SGLT2 inhibitors are now approved glucose lowering agents for individuals with type 2 diabetes and preserved kidney function. By inducing glucosuria, these drugs improve glycaemic control in all stages of type 2 diabetes, while their risk of causing hypoglycaemia is low because they naturally stop working when the filtered glucose load falls below ∼80g/day and they do not otherwise interfere with metabolic counterregulation. Through glucosuria, SGLT2 inhibitors reduce body weight and body fat, and shift substrate utilisation from carbohydrates to lipids and, possibly, ketone bodies. Because SGLT2 reabsorbs sodium along with glucose, SGLT2 blockers are natriuretic and antihypertensive. Also, because they work in the proximal tubule, SGLT2 inhibitors increase delivery of fluid and electrolytes to the macula densa, thereby activating tubuloglomerular feedback and increasing tubular back pressure. This mitigates glomerular hyperfiltration, reduces the kidney's demand for oxygen and lessens albuminuria. For reasons that are less well understood, SGLT2 inhibitors are also uricosuric. These pleiotropic effects of SGLT2 inhibitors are likely to have contributed to the results of the EMPA-REG OUTCOME trial in which the SGLT2 inhibitor, empagliflozin, slowed the progression of chronic kidney disease and reduced major adverse cardiovascular events in high-risk individuals with type 2 diabetes. This review discusses the role of SGLT2 in the physiology and pathophysiology of renal glucose reabsorption and outlines the unexpected logic of inhibiting SGLT2 in the diabetic kidney.

Cardio-renal protection with empagliflozin

Cardiovascular (CV) and kidney disease are common and significant complications in people with type 2 diabetes (T2DM). CV disease is the leading cause of death, morbidly and hospitalisations for people with T2DM. Furthermore, diabetic kidney disease is a major risk factor for CV disease and is the main reason why patients need renal replacement therapy. In this perspective, we highlight the results of the recent landmark EMPA-REG OUTCOME trial which has shown that empagliflozin, a member of the sodium-glucose co-transporter 2 (SGLT-2) inhibitor class of glucose lowering medications, reduces death from CV causes, hospitalisation for heart failure and progression to end stage kidney disease in patients with T2DM and established CV disease. The SGLT2 receptor mediates high-capacity glucose uptake in the early proximal tubule, and SGLT2 inhibitors, via their ability to promote glycosuria, have been developed as glucose lowering medications. As well as having a glucose lowering effect, SGLT-2 inhibitors also reduce blood pressure, promote weight loss and reduce uric acid levels. Potential side-effects or concerns related to the use of SGLT-2 inhibitors include increased rates of urinary tract infections, genital tract infections, postural hypotension, diabetic ketoacidosis, acute kidney injury and possible increased rates of fractures. The exact mechanisms that result in empagliflozin's dramatic CV and renal protective effects, with a very favourable safety/tolerability profile, in the EMPA-REG study remain to be fully defined. However, they are most likely distinct from the glucose lowering effects of empagliflozin. CV safety trials involving dapagliflozin and canagliflozin, members of the SGLT-2 class, are under way and the results from these studies will help to answer the question as to whether the cardio-renal benefits of empagliflozin are a class-effect or not. Without doubt, trials to investigate whether SGLT-2 inhibitors have cardio-renal protective effects in patients without diabetes will start soon.

Dapagliflozin Binds Specifically to Sodium-Glucose Cotransporter 2 in the Proximal Renal Tubule.

Kidneys contribute to glucose homeostasis by reabsorbing filtered glucose in the proximal tubules via sodium-glucose cotransporters (SGLTs). Reabsorption is primarily handled by SGLT2, and SGLT2-specific inhibitors, including dapagliflozin, canagliflozin, and empagliflozin, increase glucose excretion and lower blood glucose levels. To resolve unanswered questions about these inhibitors, we developed a novel approach to map the distribution of functional SGLT2 proteins in rodents using positron emission tomography with 4-[18F]fluoro-dapagliflozin (F-Dapa). We detected prominent binding of intravenously injected F-Dapa in the kidney cortexes of rats and wild-type and Sglt1-knockout mice but not Sglt2-knockout mice, and injection of SGLT2 inhibitors prevented this binding. Furthermore, imaging revealed only low levels of F-Dapa in the urinary bladder, even after displacement of kidney binding with dapagliflozin. Microscopic ex vitro autoradiography of kidney showed F-Dapa binding to the apical surface of early proximal tubules. Notably, in vivo imaging did not show measureable specific binding of F-Dapa in heart, muscle, salivary glands, liver, or brain. We propose that F-Dapa is freely filtered by the kidney, binds to SGLT2 in the apical membranes of the early proximal tubule, and is subsequently reabsorbed into blood. The high density of functional SGLT2 transporters detected in the apical membrane of the proximal tubule but not detected in other organs likely accounts for the high kidney specificity of SGLT2 inhibitors. Overall, these data are consistent with data from clinical studies on SGLT2 inhibitors and provide a rationale for the mode of action of these drugs.

SY 10-1 RENAL GLUCOSE HANDLING AND SGLT2

The kidneys maintain glucose homeostasis through its utilization, gluconeogenesis, and reabsorption. Glucose is freely filtered and reabsorbed in order to retain energy essential between meals. The amount of glucose reabsorbed by the kidneys is equivalent to the amount entering the filtration system. With a daily glomerular filtration rate of 180 L, approximately 180 g (180 L/day × 100 mg/dL) of glucose must be reabsorbed each day to maintain an average fasting plasma glucose concentration of 5.6 mmol/L (100 mg/dL). The reabsorption increases with increase in plasma glucose concentration up to approximately 11 mmol/L (198 mg/dL). At this threshold level, the system becomes saturated and the maximal resabsorption rate-the glucose transport maximum (Tm G ) is reached. No more glucose can be absorbed, and the kidneys begin excreting it in the urine-the beginning of glycosuria. Reabsorption of glucose occurs mainly in the proximal tubule and is mediated by 2 different transport proteins, Sodium Glucose Cotransporter (SGLT)1 and SGLT2. SGLT1, which are found in the straight section of the proximal tubule (S3), are responsible for approximately 10% of glucose reabsorption. The other 90% of filtered glucose is reabsorbed through by SGLT2, which are located in the convoluted section on the proximal tubule (S1). The SGLT2 are located on the luminal side of the early proximal tubule S1 segment. Absorption of sodium across the cell membrane creates an energy gradient that in turn allows glucose to be absorbed. On the other side of the cell, sodium is reabsorbed through sodium-potassium ATPase pump into the bloodstream. The concentration gradient within the cell, resulting from this exchange drives glucose reabsorption into the bloodstream via the Glucose transporter (GLUT) 2. The role of kidneys in glucose regulation has been well recognized in the recent years, and inhibition of glucose reabsorption by SGLT2 inhibitors has evolved as a promising target for therapeutic intervention in diabetes mellitus and an added benefit in hypertension.

Predicted consequences of diabetes and SGLT inhibition on transport and oxygen consumption along a rat nephron.

Diabetes increases the reabsorption of Na(+) (TNa) and glucose via the sodium-glucose cotransporter SGLT2 in the early proximal tubule (S1-S2 segments) of the renal cortex. SGLT2 inhibitors enhance glucose excretion and lower hyperglycemia in diabetes. We aimed to investigate how diabetes and SGLT2 inhibition affect TNa and sodium transport-dependent oxygen consumption [Formula: see text] along the whole nephron. To do so, we developed a mathematical model of water and solute transport from the Bowman space to the papillary tip of a superficial nephron of the rat kidney. Model simulations indicate that, in the nondiabetic kidney, acute and chronic SGLT2 inhibition enhances active TNa in all nephron segments, thereby raising [Formula: see text] by 5-12% in the cortex and medulla. Diabetes increases overall TNa and [Formula: see text] by ∼50 and 100%, mainly because it enhances glomerular filtration rate (GFR) and transport load. In diabetes, acute and chronic SGLT2 inhibition lowers [Formula: see text] in the cortex by ∼30%, due to GFR reduction that lowers proximal tubule active TNa, but raises [Formula: see text] in the medulla by ∼7%. In the medulla specifically, chronic SGLT2 inhibition is predicted to increase [Formula: see text] by 26% in late proximal tubules (S3 segments), by 2% in medullary thick ascending limbs (mTAL), and by 9 and 21% in outer and inner medullary collecting ducts (OMCD and IMCD), respectively. Additional blockade of SGLT1 in S3 segments enhances glucose excretion, reduces [Formula: see text] by 33% in S3 segments, and raises [Formula: see text] by <1% in mTAL, OMCD, and IMCD. In summary, the model predicts that SGLT2 blockade in diabetes lowers cortical [Formula: see text] and raises medullary [Formula: see text], particularly in S3 segments.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Sodium glucose cotransporter 2 inhibition in the diabetic kidney: an update.

The sodium glucose cotransporter 2 (SGLT2) reabsorbs most of the glucose filtered by the kidneys. SGLT2 inhibitors reduce glucose reabsorption, thereby lowering blood glucose levels, and have been approved as new antihyperglycemic drugs. Although the therapeutic strategy is very promising, many questions remain. Using validated antibodies, SGLT2 expression was localized to the brush border of the early proximal tubule in the human kidney and was found upregulated in genetic murine models of type 1 and 2 diabetes. SGLT2 may functionally interact with the Na/H exchanger NHE3 in the proximal tubule. SGLT1-mediated reabsorption explains the fractional renal glucose reabsorption of 40-50% during SGLT2 inhibition. SGLT2 is expressed on pancreatic alfa cells where its inhibition induces glucagon secretion. SGLT2 inhibition lowers glomerular filtration rate in hyperfiltering diabetic patients consistent with the tubular hypothesis of diabetic hyperfiltration. New data indicate a potential of SGLT2 inhibition for renal medullary hypoxia and ketoacidosis, but also for blood glucose effect-dependent and independent nephroprotective actions, renal gluconeogenesis inhibition, reduction in cardiovascular mortality, and cancer therapy. The findings expand and refine our understanding of SGLT2 and its inhibition, have relevance for clinical practice, and will help interpret ongoing clinical trials on the long-term safety and cardiovascular effects of SGLT2 inhibitors.

A comprehensive review of the pharmacodynamics of the SGLT2 inhibitor empagliflozin in animals and humans.

Empagliflozin (formerly known as BI 10773) is a potent, competitive, and selective inhibitor of the sodium glucose transporter SGLT2, which mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. Accordingly, empagliflozin treatment increased urinary glucose excretion. This has been observed across multiple species including humans and was reported under euglycemic conditions, in obesity and, most importantly, in type 2 diabetic patients and multiple animal models of type 2 diabetes and of type 1 diabetes. This led to a reduction in blood glucose, smaller blood glucose excursions during oral glucose tolerance tests, and, upon chronic treatment, a reduction in HbA1c in animal models and patients. In rodents, such effects were observed in early and late phases of experimental diabetes and were associated with preservation of pancreatic β-cell function. Combination studies in animals demonstrated that beneficial metabolic effects of empagliflozin may also manifest when added to other types of anti-hyperglycemic treatments including linagliptin and pioglitazone. While some anti-hyperglycemic drugs lead to weight gain, empagliflozin treatment was associated with reduced body weight in normoglycemic obese and non-obese animals despite an increased food intake, largely due to a loss of adipose tissue; on the other hand, empagliflozin preserved body weight in models of type 1 diabetes. Empagliflozin improved endothelial dysfunction in diabetic rats and arterial stiffness, reduced blood pressure in diabetic patients, and attenuated early signs of nephropathy in diabetic animal models. Taken together, the SGLT2 inhibitor empagliflozin improves glucose metabolism by enhancing urinary glucose excretion; upon chronic administration, at least in animal models, the reductions in blood glucose levels are associated with beneficial effects on cardiovascular and renal complications of diabetes.

Development of a manipulative for nephron physiology education.

some physiological concepts, such physiology of filtration and absorption in the different nephron segments, are so detailed that they can be a challenge to be memorized. This article describes an exercise that solidifies learning as students manipulate, using paper models, “transporters” and