Renal Glucose Reabsorption in Response to Dapagliflozin
Renal Glucose Reabsorption in Response to Dapagliflozin
In the current study, we examined for the first time in humans, to our knowledge, the effect of SGLT2 inhibition on the key parameters (TmG, threshold, and splay) of renal glucose handling. In individuals with established type 2 diabetes, multiple pathophysiologic abnormalities have been shown to contribute to the disturbance in glucose homeostasis. Although it long has been known that the TmG is increased in both type 1 and type 2 diabetes, until recently, this alteration in renal glucose handling has received little attention. With the development of SGLT2 inhibitors as a potential therapeutic modality for type 2 diabetes, there has been renewed interest in the mechanisms responsible for enhanced renal glucose reabsorption. In the current study, we used the SHC with octreotide and basal hormone replacement (pancreatic clamp) to progressively increase plasma glucose concentration throughout the physiologic and pathophysiologic range while clamping plasma insulin, glucagon, and growth hormone concentrations at basal levels. This study design allowed us to quantify TmG, splay in the renal glucose reabsorptive curve, and threshold at which glucose first appears in the urine before and after treatment with an SGLT2 inhibitor.
As previously demonstrated in diabetic humans and animals, the current results, obtained through the use of a state-of-the-art methodology, confirm that TmG is increased in type 2 diabetic versus healthy individuals (420 vs. 317 mg/min, P < 0.04) (Fig. 2) and extend the results of previous studies by excluding hyperinsulinemia as a confounding factor in measuring the TmG. This finding has important clinical implications, demonstrating that the kidney plays an active role in the pathogenesis of type 2 diabetes by excessively reabsorbing glucose and contributing to the maintenance of hyperglycemia rather than to the excretion of the excess filtered glucose load. Because 80–90% of the filtered glucose load is reabsorbed by the SGLT2 transporter, it is reasonable to assume that upregulation of this protein is responsible for the increased TmG in diabetic patients in the current study and is consistent with studies in cultured renal tubular cells in humans and in vivo and in vitro studies in animals. However, in a study by Rahmoune et al., the cell type used in culture experiments was not definitively identified. Farber et al. demonstrated that treatment with insulin resulted in a decrease in the TmG in type 2 diabetic patients despite a constant infusion of glucose, suggesting that hyperglycemia, not hyperinsulinemia, is responsible for the increased TmG in type 2 diabetes. In the current study, the mean HbA1c in subjects with type 2 diabetes was 6.5%, and the mean FPG level was 6.0 mmol/L (108 mg/dL), indicating good glycemic control. Thus, if hyperglycemia is responsible for the increase in TmG, only a modest deterioration in glycemic control is required to increase the TmG.
The current study is the first in our knowledge to measure the splay in the renal glucose titration curve in human type 2 diabetes before and after SGLT2 inhibition. Although the splay area was increased in diabetic versus healthy subjects (Supplementary Fig. 1), the plasma glucose concentration at which glucose first appeared in the urine was not markedly different between them (10.9 vs. 9.5 mmol/L [196 vs. 171 mg/dL], respectively). After 7 days of dapagliflozin treatment, the geometric mean TmG was significantly reduced in both diabetic (420–176 mg/min) and healthy (317–150 mg/min) subjects. Of note, the percent reduction in TmG was identical (~55%) in both groups, and the TmG after dapagliflozin treatment in diabetic subjects was lower than that in healthy subjects before dapagliflozin treatment. Dapagliflozin decreased the splay in the renal glucose titration curve in both groups. Therefore, increased splay cannot explain the glucosuric effect of the drug (Supplementary Fig. 1).
The current results demonstrate that the dramatic effect of dapagliflozin to induce glucose excretion is explained by the drug's action to reduce the threshold at which renal glucose excretion first begins. Thus, the mean threshold was reduced in both type 2 diabetic and healthy subjects to 1.2 mmol/L (21 mg/dL) and 2.1 mmol/L (37 mg/dL), respectively. This finding can best be appreciated by examining the effect of dapagliflozin in the control group. At an FPG concentration of only 5.6 mmol/L (100 mg/dL), healthy subjects excreted 20% of the filtered glucose load (Fig. 3). This observation cannot be explained by the effect of dapagliflozin to reduce the TmG. Because the SHC started at an FPG target of 5.6 mmol/L, the precise threshold at which glucose excretion begins can only be estimated. A more precise determination of the threshold would require reduction of plasma glucose concentration <2.8 mmol/L (50 mg/dL) before starting the SHC, and this is not feasible. Sha et al. indirectly examined the effect of canagliflozin on the threshold by measuring simultaneously 24-h urine glucose excretion and plasma glucose concentration at frequent intervals throughout the day. With a modeling approach, the authors concluded that SGLT2 inhibition reduced the renal threshold, which is consistent with the present results. It should be noted that the approach Sha et al. used to measure the renal threshold for glucose is indirect and never has been validated by direct measurement using the gold standard renal glucose titration curve in the same subjects. Furthermore, this approach does not allow one to examine either the splay or the TmG at which glucose excretion begins.
To our knowledge, no previous study in humans has examined the effect of SGLT2 inhibition on the TmG. The current results demonstrate that dapagliflozin reduces the TmG in both diabetic and healthy subjects. The reduction in TmG would be expected to contribute to the decrease in postprandial glucose excursion after meal ingestion.
It is notable that the increase in 24-h urine glucose excretion (60–80 g/day) observed with all orally administered SGLT2 inhibitors currently in clinical trials represents only 40–50% of filtered glucose load. This relationship between plasma glucose concentration and the percent of glucose reabsorption that is inhibited has important implications for understanding normal renal physiology with respect to glucose handling. Because studies on renal glucose reabsorption largely have included patients with extremely elevated plasma glucose values, the percent of glucose reabsorption mediated by SGLT2 reported in the literature may overestimate the relative contribution at normal plasma glucose levels. It is possible that in humans, transporters other than SGLT2 may be capable of a much greater fraction of glucose reabsorption at lower filtered glucose loads but are saturated as tubular glucose concentrations increase, as has been demonstrated in SGLT2 knockout mice. It is also possible that human SGLT2 works differently from rodent SGLT2 and that the increase in plasma and urine glucose concentrations associated with the marked increase in plasma glucose levels (i.e., in excess of 13.9 mmol/L [250 mg/dL]) alters the transport characteristics of the SGLT2 transporter, rendering it more susceptible to inhibition with dapagliflozin. Finally, it is possible that after inhibition of the SGLT2 transporter, other renal tubular transporters (i.e., SGLT1) augment their reabsorption of glucose.
There are several limitations of the current study. First, the number of subjects in each group (n = 12) was relatively small. Second, we did not observe any significant difference in the response of any renal parameter after dapagliflozin treatment between male and female subjects. However, the number of subjects most likely was too small to detect such a difference, even if it were present. Third, the control group was slightly, although not significantly, younger. We doubt that this small, insignificant difference in age could have affected the results because the effect of dapagliflozin on TmG, threshold, and splay were similar in both groups. Fourth, it is not possible to determine whether the renal response to dapagliflozin would be different in poorly controlled diabetic patients because all individuals in the current study were reasonably well controlled. Finally, the duration of therapy was short (7 days), and with a longer treatment duration, upregulation of other transporters in the kidney could alter the quantitative findings demonstrated in the present study.
In summary, the current study examined for the first time in humans the effect of dapagliflozin on the major determinants of glucose handling (TmG, splay, and threshold) in patients with type 2 diabetes. Both the TmG and the splay in the renal glucose reabsorption curve are increased in diabetic versus healthy control subjects. Dapagliflozin induced marked glucosuria, reduced TmG, and decreased splay in subjects with and without type 2 diabetes. Most notably, dapagliflozin dramatically reduced the threshold at which glucose excretion begins to plasma glucose concentrations well below fasting levels (4.7–6.0 mmol/L [85–108 mg/dL]) in subjects with and without type 2 diabetes. From a quantitative standpoint, the reduced threshold represents the major mechanism through which SGLT2 inhibitors increase glucose excretion in humans.
Conclusions
In the current study, we examined for the first time in humans, to our knowledge, the effect of SGLT2 inhibition on the key parameters (TmG, threshold, and splay) of renal glucose handling. In individuals with established type 2 diabetes, multiple pathophysiologic abnormalities have been shown to contribute to the disturbance in glucose homeostasis. Although it long has been known that the TmG is increased in both type 1 and type 2 diabetes, until recently, this alteration in renal glucose handling has received little attention. With the development of SGLT2 inhibitors as a potential therapeutic modality for type 2 diabetes, there has been renewed interest in the mechanisms responsible for enhanced renal glucose reabsorption. In the current study, we used the SHC with octreotide and basal hormone replacement (pancreatic clamp) to progressively increase plasma glucose concentration throughout the physiologic and pathophysiologic range while clamping plasma insulin, glucagon, and growth hormone concentrations at basal levels. This study design allowed us to quantify TmG, splay in the renal glucose reabsorptive curve, and threshold at which glucose first appears in the urine before and after treatment with an SGLT2 inhibitor.
As previously demonstrated in diabetic humans and animals, the current results, obtained through the use of a state-of-the-art methodology, confirm that TmG is increased in type 2 diabetic versus healthy individuals (420 vs. 317 mg/min, P < 0.04) (Fig. 2) and extend the results of previous studies by excluding hyperinsulinemia as a confounding factor in measuring the TmG. This finding has important clinical implications, demonstrating that the kidney plays an active role in the pathogenesis of type 2 diabetes by excessively reabsorbing glucose and contributing to the maintenance of hyperglycemia rather than to the excretion of the excess filtered glucose load. Because 80–90% of the filtered glucose load is reabsorbed by the SGLT2 transporter, it is reasonable to assume that upregulation of this protein is responsible for the increased TmG in diabetic patients in the current study and is consistent with studies in cultured renal tubular cells in humans and in vivo and in vitro studies in animals. However, in a study by Rahmoune et al., the cell type used in culture experiments was not definitively identified. Farber et al. demonstrated that treatment with insulin resulted in a decrease in the TmG in type 2 diabetic patients despite a constant infusion of glucose, suggesting that hyperglycemia, not hyperinsulinemia, is responsible for the increased TmG in type 2 diabetes. In the current study, the mean HbA1c in subjects with type 2 diabetes was 6.5%, and the mean FPG level was 6.0 mmol/L (108 mg/dL), indicating good glycemic control. Thus, if hyperglycemia is responsible for the increase in TmG, only a modest deterioration in glycemic control is required to increase the TmG.
The current study is the first in our knowledge to measure the splay in the renal glucose titration curve in human type 2 diabetes before and after SGLT2 inhibition. Although the splay area was increased in diabetic versus healthy subjects (Supplementary Fig. 1), the plasma glucose concentration at which glucose first appeared in the urine was not markedly different between them (10.9 vs. 9.5 mmol/L [196 vs. 171 mg/dL], respectively). After 7 days of dapagliflozin treatment, the geometric mean TmG was significantly reduced in both diabetic (420–176 mg/min) and healthy (317–150 mg/min) subjects. Of note, the percent reduction in TmG was identical (~55%) in both groups, and the TmG after dapagliflozin treatment in diabetic subjects was lower than that in healthy subjects before dapagliflozin treatment. Dapagliflozin decreased the splay in the renal glucose titration curve in both groups. Therefore, increased splay cannot explain the glucosuric effect of the drug (Supplementary Fig. 1).
The current results demonstrate that the dramatic effect of dapagliflozin to induce glucose excretion is explained by the drug's action to reduce the threshold at which renal glucose excretion first begins. Thus, the mean threshold was reduced in both type 2 diabetic and healthy subjects to 1.2 mmol/L (21 mg/dL) and 2.1 mmol/L (37 mg/dL), respectively. This finding can best be appreciated by examining the effect of dapagliflozin in the control group. At an FPG concentration of only 5.6 mmol/L (100 mg/dL), healthy subjects excreted 20% of the filtered glucose load (Fig. 3). This observation cannot be explained by the effect of dapagliflozin to reduce the TmG. Because the SHC started at an FPG target of 5.6 mmol/L, the precise threshold at which glucose excretion begins can only be estimated. A more precise determination of the threshold would require reduction of plasma glucose concentration <2.8 mmol/L (50 mg/dL) before starting the SHC, and this is not feasible. Sha et al. indirectly examined the effect of canagliflozin on the threshold by measuring simultaneously 24-h urine glucose excretion and plasma glucose concentration at frequent intervals throughout the day. With a modeling approach, the authors concluded that SGLT2 inhibition reduced the renal threshold, which is consistent with the present results. It should be noted that the approach Sha et al. used to measure the renal threshold for glucose is indirect and never has been validated by direct measurement using the gold standard renal glucose titration curve in the same subjects. Furthermore, this approach does not allow one to examine either the splay or the TmG at which glucose excretion begins.
To our knowledge, no previous study in humans has examined the effect of SGLT2 inhibition on the TmG. The current results demonstrate that dapagliflozin reduces the TmG in both diabetic and healthy subjects. The reduction in TmG would be expected to contribute to the decrease in postprandial glucose excursion after meal ingestion.
It is notable that the increase in 24-h urine glucose excretion (60–80 g/day) observed with all orally administered SGLT2 inhibitors currently in clinical trials represents only 40–50% of filtered glucose load. This relationship between plasma glucose concentration and the percent of glucose reabsorption that is inhibited has important implications for understanding normal renal physiology with respect to glucose handling. Because studies on renal glucose reabsorption largely have included patients with extremely elevated plasma glucose values, the percent of glucose reabsorption mediated by SGLT2 reported in the literature may overestimate the relative contribution at normal plasma glucose levels. It is possible that in humans, transporters other than SGLT2 may be capable of a much greater fraction of glucose reabsorption at lower filtered glucose loads but are saturated as tubular glucose concentrations increase, as has been demonstrated in SGLT2 knockout mice. It is also possible that human SGLT2 works differently from rodent SGLT2 and that the increase in plasma and urine glucose concentrations associated with the marked increase in plasma glucose levels (i.e., in excess of 13.9 mmol/L [250 mg/dL]) alters the transport characteristics of the SGLT2 transporter, rendering it more susceptible to inhibition with dapagliflozin. Finally, it is possible that after inhibition of the SGLT2 transporter, other renal tubular transporters (i.e., SGLT1) augment their reabsorption of glucose.
There are several limitations of the current study. First, the number of subjects in each group (n = 12) was relatively small. Second, we did not observe any significant difference in the response of any renal parameter after dapagliflozin treatment between male and female subjects. However, the number of subjects most likely was too small to detect such a difference, even if it were present. Third, the control group was slightly, although not significantly, younger. We doubt that this small, insignificant difference in age could have affected the results because the effect of dapagliflozin on TmG, threshold, and splay were similar in both groups. Fourth, it is not possible to determine whether the renal response to dapagliflozin would be different in poorly controlled diabetic patients because all individuals in the current study were reasonably well controlled. Finally, the duration of therapy was short (7 days), and with a longer treatment duration, upregulation of other transporters in the kidney could alter the quantitative findings demonstrated in the present study.
In summary, the current study examined for the first time in humans the effect of dapagliflozin on the major determinants of glucose handling (TmG, splay, and threshold) in patients with type 2 diabetes. Both the TmG and the splay in the renal glucose reabsorption curve are increased in diabetic versus healthy control subjects. Dapagliflozin induced marked glucosuria, reduced TmG, and decreased splay in subjects with and without type 2 diabetes. Most notably, dapagliflozin dramatically reduced the threshold at which glucose excretion begins to plasma glucose concentrations well below fasting levels (4.7–6.0 mmol/L [85–108 mg/dL]) in subjects with and without type 2 diabetes. From a quantitative standpoint, the reduced threshold represents the major mechanism through which SGLT2 inhibitors increase glucose excretion in humans.
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