abstract: # Background
  .
  An imbalance between pro-oxidants and antioxidant systems has been suggested to be implicated in the physiopathology of acute myocardial infarction (AMI). We aimed to evaluate the antioxidant capacity in Tunisian patients and to assess the possible relationship between erythrocyte catalase enzyme activity and hyperhomocysteinaemia.
  .
  # Methods
  .
  108 patients with AMI and 81 healthy subjects were enrolled in this study. Catalase erythrocyte enzyme activity was determined spectrophotometrically whereas "total antioxidant status" (TAS) concentration was measured by a commercially available method. Serum total homocysteine (tHcy) level was determined by a fluorescence polarization immunoassay (FPIA). Lipid peroxidation was measured with a fluorimetric method as "thiobarbituric acid reactive substances" (TBARS).
  .
  # Results
  .
  Compared with healthy subjects, patients with AMI had significantly lower catalase activity (*P\<0.001*), TAS concentrations (*P\<0.001*), and significantly higher serum tHcy (*P\<0.001*) and TBARS levels (*P\<0.001*). Erythrocyte catalase enzyme activity was negatively correlated with serum tHcy and TBARS while serum tHcy and TBARS were in positive correlation. Furthermore, the unbalance between pro-oxidants and antioxidants seems to be more aggravated in patients with Q wave AMI compared to patients with non-Q wave AMI.
  .
  # Conclusion
  .
  Our results suggest the involvement of hyperhomocysteinaemia in the drop of erythrocyte catalase activity related to myocardial ischemia reperfusion. Hyperhomocysteinaemia may increase the myocardial wall dysfunction under ischemia reperfusion by excessive production of reactive oxygen species which is made evident by increased lipid peroxidation.
  .
  # Virtual slides
  .
  The virtual slide(s) for this article can be found here: <http://www.diagnosticpathology.diagnomx.eu/vs/1623509866881834>
author: Yosri Noichri; Abdelkader Chalghoum; Latifa Chkioua; Bruno Baudin; Samia Ernez; Salima Ferchichi; Abdelhédi Miled
date: 2013
institute: 1Biochemistry Laboratory CHU Farhat HACHED, Street Doctor Moreau, 4000 Sousse, Tunisia; 2Department of Biochemistry, Hospital Saint-Antoine, 184 Street Faubourg Saint-Antoine, 75571 Paris Cedex 12, France; 3Department of Cardiology, CHU Farhat Hached, Street Doctor Moreau, 4000 Sousse, Tunisia
references:
title: Low erythrocyte catalase enzyme activity is correlated with high serum total homocysteine levels in tunisian patients with acute myocardial infarction

# Background

Myocardial infarction is a major cause of morbidity and mortality worldwide. Recently, an increase in the incidence of coronary heart disease (CHD) has been recorded in Tunisian cardiovascular disease register. Among diabetes, hypertension, abdominal obesity and smoking, a family lipoprotein disorder such as a high level of serum apolipoprotein B (Apo B) or/and a lower level of serum apolipoprotein A-1 accounted for almost all the population attributable risk of AMI \[1,2\]. Similarly, hyperhomocysteinaemia related to nutritional or genetic factors, such as methylenetetrahydrofolate reductase, endothelial nitric oxide synthase genes or with low paraoxonase activity led to increased risk of CHD severity. It can induce sustained injury of arterial endothelial cells and proliferation of arterial smooth muscle cells \[3\].

Atherosclerotic plaques are the major cause of Myocardial infarction. Other unusual causes of MI can be related to myocardial necrosis, or Thiamine deficiency causing alterations in heart metabolism \[4,5\]. Coronary artery occlusion can result in a reduction in myocardial blood flow \[6\]. In fact, myocardial ischemia occurs when myocardial oxygen demand exceeds the oxygen supply. Reperfusion of the ischemic myocardium can restore, afterwards, the blood flow but sudden massive increase in oxygen supply can lead to additional myocardial cell dysfunction and cell necrosis. Excessive production of Reactive Oxygen Species (ROS) has been most importantly proposed to mediate ischemia reperfusion injury. These species are toxic and may cause potential biological damage to all cellular components \[7\]. This occurs when there is an overproduction of ROS on one side and a deficit or inadequate availability of antioxidant systems on the other \[6\]. ROS generation may induce irreversible myocardial cell necrosis or apoptosis through triggering DNA fragmentation and caspase activation and causing myocardial injury \[8,9\]. Exposure to ROS from a variety of sources led to develop a series of defense mechanisms to neutralize these species and so protect cells against their toxic effects. This is achieved mainly by enzymatic antioxidants such as catalase, or by non-enzymatic antioxidants such as vitamins. Catalase is believed to play a major role in the first line of enzymatic antioxidant defense. It is a tetrameric enzyme, consisting of four identical subunits that contain a single ferriprotoporphoryn group per subunit. Catalase reacts efficiently in peroxisomes with hydrogen peroxide (H202) to form water and molecular oxygen \[10,11\]. Hyperhomocysteinaemia and other cardiovascular risk factors have been suggested to be implicated in the imbalance aggravation between pro-oxidants and antioxidants linked to its pro-oxidant properties or in the impairment of antioxidant systems. The aim of our study is to evaluate the total antioxidant capacity and erythrocyte catalase activity in patients hospitalized for AMI. We aimed to access the possible relationships between erythrocyte catalase activity, hyperhomocysteinaemia and the severity of myocardial dysfunction under ischemia reperfusion.

# Materials and methods

## Study population

The study consisted of 108 patients with a mean age of 63 *±* 12 years admitted to the department of cardiology in Farhad Hached hospital in Tunisia. The diagnostic of AMI was established according to universal clinical criteria: chest pain which lasted for up 3 hours, ECG changes and serum troponin elevation. The control group consisted of 81 healthy volunteers with no history of coronary artery disease, diabetes, hypertension or inflammatory disease. Their mean of age was 59 *±* 9 years.

The electrocardiographic changes were recorded and assessed by 2 cardiologists who were unaware that the patients are included in our study.

Coronary risk factors have been referred in our study population according to universal definitions. Dyslipidemia was defined when total cholesterol concentration was ≥ 5,68 mmol/l, or triglyceride concentration ≥ 2,28 mmol/l. Hyperhomocysteinaemia was defined by a raised blood total homocysteine, exceeding 15 μmol/l.

Informed written consent was obtained from each patient and healthy subject according to the guidelines of our ethics' committee.

## Blood collection and biochemical methods

Venous blood was collected after overnight fasting and within 24 hours after admission for chest pain. Serum was separated by centrifugation at 1500 g for 10 min, and then stored at −80°C until the day of analysis. Serum was used for the estimation of lipid peroxidation, total homocysteine (tHcy), apolipoproteins (Apo A-1 and Apo B) levels and total antioxidant status (TAS) concentration.

Total homocysteine in serum was measured by the "Abbott homocysteine assay", a fully automated fluorescence polarization immunoassay (FPIA) from Abbott diagnostics. Serum apolipoproteins (Apo A-1 and Apo B) were measured by immunonephelometry (Dade Behring, Marburg, Germany). Plasma TAS was measured with commercial kit (Randox, Antrim, UK) according to the method of Miller et al. \[12\]. It can evaluate the total capacity of all antioxidants found in serum to neutralize the oxidative action of free radicals. Lipid peroxidation level was estimated by measurement of thiobarbituric acid reactive substances (TBARS) in serum according to the fluorimetric method of Yagi \[13\]. The pink chromogen produced by the reaction of thiobarbituric acid with lipid peroxidation products such as malondialdehyde (MDA) was estimated using 1,1,3,3-tetraethoxypropane as standard MDA. Serum TBARS levels were measured at 515 nm excitation and 553 nm emission.

Catalase activity was determined in washed red cells prepared immediately after sampling from whole blood anticoagulant with heparin. Then, cell lysates were stored frozen. Catalase activity was measured according to the spectrophotometric method of Goth \[14\]. This assay is based on the ability of hydrogen peroxide to form a stable stained complex with ammonium molybdate measured at 405 nm.

## Statistical analysis

Database management and statistical analyses were carried out using SPSS (Statistical Package for the Sociological Sciences), version 17.0. Results are presented as means ± SD, or percentages. Means were compared using Student for independent samples. The relations between variables were assessed with Pearson's correlation analysis. The significance threshold was set at 5% (p\<0.05).

# Results

Clinical characteristics of patients with AMI and control groups are illustrated in Table 1. No difference was found between the 2 groups for the mean of age and sex. Among cardiovascular factors risk, diabetes, hypertension and dyslipidemia are greatly mentioned in patients compared with healthy subjects. Apolipoproteins disorder was observed in patients. There was a significant increase in serum Apo B level (*\<0.001)* and a decreased serum Apo A-1 level (*\<0.001)* compared with serum apolipoprotein levels in healthy subjects. In other hand, tHcy and TBARS, as a marker of lipid peroxidation, tended to be increased in patients whereas serum TAS concentrations and erythrocyte catalase activity were found lower than in controls.

Clinical and biochemical features of patients with acute myocardial infarction and controls

|   | ***Patients (n= 108)*** | ***Controls (n =81)*** | ***P*** |
|:---|:--:|:--:|:--:|
| *Age (years)* | 63 *±* 12 | 59 *± 9* | *NS* |
| *Men (%)* | 70 | 38 |   |
| *BMI (kg/m*^*2*^*)* | 27 *± 6* | *23 ± 3* | *NS* |
| *Diabetes mellitus (%)* | 61 | 0 |   |
| *Dyslipidemia (%)* | 45 | 0 |   |
| *Hypertension (%)* | 70 | 0 |   |
| *Smokers (%)* | 50 | 6 |   |
| *Alcohol (%)* | 25 | 0 |   |
| *Apo A1 (g/L)* | 1.26 ± 0.19 | 1.5 ± 0.17 | *\<0.001* |
| *Apo B (g/L)* | 1.29 ± 0.36 | 0.92 ± 0.22 | *\<0.001* |
| *TAS (mmol/L)* | 1.47 ± 0.38 | 1.71 ± 0.22 | *\<0.001* |
| *Catalase (kU/g Hb)* | 283 ± 141 | 652 ± 104 | *\<0.001* |
| *Homocysteine (μmol/L)* | 26.9 ± 15.47 | 14.75 ± 2.69 | *\<0.001* |
| *TBARS (μmol/L)* | 1.31 ± 0.36 | 0.71 ± 0.12 | *\<0.001* |
| *Troponine TnIc (μg/L)* | *5,04* ± 2,90 | 0 |   |
|   Q wave AMI | 9,13 ± 2,18 |   |   |
|   Non Q wave AMI | 4,09 ± 2,03 |   |   |
| *Treatment* |   |   |   |
| *Statins* (%) | 38 | 0 |   |
| *Beta-Blockers* (%) | 40 | 0 |   |
| *Ca-Blockers* (%) | 28 | 0 |   |
| *Diuretics* (%) | 37 | 0 |   |

Values are expressed as mean ± *SD* or percentage (%).

NS: not significant, BMI: body mass index, TAS: total antioxidant status.

We showed in Table 2 that erythrocyte catalase activity tended to decrease with smoking and/or arterial hypertension as cardiovascular risk factors. Serum tHcy level was significantly higher in hypertensive patients compared to the normotensive patients.

Erythrocyte catalase activity and serum total homocysteine in patients with different cardiovascular risk factors

| ***Risk factor*** |   | ***Catalase (kU/g Hb)*** | ***P*** | ***Homocysteine (μmol/L)*** | ***P*** |
|:---|:---|:---|:---|:---|:---|
| *Sex* | Men | 294 ± 129 | NS | 25.8 ± 11.6 | NS |
|  | *Women* | 271 ± 152 |  | 28 ± 18.4 |  |
| *Diabetes* | *Yes* | 290 ± 152 | NS | 26.3 ± 15.9 | NS |
|  | *No* | 268 ± 121 |  | 27.8 ± 14.9 |  |
| *Dyslipidemia* | *Yes* | 282 ± 151 | NS | 26.5 ± 12.3 | NS |
|  | *No* | 279 ± 124 |  | 27.6 ± 19.6 |  |
| Obesity | *Yes* | 298 ± 131 | NS | 24.7 ± 13.2 | NS |
|  | *No* | 268 ± 150 |  | 29.1 ± 17.2 |  |
| *Hypertension* | *Yes* | *211* ± 90 | \< 0.001 | 30 ± 17 | \< 0.001 |
|  | *No* | *412* ± 95 |  | 20.3 ± 8.2 |  |
| *Smoking* | *Yes* | *224* ± 105 | \< 0.001 | 28.4 ± 13.1 | NS |
|  | *No* | *377* ± 142 |  | 24.5 ± 18.7 |  |
| *Alcohol* | *Yes* | *275* ± 148 | NS | 24.5 ± 18.7 | NS |
|   | *No* | *387* ± 135 |   | 28.6 ± 11 |   |

Values are expressed as mean ± SD.

NS: not significant.

## Erythrocyte catalase activity and serum tHcy level according to the severity of the myocardial dysfunction

Thirty one patients had a distinct increase of Q wave in the electrocardiogram established at the diagnostic. Patients with Q wave AMI had a significantly lower erythrocyte catalase activity, higher serum tHcy and TBARS levels compared to patients with non Q wave AMI (Figure 1).

## Results of Pearson correlation

We studied the Pearson correlation between serum tHcy, erythrocyte catalase activity and serum TBARS in patients. We found that erythrocyte catalase activity is negatively correlated with both serum tHcy and TBARS (r=−0.38, p\<0.001; r=−0.41, p\<0.001) (Figures 2 and 3). Furthermore, serum tHcy was found positively correlated with serum TBARS level (r=0.4; p\<0.001) (Figure 4).

# Discussion

Myocardial infarction is a complex and multifactor disease in which the cellular and molecular mechanisms contributing to myocardial injury need to be more defined. In our data, investigation about oxidative stress parameters shows an increased serum TBARS level, as a lipid peroxidation marker, and a drop in the total antioxidant capacity in patients with AMI compared to healthy subjects. According to our observation, Pasupathi et al. have considered that the increased serum TBARS is a consequence to excessive ROS generation. The authors reported it to the raised xanthine oxidase activity under ischemia reperfusion \[15\]. Other enzymatic and cellular sources are suggested to be implicated in ROS generation. Some of these are related to the uncoupling of mitochondrial electron transport in consequence to the lack of oxygen supply under ischemia, or to the impairment of NO synthase activity \[7\].

In our study, we observed a decrease in erythrocyte catalase activity in patients compared to controls. Accumulation of hydrogen peroxide under ischemia reperfusion can also inhibit antioxidant enzymes (high substrate inhibition) and alter its enzymatic conformation. According to Senthil et al., the decreased activity of catalase in patients with CHD could be due to the inactivation of this enzyme by cross linking or to the impairment of NO synthase. Nitrite oxide (NO) can bind reversibly to ferric iron, inhibiting afterwards catalase activity \[16,17\]. Erythrocyte catalase activity tended to decrease in patients with hypertension and smoking as the major cardiovascular risk factors. Cigarette smoke is an abundant source of free radicals. It contains more than 10^15^ free radicals including superoxide anion and NO. Chemical oxidants in cigarette smoke can cause the oxidation of DNA encoding for antioxidant proteins \[18\]. On the other hand, essential Hypertension is subjected to increased oxidative stress. It may damage the endothelium and impair endothelium-dependent vascular relaxation. ROS can act on angiotensin converting enzyme to increase its catalytic activity resulting in the increase in angiotensin II production, which, in turn, is a major endogenous inducer of NADPH oxidase. Oxidative stress, endothelial dysfunction and inflammation represent a key triad for the development and progression of Coronary heart disease \[19,20\].

The imbalance between pro-oxidants and antioxidants occurred under ischemia reperfusion can be aggravated by hyperhomocysteinaemia. The moderate hyperhomocysteinaemia revealed in patients can be reported to a hereditary defect of any of the Hcy metabolic enzymes (cystathionine β-synthase, methylene tetrahydrofolate reductase) or to the depletion in folic acid and vitamin B6 or B12 \[3\]. Increased serum tHcy may cause endothelial dysfunction by promoting free radicals. Homocysteine is readily oxidized. Its auto-oxidation is catalyzed by transition metal ions, such as copper leading to homocystine, homocysteine mixed disulfides and homocysteine thiolactone formation \[21,22\]. Homocysteine reduces the transition metal ion (M^n+^) to generate a thioyl radical (Hcy°). It is thought to react with homocysteine to generate a free radical intermediate that reduces oxygen to superoxide anion (O~2~°) and then to peroxide hydrogen generation \[3,23\]. In our data, we have shown a negative correlation between hyperhomocysteinaemia and erythrocyte catalase activity in patients with AMI. Homocysteine has the ability to bind proteins and to form disulphide bridges with cysteine residues within proteins. Milton N et al. suggest that modification of cysteine residues by Homocysteine may alter the enzymatic activity of catalase \[24\]. The excessive generation of ROS under ischemia reperfusion can affect the red cell metabolism and the possible hemolysis when more than 98% in blood catalase is located in erythrocytes \[25\]. Now this is in line with other works suggesting that Hcy can affect the antioxidant enzyme expression. Nanako et al. reported that homocysteine reduced the expression of superoxide dismutase (SOD) mRNA in cultured rat smooth muscle cells \[26\]. Other studies showed a positive correlation between plasma Hcy and genomic damage related to DNA hypomethylation which let us to suggest that Hcy can exert genotoxic effects on DNA genes encoding for antioxidant proteins \[27\].

Reactive oxygen species generated by Hcy auto-oxidation are involved through Fenton type reaction in lipid peroxidation. The serum tHcy and TBARS levels were found higher in patients' with electrocardiogram presenting Q wave AMI compared to patients with non Q wave MI. Myocardial Infarction with Q wave can be used as a predictor of morbidity and mortality patterns after Myocardial events. Desmarais PL and al showed that individuals who had non-Q wave MI had better survival rates for the first 3 years after myocardial rehabilitation than did those who had Q wave MI \[28\]. Excessive lipid peroxidation can be directly involved in the myocardial necrosis manifesting as a myocardial wall dysfunction. Polyunsaturated fatty acids (PUFAs) such as arachidonic, linolenic and linoleic acids present the major targets for free radical attack. It has also been suggested that lipid peroxidation might proceed not only in plasma membranes but also in the nuclear membranes close to chromosomes, due to the loss of membrane integrity in cell membranes consisting of phospholipids. Lipid peroxidation products, such as 4-hydroxynonenal (HNE) and Malondialdehyde (MDA) are toxic. HNE leads to a decrease in protein thiols, disturbance of calcium homeostasis, inhibition of DNA, RNA and protein synthesis, inhibition of respiration and glycolysis \[29\]. Furthermore, HNE leads to mammalian cell death. It increases the expression of p53 family members as well as an increase of expression of the Bax pro-apoptotic gene \[30\]. MDA is able to interact with nucleic acid bases to form several different adducts and to exacerbate DNA oxidative damage, including genes encoding for antioxidant proteins such as catalase, glutathione peroxidase and SOD.

# Conclusion

Molecular mechanisms of myocardial necrosis under ischemia reperfusion need to be more defined. We have shown that hyperhomocysteinaemia, as a cardiovascular risk factor, may be involved in the drop of the enzymatic antioxidant activity. Hyperhomocysteinaemia can be involved in the excessive lipid peroxidation due to its pro-oxidant proprieties, aggravating the magnitude of the oxidative stress and the severity of the myocardial wall dysfunction. Further insights into the molecular mechanisms of the metabolic basis of hyperhomocysteinaemia will prove invaluable in the treatment and the prevention of cardiovascular diseases related to atherosclerosis.

# Abbreviations

AMI: Acute myocardial infarction; TAS: Total antioxidant status; tHcy: Total homocysteine; TBARS: Thiobarbituric acid reactive substances; ROS: Reactive oxygen species; ECG: Electrocardiogram; NO: Nitric oxide; SOD: Superoxide dismutase

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

All authors have done all the work in the laboratory. All authors have done the analysis of the results. All authors have given final approval of the version to be published. All authors read and approved the final manuscript.

## Acknowledgments

The authors thank the staff of the Biochemistry Laboratory and the Cardiology Department of CHU Farhat Hached of Sousse and Mrs Imen ANNABI in French Institute (Tunis) for their valuable contribution in this work.


abstract: Greg's two dogs are impressed that only a few genes control the different lengths, growth patterns and curl of their coats.
author: Gregory A Petsko
date: 2009
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: The dog days of autumn

*In the world which we know, among the different and primitive geniuses that preside over the evolution of the several species, there exists not one, excepting that of the dog, that ever gave a thought to the presence of man*. Maurice Maeterlinck

*Greg Petsko is tied up with teaching, so, by popular demand - actually, he wonders why there never seems to be a demand for more from* him- *his column will be guest-written this month by his two dogs, the mixed poodle/spaniel Clifford and the chocolate Labrador retriever Mink (Figure* 1). *They are not strangers to these pages, having written before, to much acclaim. Precisely how they manage to type their text is unclear*.

**Mink**: Did you see the paper in the issue of *Science* for 2 October 2009 (**326:**150-153) by Cadieu and coworkers? It's entitled *Coat variation in the domestic dog is governed by variants in three genes*.

**Clifford**: What's a domestic dog?

**Mink**: I'm not sure. I think maybe it's the opposite of a foreign dog.

**Clifford**: Are you a foreign dog? After all, you're from Labrador.

**Mink**: No, I'm from New England. My ancestors were from Labrador. And yours were from France and England.

**Clifford**: Does that mean I'm not a domestic dog? I don't want to be a foreign dog! I don't speak French!

**Mink**: Calm down. We're both domestic dogs, I'm sure. But we're getting off the subject here. Did you see that paper?

**Clifford**: No, I didn't. Was it written in French?

**Mink**: Will you forget about French! It was written in scientific English, which means it's not easy for a little puppy to understand, but I'll explain it to you. It's about the genes that control different coats in dogs.

**Clifford**: You mean like how your coat is dark brown and mine is like wheat?

**Mink**: No, the genes that govern coat color have been known for quite a while. This paper is about the genes that control coat length, growth pattern, and curl. For example, I have a fur coat that's all one color, and it only grows to a certain length and then it stops. I shed in winter -

**Clifford**: I'll say you do! I've never seen so much brown fur flying around! Why, the carpet in the family room is covered with little mounds of -

**Mink**: Yes, yes, I know. I can't help it. But as I was saying, I have solid brown, straight fur while you have patchy off-white and beige curly hair. Your coat would just keep growing forever and curl into huge mats if you didn't get taken to the groomer for -

**Clifford**: I hate the groomer! Hate them!

**Mink**: Can we stay focused here? I know you hate the groomer. You make that perfectly plain every time Greg tries to take you there. I haven't seen such a performance of suffering since we watched that television broadcast of *King Lear* with Greg last spring.

**Clifford (sotto voce)**: Hate them!

**Mink**: OK, we've established that. But the point I'm trying to make is: look how different our coats are.

**Clifford**: Mine's better. Except for having to go to the groomer. I hate -

**Mink (quickly)**: I'm glad you like your coat. I think mine is perfectly fine, too. And I don't have to go to the groomer. So there.

**Clifford (sullenly)**: What was your point about the paper?

**Mink**: Oh, yes. The paper. Well, our coats are so completely different, you would think that there would be many genes that were involved in determining those different properties. But the authors of this paper found that's simply not the case. They carried out what are called genome-wide association studies (which is basically just looking for variations in gene sequence that correlate with changes in some property) of more than 1,000 dogs from 80 domestic breeds to identify genes associated with canine fur phenotypes. They were able to take advantage of both inter- and intrabreed variability.

**Clifford**: What does that mean?

**Mink**: I think it means that, although dogs' coats vary a lot from breed to breed, like with you and me, they also vary a bit within breeds. Not all poodles have the same kinds of coat, as any groomer can tell you.

**Clifford**: I hate the groomer!

**Mink**: Right. Nothing more about groomers, I promise. Anyway, it's an advantage when you have small variations within a breed, because you can use that to find the small number of genes that most likely account for those variations (they stand out against a background that doesn't vary so much since all the dogs are from the same breed), and then you can pay particular attention to those genes when you look for what controls the much larger variations between breeds. That makes genome-wide association studies in dogs much easier and more rewarding than genome-wide association studies in people, where it's harder to find candidate genes, so you have to look at thousands of individuals and it's very expensive.

**Clifford (proudly)**: Dogs are better than people.

**Mink**: Of course we are. But as I was saying, Greg has talked about this before. He is convinced that, for association studies in people, it would be smart to use the relatively common mutations that give rise to autosomal recessive diseases and examine the carriers for association with other diseases. For example, people with Gaucher Disease are much more likely to get multiple myeloma, so an obvious thing to do would be to see if Gaucher carriers are overrepresented among myeloma patients. Greg thinks that's what the human genome people ought to be doing if they want to make rapid progress on diseases, because the carrier mutations are known to affect the functions of those proteins, so they're much more likely to do something than the common variants that the gene association studies mostly look at. Greg says those people are barking up the wrong tree.

**Clifford**: Barking up the wrong tree? Why would anybody bark up the wrong tree?

**Mink**: I have no idea.

**Clifford**: Can we get back to talking about dogs?

**Mink**: Sorry. As I was saying, with dogs you can get a good idea what genes to look at as well, from variations within a breed. That's how the people in this paper started their project. The team of scientists, which was headed by Elaine Ostrander of the National Institutes of Health -

**Clifford**: I've heard of her! She's a genome biologist. We like her. She works on genes responsible for cancer susceptibility in people and dogs. Cancer is the number one killer of dogs. We hate cancer! We hate it almost as much as we hate the gr -

**Mink (even more quickly)**: Yes, she is a great benefactor of the canine race. You may remember that, about two years ago, she headed the team that studied height variation in dogs (*Science*, **316:**112-115, 2007). Dogs have the greatest variation in height of any mammalian species. She discovered that the default for dogs is to be tall, like me, but that a mutation in a single gene, insulin-like growth factor 1, could account for the fact that many dogs are quite small, like Chihuahuas, fox terriers, and, well, like you.

**Clifford**: I'm not small! I just have short legs for my body height.

**Mink**: Whatever. The point is, it was a big surprise that one gene could account for such big differences.

**Clifford**: How did they find that gene? I forget.

**Mink**: Exactly the same way they found the genes in this study. They first looked at variation in height within a breed where it varies a lot: Portuguese water dogs. That allowed them to home in on the likely gene. Then they checked it across breeds.

**Clifford**: President Barack Obama has a Portuguese water dog named Bo, doesn't he? I wonder why he didn't pick a poodle/spaniel mix.

**Mink**: Or a chocolate Lab. Well, nobody's perfect. Anyway, that discovery sort of made sense because insulin-like growth factor is one of the genes that controls cell growth and lifespan.

**Clifford (musing)**: I'd like to meet Bo. Do you think President Obama would let him play with us?

**Mink**: Can we stay on the topic here? This column'll be over soon.

**Clifford**: OK. Did they use Portuguese water dogs in this new study about coat variation too?

**Mink**: As a matter of fact, they did. One of their same-breed groups comprised 76 Portuguese water dogs, because it's a breed that varies a lot in hair curl. They looked at three phenotypes, actually: hair curl, hair length, and the presence or absence of what they call 'furnishings' - you know, that little moustache and bushy eyebrows you have.

**Clifford (proudly)**: I am well furnished.

**Mink**: Of course you are. Well, after they looked at a few same-breed groups, they then examined genetic variation across 903 dogs from 80 different breeds. They found that distinct mutations in just three genes, *RSPO2*, *FGF5*, and *KRT71*, together account for most coat phenotypes in pure-bred dogs in the United States.

**Clifford**: You mean my coat is controlled by just three genes?

**Mink**: Maybe not. They only looked at purebreds, and you're a mixture of two breeds.

**Clifford**: Are you insulting my mother? I'm just as pure as -

**Mink**: No, not at all. It's just that, er, uh, more sophisticated dogs like you are too complex for simple genetic analysis.

**Clifford**: That's me, all right. I'm complicated.

**Mink**: You can say that again. Anyway, *RSPO2* largely controls furnishing, which is interesting, because the gene codes for a protein called R-spondin-2, which is a signaling regulator that synergizes with the Wnt pathway to activate β-catenin, and Wnt signaling is required for the establishment of hair follicles in mammals. The mutation doesn't seem to change the protein sequence; it probably affects the mRNA level. You know, this same pathway is involved in the development of hair-follicle tumors, or pilomatricomas, which occur most frequently in breeds that have furnishings. Recent studies have shown that a mutation in the *EDAR* gene, also involved in the Wnt pathway, is responsible for a coarse East-Asian hair type found in humans, and as you know, that hair type has some similarity to canine wirehair.

**Clifford**: Do you think this pathway controls Greg's hair?

**Mink**: He's a middle-aged man. What hair?

**Clifford**: How about the other two phenotypes?

**Mink**: Curl seems to be determined by the *KRT71* gene, which codes for one of the forms of keratin, the major protein component of hair.

**Clifford**: That makes sense. Does the mutation change the protein sequence?

**Mink**: Yes, it does. It replaces one amino acid, an arginine, with a tryptophan. But why that leads to curly hair is not obvious. The third gene, *FGF5*, is involved in hair length.

**Clifford**: What does that protein do?

**Mink**: It makes one of the fibroblast growth factors. Makes sense, right?

**Clifford**: It does. Amazing. And if a dog has all three genes mutated...

**Mink**: He's a wire-hair.

**Clifford**: Like our friend Max in the park. Cool. But why is this important - besides the fact that it refers to dogs, of course?

**Mink**: Isn't that enough? Well, I guess one other reason is that it explains how so many different sizes, shapes and appearances of dog could have arisen in only about 15,000 years of accidental and deliberate breeding. If combinations of only a few genes can have a big effect on morphology and so forth, it won't take that many generations to produce a large number of possibilities. In fact, it's thought that most of the breeds we see today originated since about 1800, so it really can happen fast. Dog evolution is much faster than evolution of other mammals in the wild.

**Clifford**: That's because we're a superior species.

**Mink**: Obviously. After all, who lies around all day and gets fed, while the other species works to support us?

**Clifford**: Isn't evolution wonderful?

**Mink**: It is, but in our case, I prefer the term intelligent design.


abstract: Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.
author: Jiwen (Jim) Liu; TaeWeon Lee; Ralph A. DeFronzoCorresponding authors: Jiwen (Jim) Liu, <email@example.com>, and Ralph A. DeFronzo, <firstname.lastname@example.com>.
date: 2012-09
references:
title: Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

Type 2 diabetes is a serious global health issue that has reached epidemic proportions in both developed and developing countries over the last two decades (1). With currently available medicines, many diabetic patients fail to achieve optimal glycemic control (HbA~1c~ \<6.5–7.0%). With the exception of the glucagon-like peptide 1 analogs and the thiazolidinediones (2), other antidiabetic medications lose their effectiveness to control hyperglycemia over time, partially due to the progressive decline of β-cell function (2–4). As a consequence, many patients receive multiple antidiabetic medicines and eventually require insulin therapy, which often fails to achieve the desired glycemic goal and is associated with weight gain and hypoglycemia (5,6). Failure to achieve glycemic targets is the primary factor responsible for the microvascular complications (retinopathy, neuropathy, nephropathy) and, to a lesser extent, macrovascular complications (2,7). In addition, the majority of diabetic patients are overweight or obese, and many of the current therapies are associated with weight gain, which causes insulin resistance and deterioration in glycemic control (2).

Given the difficulty in achieving optimal glycemic control (8,9) for many diabetic patients using current therapies, there is an unmet medical need for new antidiabetic agents. Although it has been known for 50 years (10,11) that renal glucose reabsorption is increased in type 2 diabetic patients, only recently have the clinical therapeutic implications of this observation been recognized (2,12). Inhibition of renal tubular glucose reabsorption, leading to a reduction in blood glucose concentration through enhanced urinary glucose excretion, provides a novel insulin-independent therapy (2,12) that in animal models of diabetes has been shown to reverse glucotoxicity and improve insulin sensitivity and β-cell function (13,14). The majority (∼80–90%) of filtered plasma glucose is reabsorbed in the early proximal tubule by the high-capacity, low-affinity sodium glucose cotransporter (SGLT) 2 (15,16). The remaining 10–20% of filtered glucose is reabsorbed by the high-affinity, low-capacity SGLT1 transporter in the more distal portion of the proximal tubule. After glucose is actively reabsorbed by SGLT2 and SGLT1 into the proximal tubular cells, it is diffused out of the cells from the basolateral side into blood through facilitative GLUT 2 and 1 (15). Because the majority of glucose reabsorption occurs via the SGLT2 transporter, pharmaceutical companies have focused on the development of SGLT2 inhibitors, and multiple SGLT2 inhibitors currently are in human phase II and III clinical trials (17). This class of antidiabetic medication effectively lowers blood glucose levels and offers additional benefits, including weight loss, low propensity for causing hypoglycemia, and reduction in blood pressure. The SGLT2 inhibitors are effective as monotherapy and in combination with existing therapies (2,12,14,15,17), including insulin (18). Because of their unique mechanism of action (12,15), which is independent of the severity of insulin resistance and β-cell failure, type 2 diabetic individuals with recent-onset diabetes (\<1 year) respond equally well as type 2 diabetic patients with long-standing diabetes (\>10 years) (19).

Dapagliflozin is the most advanced SGLT2 inhibitor in clinical trials (12,17,20). In addition, multiple other SGLT2 inhibitors are in phase II to III trials (Fig. 1) (17,21). However, none of these SGLT2 inhibitors are able to inhibit \>30–50% of the filtered glucose load, despite in vitro studies indicate that 100% inhibition of the SGLT2 transporter should be achieved at the drug concentrations in humans (22,23). In this perspective, we shall examine potential explanations for this apparent paradox. Resolution of the paradox has important clinical implications with regard to the efficacy of this class of drugs and the development of more efficacious SGLT2 inhibitors.

# PUZZLE ABOUT SGLT2 INHIBITORS

In healthy nondiabetic humans, ∼160–180 g of plasma glucose is filtered daily (glomerular filtration rate \[GFR\] = 180 L/day × plasma glucose = 900–1000 mg/L), and essentially all of the filtered glucose is reabsorbed in the proximal tubule of the kidneys. It is generally believed that SGLT2 reabsorbs 80–90% of the filtered glucose load (15,16). However, SGLT2 inhibitors in clinical development induce a maximum of 50–80 g of urinary glucose excretion (UGE) per day (i.e., only 30–50% of the filtered glucose load) in healthy volunteers. Some SGLT2 inhibitors cause a maximum daily UGE at a low dose and cannot augment UGE even with a \>10-fold increase in dose (22,23). For example, dapagliflozin produces a maximum UGE of ∼60 g/day at a dose of 20 mg/day in healthy human volunteers, and UGE remains at 60 g/day when the dose is increased to 500 mg/day (23). Why can these inhibitors not block 90% of the filtered glucose load in humans?

A number of explanations have been proposed to explain this paradox (Table 1, explanations 1–5), but they are insufficient to account for many of the data and observations. In this perspective, we will analyze these explanations and attempt to provide a more satisfactory solution to this puzzle. We will start by examining the pharmacokinetic and pharmacodynamic (PK/PD) characteristics of SGLT2 inhibitors, which are varied and very unique.

Potential explanations for why SGLT2 inhibitors cannot inhibit \>30–50% of the filtered glucose load

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# PK/PD CHARACTERISTICS OF SGLT2 INHIBITORS

First, in healthy human volunteers, there are large differences in the dose-dependent PD responses between empagliflozin (BI 10773) and ipragliflozin (ASP-1941) (Fig. 1), despite sharing many similarities (22,24). Both SGLT2 inhibitors have similar chemical structures, human PK profiles, and in vitro potencies. The in vitro SGLT2 half-maximal inhibitory concentration (IC~50~) of empagliflozin and ipragliflozin is 3.1 and 7.4 nmol/L, respectively. The half-lives (*t*~1/2~) of empagliflozin and ipragliflozin in the blood are 8.6–13 and 10–14 h, respectively, and the time to maximum plasma concentration (*t*~max~) is 1.5–2.5 and 1.0–2.3 h, respectively. The area under the curve (AUC) of empagliflozin at the 2.5-mg dose is 396 nmol·h/L and that of ipragliflozin at the 5-mg dose is 810 nmol·h/L. However, UGE over 24 h of empagliflozin at 2.5-mg dose is 30.6 g, whereas that of ipragliflozin at 5-mg dose is only 3.1 g. Differences in plasma protein binding (PPB) can result in different PD responses, but it is likely (although not measured) that the PPB of the two compounds is similar, because the time course PK profiles of the two compounds (as characterized by their *t*~1/2~, *t*~max~, and AUC) are almost identical. A difference in PPB would be expected to change the time course PK profile, because this would change the clearance and volume of distribution of the drugs and thus the *t*~1/2~. Further, there are no reports in the literature that describe a major difference in PPB of the SGLT2 inhibitors in current clinical development.

Second, the unbound plasma concentrations of dapagliflozin do not seem to account for the drug's effect on UGE. In phase I single and multiple ascending-dose studies of dapagliflozin, a near-maximal UGE was maintained in healthy volunteers for 24 h following a dose of 20 mg, whereas the plasma concentration decreased to a range of 10–20 nmol/L at 24 h from a maximum concentration (*C*~max~) of 600–700 nmol/L (Fig. 2) (21). Taking into account PPB, this corresponds to an unbound dapagliflozin plasma concentration of \<2 nmol/L 24 h after the dose. This concentration is in the vicinity of the drug's in vitro SGLT2 IC~50~ (1.1 nmol/L), yet dapagliflozin is still capable of generating a near maximum UGE response. To our knowledge, there are no published data that suggest that in vitro potency overestimates the in vivo drug concentration needed to inhibit SGLT2. To the contrary, much higher concentrations of T-1095A (an *O*-glucoside inhibitor) were required to inhibit glucose uptake into brush border membrane vesicles prepared from animal kidneys than into cells overexpressing SGLT2 (25). It is possible the SGLT2 expression levels in the renal proximal tubule are higher than those on SGLT2-transfected cells. This possibility is supported by studies that have measured the binding of tritium-labeled dapagliflozin to the same amount of protein from mouse kidneys or transfected cells (Amgen, unpublished data). It was found that SGLT2 expression (calculated from the amount of radioactivity bound) in the mouse kidney tissue was several fold higher than in the proteins of transfected cells ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0052/-/DC1); T.L., unpublished observations). Because proximal tubular proteins only comprise a small portion of the kidney tissue, the SGLT2 expression levels in the proximal tubule could be substantially higher than those of SGLT2-transfected cells. Considering dapagliflozin's high selectivity and specificity for SGLT2, nonspecific binding is unlikely to be a significant contributing factor.

Third, there are disconnects between the UGE effect of empagliflozin and its plasma concentration. After administration of single doses of empagliflozin to healthy humans, a rapid onset of UGE response occurred and was maintained long after the plasma concentrations diminished to very low levels (Fig. 3) (22,26). The empagliflozin plasma concentrations peaked ∼2 h postdose, whereas the maximal UGE rate occurred at about 7 h across a range of doses (10 to 800 mg). Further, the UGE rates did not decline nearly as rapidly as the decrease in plasma concentrations. Following the 100- and 400-mg doses of empagliflozin, the UGE rates at 60 h postdose dropped about threefold from the peak values, whereas the plasma concentrations at the same doses dropped \>50-fold from their peak levels during the same period of time.

The unbound plasma drug concentration usually correlates well with PD response. In the case of SGLT2 inhibitors, it also appears reasonable to assume that the unbound plasma concentration reflects the concentration in the location of the SGLT2 transporters. Unbound SGLT2 inhibitors are filtered through glomeruli and travel a short distance to reach the SGLT2 transporters, which are located in the early segment of the renal proximal tubule. However, the three observations above indicate that the UGE responses induced by the SGLT2 inhibitors do not correlate with the drug's unbound plasma concentrations. Why? In the following discussion, we provide a possible answer to this question, followed by an explanation for the puzzle described in the title.

# EXPLANATION OF THE PARADOX

As described above, SGLT2 inhibitors, such as dapagliflozin and empagliflozin, demonstrate unique PK/PD relationships. Robust UGE was maintained long after the plasma drug concentrations diminished (22,23) (Fig. 3). If SGLT2 inhibitors are actively secreted (in addition to filtered) into the renal proximal tubule, the renal secretion can deliver higher drug concentrations to the area of the SGLT2 transporter and thus contribute to the robust UGE effect, especially when the plasma concentrations are low. Another possible contributing factor that may explain the prolonged UGE effect is a slow off rate of SGLT2 inhibitors. Compounds with a slow off rate can maintain their PD effect even when their plasma concentrations are low. Empagliflozin has a slow off rate with a dissociation *t*~1/2~ of 1 h both in the absence and presence of 20 mmol/L glucose (27). If SGLT2 inhibitors also block the transporter from inside the cells, their inhibition effect can last longer than indicated by their dissociation *t*~1/2~ (28).

To the best of our knowledge, renal secretion has not been considered as a factor for the PK/PD relationship in the literature. There are several potential reasons for the lack of consideration of renal secretion. First, the reported renal clearance of SGLT2 inhibitors is low across species and insignificant compared with their hepatic clearance (29,30). Dapagliflozin has a low renal clearance of 5.6 mL/min in humans, half of its GFR\*fu (10.8 mL/min), in which fu is fraction unbound (29). When renal clearance is insignificant and less than the product of GFR\*fu, renal secretion is not considered to be a significant factor for drug–drug interaction (DDI), so it is not critical to study renal secretion and the transporters involved for compounds with low renal clearances (31). Second, even if renal transporters are studied, the ones most often examined for DDI are limited, such as P-glycoprotein, breast cancer–resistant protein, organic anion transporters, and organic cation transporters (OCTs) (31). There are many more transporters (peptide transporters 1 and 2, multidrug resistance–associated protein 2 and 4, OCTN1, and OCTN2, etc.) involved in renal secretion, but often they are not studied for DDI (31).

Low renal clearance does not mean lack of renal secretion, because significant renal secretion can be masked by renal reabsorption, if compounds have good passive permeability (32–34). SGLT2 inhibitors generally have good permeability, as indicated by high oral bioavailability and permeability measurement (29). Although there is no direct evidence in the literature to support renal secretion of SGLT2 inhibitors, indirect evidence supports such a mechanism. It is interesting to note that following oral administration of a 1 mg/kg dose of TS-071 (Fig. 1), rats exhibited kidney/plasma ratios of 35 at 4 h postdose, despite the fact that TS-071 primarily is excreted by hepatic metabolism (35). After 24 h, TS-071 was no longer detected in rat kidneys. Renal secretion and slow off rate from targets in the kidney can make compounds preferentially distributed into the kidney.

Neutral drugs that have high lipophilicity (indicating good permeability), like dapagliflozin, tend to have a low renal clearance (33). Even though the renal clearance of dapagliflozin is low, many drugs that have similar PPB (indicated by GFR\*fu) and similar lipophilicity (indicated by cLogD or cLogP) to those of dapagliflozin actually have a much lower renal clearance than dapagliflozin (34). The reason why dapagliflozin does not have a lower renal clearance may be due to its renal secretion.

The sustained robust UGE and different timing of peak UGE and *C*~max~ of empagliflozin also suggest renal secretion (Fig. 3) (22,26). If glomerular filtration were the only way for SGLT2 inhibitors to reach the site of action, one would expect the timing of peak UGE to match the *t*~max~ of the plasma concentration and UGE to decrease in concert with the reduction of the plasma concentration. The onset of UGE response is rapid after SGLT2 inhibition. Although slow off rate of SGLT2 inhibitors can contribute to the slow decrease of UGE response, it cannot explain the vastly slower decline of UGE response compared with the plasma concentration.

The human metabolite profile of dapagliflozin suggests that active metabolites do not significantly contribute to the PD response, because dapagliflozin is primarily eliminated as a pharmacologically inactive glucuronide metabolite (29). The possibility of superpotent minor metabolites contributing to the PD effects cannot be ruled out, but there is no evidence to support this.

If SGLT2 inhibitors are secreted by the renal tubule, this could provide a solution to the puzzle as to why SGLT2 inhibitors are unable to inhibit 80–90% of the filtered glucose load in humans. First, even though renal secretion may deliver sufficient drug to achieve robust UGE for a longer duration, it can saturate at high doses and thus limit SGLT2 inhibitors from maximally inhibiting renal glucose reabsorption (Table 1, explanation 6). Second, location of renal secretion relative to that of the SGLT2 transporters can affect the efficiency of SGLT2 inhibition. Downstream renal secretion of drug cannot affect glucose reabsorption, if the SGLT2 transporters are located upstream (Table 1, explanation 7). Third, full inhibition of the SGLT2 transporter may require significantly higher concentrations of the inhibitors than suggested by the in vitro SGLT2 potency, because the expression levels of SGLT2 in the renal proximal tubule may be much higher than those on the transfected cells used in the in vitro assays (Table 1, explanation 8). Fourth, SGLT2 inhibitors delivered through glomerular filtration may have an impact on UGE during the initial few hours after drug administration, but exert limited effect at later times even at high doses, because the plasma concentration declines rapidly after the *C*~max~ is reached. Taken collectively, it is possible that glomerular filtration cannot deliver impactful levels of the SGLT2 inhibitors over a long duration even when high doses of the drugs are given. Rather, renal tubular secretion and slow off rate of the inhibitors may be responsible for the sustained drug levels in the proximal tubule, which in turn results in a sustained UGE response. Full inhibition of SGLT2 may be limited by the capacity of renal secretion and/or the proximity of the site of renal secretion and the SGLT2 transporters.

It also is possible that excessive reabsorption of SGLT2 inhibitors in the proximal tubule contributes to the inability of SGLT2 inhibitors to block \>30–50% of glucose reabsorption (Table 1, explanation 9). Excessive active reabsorption through transporters could result in significantly lower drug concentrations in the proximal tubule than the plasma unbound concentration and thus limit the inhibition. But the lower tubular concentration is not consistent with the prolonged robust UGE effect, unless inhibition of the SGLT2 transporter also can take place intracellularly. Active reabsorption can accumulate SGLT2 inhibitors inside the tubular cells and result in the prolonged UGE effect (especially in combination with slow off rate), if the inhibitors also block from inside the cells. In this case, the capacity of the active reabsorption and/or its location relative to the SGLT2 transporters dictates the extent of SGLT2 inhibition. However, there is no evidence in the literature regarding possible intracellular inhibition of the SGLT2 transporter by the SGLT2 inhibitors in clinical trials. Passive reabsorption does not have the same effect as active reabsorption, because it is driven by the drug concentration gradient from the renal tubule to the bloodstream. Therefore, it only makes the drug tubular concentration approach the plasma unbound concentration.

Renal secretion (and/or reabsorption) also can explain several other important observations. First, as discussed above, empagliflozin and ipragliflozin have very different dose-dependent UGE responses, even though they share many similar properties, such as potency and PK properties. It is possible that differences in renal secretion (and/or reabsorption) and dissociation *t*~1/2~ are responsible for the different UGE responses. Second, in contrast to humans, SGLT2 inhibitors can block \>70% of the glucose reabsorption in Sprague-Dawley rats. In these rats, about 3.6 g/day/200 g body weight of glucose is filtered through glomeruli (GFR = 1.4 mL/min/100 g body weight = 4 L/day/200 g body weight × 0.9 g/L = plasma glucose level) (36). The maximum UGE/day/200 g body weight induced by SGLT2 inhibitors in Sprague-Dawley rats is 2.6 g for dapagliflozin and PF-04971729 (37,38), 2.9 g for EGT1442 (39), and 3.7 g for canagliflozin (40). Potency and PK cannot explain this species difference. This difference may be explained by a greater renal secretory (and/or reabsorption) capacity and/or more upstream renal secretion (and/or reabsorption) in rodents compared with humans.

There are other potential explanations (Table 1, explanations 1–5) for this puzzle, but they are unlikely to explain the paradox. The first explanation in Table 1 is that increased levels of glucose in the proximal tubule, as a result of SGLT2 inhibition, compete with the drug and thus prevent the full inhibition of SGLT2. However, this is not consistent with the observation that a \>20-fold increase in the dose of dapagliflozin from 20 to 500 mg/day only prolongs the PD effect and does not further increase the level of inhibition (∼40% inhibition of the glucose reabsorption) (23). The second explanation in Table 1 is that GLUT other than SGLT2 may be responsible for a much greater fraction of the glucose reabsorption. However, this cannot explain why some patients with severe SGLT2 mutations excrete \>150 g/day of glucose into the urine, unless the SGLT2 mutation also affects other glucose transporters (i.e., SGLT5 or SGLT1, for which genes are not located on the same chromosome as SGLT2). In addition, LX-4211, a SGLT2/SGLT1 dual inhibitor in phase II clinical trials, does not generate a larger glucosuric effect than selective SGLT2 inhibitors (41). SGLT5 has been suggested as a transporter that may be responsible for a greater fraction of glucose reabsorption, because of its almost exclusive expression in the renal tubule and its unidentified function. However, recent studies have demonstrated that SGLT5 is a mannose fructose transporter (27). Further, remogliflozin is a SGLT2/SGLT5 dual inhibitor with IC~50~ for the two transporters of 12 and 480 nmol/L, respectively (42). In healthy humans, remogliflozin etabonate (prodrug of remogliflozin) could not induce more than ∼40 g/day of UGE, even when its dose was increased from 150 to 1000 mg (43). Based upon these observations, the role of SGLT5 in renal glucose reabsorption appears to be insignificant. Furthermore, knockout of SGLT2 in the mouse results in the excretion of 70–80% of the filtered glucose load (16), although one could argue that other GLUTs play a more dominant role in man. Last and most important, SGLT2 antisense oligonucleotides, which knock down SGLT2 mRNA expression up to 80%, have a much greater glucosuric effect than the orally administered SGLT2 inhibitors in multiple species, including rats and dogs as well as monkeys (44,45).

Explanations 3–5 in Table 1 also are unlikely. Based upon animal studies that have localized the SGLT2 transporter to the brush border membrane of the proximal tubule (16), inability of the filtered SGLT2 inhibitor to interact with the SGLT2 transporters because of their anatomical location seems unlikely (Table 1, explanation 3). The high PPB of some SGLT2 inhibitors should not limit the extent of inhibition if the dose is increased (Table 1, explanation 4). High PPB limits the percentage of unbound drug and thus limits the amount of drug filtered through the glomeruli. However, the amount of filtered (unbound) drug can be increased, if the dose is increased. For example, unbound *C*~max~ of dapagliflozin increases from ∼60 nmol/L to \>1 μmol/L (calculated using 91% PPB and its PK in healthy humans) (23,29) when its dose is increased from 20 to 500 mg. The similar magnitude of glucosuria on days 1–3 versus day 14 after administration of dapagliflozin strongly argues against upregulation of other SGLTs or other GLUTs distal to the SGLT2 transporter (Table 1, explanation 5) (46). Moreover, phlorizin-treated diabetic rats do not upregulate GLUT2 (47). Although SGLT2 inhibition does not appear to cause upregulation of SGLTs or other GLUTs in humans, there is evidence for increased GLUT2 expression in diabetic rodent models (17,19). However, in healthy humans in whom upregulation of GLUTs and/or SGLTs does not occur, SGLT2 inhibitors still cannot block \>30–50% of renal glucose reabsorption. In addition, if there were significant contribution to glucose reabsorption from GLUTs or SGLTs other than SGLT2, it would not explain the unique PD effects of SGLT2 inhibitors, such as prolonged maximum UGE response at diminished plasma levels and different timing between the maximum PD response of empagliflozin and its maximum plasma concentration.

# CONCLUSIONS

The SGLT2 inhibitors in human clinical trials have good efficacy, but they do not inhibit \>30–50% of the filtered glucose load. Based upon their PK/PD relationship, we postulate that their mechanism of action is related to secretion and/or active reabsorption in the proximal tubule and slow off rate from the SGLT2 target. Renal micropuncture studies with radiolabeled SGLT2 inhibitors and comparison of drug radioactivity in the glomerular filtrate and proximal tubule will help to define the relative contributions of tubular secretion and/or reabsorption versus glomerular filtration in establishing the drug concentration at the site of the SGLT2 transporters in the proximal tubule. A better understanding of the renal handling of the SGLT2 inhibitors will help to develop more effective medications that are capable of inhibiting a greater percentage of filtered glucose load and causing a greater reduction in HbA~1c~.

## ACKNOWLEDGMENTS

R.A.D. received grants from Amylin and Takeda; served on the advisory boards of Amylin, Takeda, BMS, Novo Nordisk, Jansen, and Boehringer-Ingelheim; and served as a member of the Speakers Bureau at Novo Nordisk. No other potential conflicts of interest relevant to this article were reported.

J.L. and R.A.D. wrote, reviewed, and edited the manuscript. T.L. researched data and reviewed and edited the manuscript. J.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Dr. Ernest M. Wright of David Geffen School of Medicine at University of California Los Angeles and Dr. Jeff Jasper of Cytokinetics for editorial comments.

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