Rosiglitazone reverses tenofovir-induced nephrotoxicity

original article

http://www.kidney-international.org & 2008 International Society of Nephrology

Rosiglitazone reverses tenofovir-induced nephrotoxicity Alexandre B. Libo´rio1, Lu´cia Andrade1, Leonardo V.B. Pereira1, Talita R.C. Sanches1, Maria H. Shimizu1 and Antonio C. Seguro1 1

Laboratory of Basic Science, Department of Nephrology, University of Sa˜o Paulo School of Medicine, Sa˜o Paulo, Brazil

Tenofovir disoproxil fumarate (TDF) is a first-line drug used in patients with highly active retroviral disease; however, it can cause renal failure associated with many tubular anomalies that may be due to down regulation of a variety of ion transporters. Because rosiglitazone, a peroxisome proliferator-activated receptor-c agonist induces the expression of many of these same transporters, we tested if the nephrotoxicity can be ameliorated by its use. High doses of TDF caused severe renal failure in rats accompanied by a reduction in endothelial nitric-oxide synthase and intense renal vasoconstriction; all of which were significantly improved by rosiglitazone treatment. Low-dose TDF did not alter glomerular filtration rate but produced significant phosphaturia, proximal tubular acidosis, polyuria and a reduced urinary concentrating ability. These alterations were caused by specific downregulation of the sodium-phosphorus cotransporter, sodium/hydrogen exchanger 3 and aquaporin 2. A Fanconi’s-like syndrome was ruled out as there was no proteinuria or glycosuria. Rosiglitazone reversed TDF-induced tubular nephrotoxicity, normalized urinary biochemical parameters and membrane transporter protein expression. These studies suggest that rosiglitazone treatment might be useful in patients presenting with TFV-induced nephrotoxicity especially in those with hypophosphatemia or reduced glomerular filtration rate. Kidney International (2008) 74, 910–918; doi:10.1038/ki.2008.252; published online 18 June 2008 KEYWORDS: reverse transcriptase inhibitors; vasodilator agents; adenine/tenofovir; thiazolidinediones/rosiglitazone; kidney failure; acute

Correspondence: Antonio C. Seguro, Laborato´rio de Pesquisa Ba´sica LIM-12, Faculdade de Medicina da USP, Av. Dr. Arnaldo 455, sala 3310, CEP 01246-903, Sa˜o Paulo, SP, Brazil. E-mail: [email protected] Received 15 May 2007; revised 14 April 2008; accepted 15 April 2008; published online 18 June 2008 910

Tenofovir disoproxil fumarate (TDF) is the first nucleotide reverse transcriptase inhibitor approved for the treatment of HIV.1 Presenting low protein binding, TDF is freely filtered through the glomerulus without being degraded in the body. Similar to cidofovir and adefovir, TDF is eliminated through active tubular secretion via the human organic anion transporter 1.2 High intracellular concentrations of TDF in tubular cells can interfere with cell function.3 Currently, TDF is considered as a first-line drug for patients who are highly active antiretroviral therapy (HAART)-naı¨ve and is an option in many HAART combinations.4 It has recently been shown that, in such antiretroviral-naı¨ve patients, the TDF–emtricitabine–efavirenz combination is superior to the zidovudine–lamivudine– efavirenz combination in terms of virological suppression and CD4 response.5 In HIV-infected patients, treatment with TDF has been associated with good virological suppression and favorable clinical outcomes. However, TDF has serious side effects, especially with long-term use. Among the principal side effects associated with TDF use are hypophosphatemia,6–8 renal failure,9,10 and tubular toxicity. The tubular toxicity induced by TDF can manifest as renal tubular acidosis,11 Fanconi syndrome,7,12,13 or nephrogenic diabetes insipidus (NDI).7,13,14 Another significant side effect is reduced bone mineral density.11,15 Through bone histology in animals, it has been demonstrated that TDF treatment causes osteomalacia,16 possibly by reducing serum phosphorus levels. Although the incidence of adverse effects is low, the number of patients presenting with such side effects is likely to grow in parallel with the increasing use of TDF. The peroxisome proliferator-activated receptor-g (PPAR-g) is a member of the nuclear receptor superfamily of ligandactivated transcription factors. To regulate the transcription of numerous target genes, ligand-activated PPAR-g binds to a specific DNA site known as the peroxisome proliferator response element.17,18 The PPAR-g is widely distributed throughout the tissues, especially in adipose tissue. In renal tissue, PPAR-g is expressed in the medullary collecting ducts, proximal tubules, mesangial cells, and renal microvasculature.19 Song et al.20 demonstrated the effects that a PPAR-g agonist has on transporters in the renal tubular epithelium. The authors found that, in normal rats, the administration of the PPAR-g agonist rosiglitazone (RSG) was associated with Kidney International (2008) 74, 910–918

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AB Libo´rio et al.: Rosiglitazone reverses tenofovir effects

increased expression of the bumetanide-sensitive Na–K–2Cl cotransporter (NKCC2), the sodium/hydrogen exchanger 3 (NHE3), and the sodium-phosphate cotransporter subtype IIa (NaPi-IIa), as well as aquaporin 2 (AQP2). In addition, RSG administration increased the expression of endothelial nitric-oxide synthase (eNOS). In this study, we investigated the mechanisms of TDF-induced nephrotoxicity, focusing on its tubular effects. We also tested the hypothesis that RSG protects against TDF-induced renal impairment. RESULTS

Three sets of experiments were performed. The first was designated as the high-TDF (Hi-TDF) set. The Hi-TDF experiments involved two groups: Hi-TDF (rats fed with a diet containing 300 mg of TDF/kg of food for 30 days); and Hi-TDF þ RSG (rats fed with a diet containing 300 mg of TDF/kg of food for the first 15 days and, for the subsequent 15 days, 300 mg of TDF plus 92 mg of RSG/kg of food). The second set of experiments was designated as the low-TDF (Lo-TDF) set. The Lo-TDF experiments involved two groups: Lo-TDF (rats fed with a diet containing 50 mg of TDF/kg of food for 30 days); and Lo-TDF þ RSG (rats fed with a diet containing 50 mg of TDF/kg of food for the first 15 days and, for the subsequent 15 days, 50 mg of TDF plus 92 mg of RSG/kg of food). For the Hi-TDF and Lo-TDF experiments, we included a control group (rats fed with a normal diet for

30 days). The third set of experiments was designated as the RSG-only set and involved a group designated as RSG (rats fed with a diet containing 92 mg of RSG/kg of food for 15 days). For the RSG-only experiments, we included an additional control group (rats fed with a normal diet for 15 days). In all three sets of experiments, each rat was provided with 25 g of food per day, and all presented with similar ingestion (E23 g). As can be seen in Tables 1 and 2, the TDF-induced reduction in creatinine clearance was dose dependent. In the Lo-TDF-treated rats, there were only minimal alterations in renal blood flow (RBF), which were not accompanied by any significant changes in creatinine clearance. Conversely, creatinine clearance was significantly lower in the Hi-TDF group rats than in the corresponding control rats. High-dose TDF deteriorates renal function and induces downregulation of eNOS protein abundance

The results of clearance studies and RBF measurements in the Hi-TDF experiments are shown in Table 1. There were no differences among the three groups in terms of body weight. The rats in the Hi-TDF group presented with higher blood pressure and significantly impaired renal function. These alterations were accompanied by intense renal vasoconstriction (reduced RBF and increased renal vascular resistance). In addition, the Hi-TDF group rats presented with markedly lower eNOS expression than the corresponding control rats (Figure 1).

Table 1 | Renal function and hemodynamic measurements in normal (control) rats, in rats treated for 30 days with a high dose of TDF (300 mg/kg of food; Hi-TDF group), and in rats treated for 30 days with the same high dose of TDF, to which RSG (92 mg/kg of food) was added on day 15 and continued throughout (Hi-TDF+RSG group) Group Control (n=4) Hi-TDF (n=5) Hi-TDF+RSG (n=5)

BW

UO

CrCl

MAP

RBF

RVR

272±18 259±15 267±8

11.5±1.1 13.9±2.1 11.8±1.3

0.83±0.07 0.19±0.01a 0.58±0.04e

117±3.8 140±4.2b 125±3.5

6.1±0.15 2.3±0.20c,d 4.3±0.35e

19.4±0.8 60.9±1.1a 29.7±0.9e

BW, body weight (g); CrCl, creatinine clearance (ml/min/100 g); MAP, mean arterial pressure (mm Hg); RBF, renal blood flow (ml/min); RSG, rosiglitazone; RVR, renal vascular resistance (mm Hg/ml/min); TDF, tenofovir disoproxil fumarate; UO, urine output (ml/day). Data are expressed as mean±s.e.m. a Po0.001 vs other groups. b Po0.05 vs other groups. c Po0.001 vs control. d Po0.05 vs Hi-TDF+RSG. e Po0.01 vs control by analysis of variance and Tukey’s multiple comparison test.

Table 2 | Renal function and hemodynamic measurements in normal (control) rats, in rats treated for 30 days with a low dose of TDF (50 mg/kg of food; Lo-TDF group), and in rats treated for 30 days with the same low dose of TDF, to which RSG (92 mg/kg of food) was added on day 15 and continued throughout (Lo-TDF+RSG group) Group Control (n=4) Lo-TDF (n=6) Lo-TDF+RSG (n=5)

BW

UO

CrCl

MAP

RBF

RVR

272±18 274±12 262±6

11.5±1.1 23.4±1.6a 8.4±1.1

0.83±0.07 0.72±0.08 0.74±0.01

117±3.8 128±3.1 117±2.8

6.1±0.15 5.0±0.19b 5.9±0.35

19.4±0.8 25.6±1.3c 19.8±0.9

BW, body weight (g); CrCl, creatinine clearance (ml/min/100 g); MAP, mean arterial pressure (mm Hg); RBF, renal blood flow (ml/min); RSG, rosiglitazone; RVR, renal vascular resistance (mm Hg/ml/min); TDF, tenofovir disoproxil fumarate; UO, urine output (ml/day). Data are expressed as mean±s.e.m. a Po0.01 vs other groups. b Po0.05 vs other groups. c Po0.01 vs other groups by analysis of variance and Tukey’s multiple comparison test.

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40

Table 3 | Functional parameters in normal (control) rats, in rats treated for 30 days with a low dose of TDF (50 mg/kg of food; Lo-TDF group), and in rats treated for 30 days with the same low dose of TDF, to which RSG (92 mg/kg of food) was added on day 15 and continued throughout (Lo-TDF+RSG group)

20

Control

120 100

%

80 60

*

0 e-NOS Actin Control

Hi-TDF

Hi-TDF+RSG

Figure 1 | Semiquantitative immunoblotting of kidney fractions. (a) Densitometric analysis of all samples from control, high-dose TDF (Hi-TDF) and Hi-TDF þ rosiglitazone (Hi-TDF þ RSG) rats. The Hi-TDF group rats presented with decreased endothelial nitric oxide synthase (eNOS) expression. Levels of eNOS expression were completely restored in response to RSG. (b) Immunoblots that reacted with anti-eNOS. *Po0.001 vs control and Hi-TDF þ RSG group.

RSG ameliorates renal function and restores eNOS protein abundance in rats treated with high doses of TDF

Administration of RSG improved renal function, as evidenced by the fact that creatinine clearance was significantly greater in the Hi-TDF þ RSG group than in the Hi-TDF group. There was no difference in creatinine clearance between the rats in the RSG group and those in the corresponding control group (0.58±0.03 vs 0.68±0.05 ml/ min/100 g body weight). Renal vasoconstriction was lower in the Hi-TDF þ RSG group than in the Hi-TDF group (Table 1). In addition, RSG inhibited the TDF-induced downregulation of eNOS expression (Figure 1). Tubular dysfunction associated with the administration of low doses of TDF

In terms of creatinine clearance, there was no significant difference between the Lo-TDF group rats and the corresponding control group rats (Table 2). In the Lo-TDF group rats, urine output was significantly greater than that observed in the control rats (Table 3). The Lo-TDF group rats also presented with markedly lower urine osmolality than did the control rats. Proximal tubular function was impaired in Lo-TDF-treated rats, as evidenced by increased urinary phosphorus excretion and a considerable reduction in serum bicarbonate. Other markers of proximal tubular dysfunction, such as glycosuria and proteinuria, were absent (data not shown). No differences were observed in urinary sodium excretion or urinary potassium excretion. In addition, we observed no differences between the Lo-TDF-treated rats and the corresponding control rats in terms of urinary calcium excretion or urinary magnesium excretion. Administration of RSG reversed the hyperphosphaturia presented by the rats in the Lo-TDF group. In Lo-TDF þ RSG-treated rats, urinary phosphorus excretion returned to normal levels by the end of the experiment (Table 3). The acidosis presented by rats in the Lo-TDF group was not observed in the Lo-TDF þ RSG 912

Urine output (ml/day) UOsm (mOsm/kg H2O) UPV (mmol/day) UNaV (mEq./day) UKV (mEq./day) UMgV (mg/day) UCaV (mg/day) PH Serum bicarbonate (mEq./l) Serum anion gape

11.5±0.5 988±101 316±33 1.30±0.22 1.16±0.21 0.96±0.26 354±25 7.42±0.01 24.6±1.2 18.3±1.8

Lo-TDF

Lo-TDF+RSG a

23.4±1.6 520±85b,c 648±41a 1.45±0.14 1.65±0.38 0.92±0.28 298±22 7.31±0.02a 18.3±0.8d 17.9±2.0

8.4±1.1 1360±138c 351±36 1.46±0.15 1.48±0.18 1.04±0.13 362±32 7.42±0.02 23.7±1.0 18.5±1.6

RSG, rosiglitazone; TDF, tenofovir disoproxil fumarate; UOsm, urinary osmolality; UNaV, urinary sodium; UKV, urinary potassium; UPV, urinary phosphorus; UMgV, urinary magnesium; UCaV, urinary calcium. a Po0.001 vs control and Lo-TDF+RSG. b Po0.001 vs Lo-TDF+RSG. c Po0.05 vs control. d Po0.05 vs control and Lo-TDF+RSG. e Anion gap is calculated as sodium-chloride-bicarbonate.

group. The Lo-TDF group rats presented with partial NDI, by which time urine output and osmolality had normalized in the Lo-TDF þ RSG-treated rats (Table 3). Interestingly, compared with the corresponding control group rats, the RSG group rats presented with an increase in body weight, as well as an increase in urinary osmolality. However, there was no difference between the two groups in terms of urine volume. Surprisingly, the RSG group rats also presented significantly greater urinary excretions than those in the corresponding control group, including urinary excretion of sodium, potassium, and phosphorus (Table 4). NaPi-IIa cotransporter protein abundance is decreased in TDF-treated rats

Tubular reabsorption of phosphorus is largely performed in the proximal tubules via NaPi-IIa. To investigate the cause of hyperphosphaturia in TDF-treated rats, we examined NaPi-IIa expression in the renal cortex. As shown in Figure 2a, NaPi-IIa protein expression in TDF-treated rats was significantly lower than that seen in control rats (67.7±1.4 vs 99.5±0.5%, Po0.01). Treatment with TDF induces renal tubular acidosis through downregulation of NHE3

Although rats treated with TDF presented with low serum bicarbonate levels and low serum pH, urine pH also remained low (Table 3), suggesting proximal tubular dysfunction. To thoroughly investigate the cause of the serum acidosis, we examined NHE3 protein expression. In comparison with control rats, those treated with TDF presented with lower NHE3 protein expression (100±1 vs 59±2.1%, Po0.01; Figure 2b), which is consistent with the proximal type of renal tubular acidosis. Kidney International (2008) 74, 910–918

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Table 4 | Functional parameters in normal (control) rats and in rats treated for 15 days with RSG (92 mg/kg of food, RSG group)

Control group RSG group

BW (g)

UOsm (mOsm/kg H2O)

Urine output (ml/day)

UNaV (mEq./day)

UKV (mEq./day)

UPV (lmol/day)

252±7 293±6a

410±73 627±55b

26±3.2 22±3.1

0.6±0.05 1.48±0.15a

1.2±0.04 1.4±0.08b

524±25 848±35.4a

BW, body weight, RSG, rosiglitazone; UOsm, urinary osmolality; UNaV, urinary sodium; UKV, urinary potassium; UPV, urinary phosphorus. a Po0.001 vs control. b Po0.05 vs control.

a

120 100

%

80

%

×

60 40 20

180 160 140 120 100 80 60 40 20 0 Control

0 Control

Lo-TDF Lo-TDF+RSG

•×

•

Lo-TDF Lo-TDF+RSG

• P < 0.001 vs control and vs Lo-TDF × P < 0.05 vs Lo-TDF+RSG

RSG

× P < 0.01 vs control, Lo-TDF+RSG and RSG

ENaC

Na-Pi

Actin

Actin Control

b

Lo-TDF

Lo-TDF+RSG

RSG

Control

100

%

Lo-TDF

Lo-TDF+RSG

RSG

Figure 3 | Semiquantitative immunoblotting of membrane fractions prepared from renal cortices. Densitometric analysis of all samples from control, low-dose TDF (Lo-TDF), Lo-TDF þ rosiglitazone (Lo-TDF þ RSG), and rosiglitazone (RSG) rats. The Lo-TDF group rats presented with normal expression of a-ENaC. Levels of a-ENaC expression were increased in response to RSG. Immunoblots reacted with anti-a-ENaC, revealing an 86-kDa band.

120

80

RSG

•×

60 40 20 0 Control

Lo-TDF Lo-TDF+RSG

RSG

• P < 0.001 vs RSG × P < 0.01 vs control and vs Lo-TDF+RSG NHE3

Actin Control

Lo-TDF

Lo-TDF+RSG

RSG

Figure 2 | Semiquantitative immunoblotting of membrane fractions prepared from renal cortices. Densitometric analysis of all samples from control, low-dose TDF (Lo-TDF), Lo-TDF þ rosiglitazone (Lo-TDF þ RSG), and rosiglitazone (RSG) rats. (a) The Lo-TDF group rats presented with decreased expression of sodium-phosphate cotransporter subtype IIa (NaPi-IIa). Levels of NaPi-IIa expression were completely restored in response to RSG. Immunoblots reacted with anti-NaPi-IIa, revealing an 85-kDa band. (b) The Lo-TDF group rats presented decreased expression of the sodium/hydrogen exchanger 3 (NHE3). Levels of NHE3 expression were completely restored in response to RSG. Immunoblots reacted with anti-NHE3 revealing an 84-kDa band.

of the epithelial sodium channel (a-EnaC). Abundance of a-ENaC was unaffected by TDF treatment (Lo-TDF: 108.3%±1.7; control: 100%, P ¼ NS). NKCC2 protein expression is increased in rats treated with TDF: a compensatory mechanism

Protein expression of NHE3 was reduced in TDF-treated rats. As no increase in urinary sodium excretion was observed in the treated rats, we attempted to determine whether distal segments of the nephron play a compensatory role by reabsorbing the excess sodium delivered. It is well known that apical NKCC2 is the major transporter for sodium reabsorption by the thick ascending limb. As indicated in Figure 4a, NKCC2 protein expression was significantly higher in the medullae of Lo-TDF group rats than in those of control rats (136±2.2 vs 99±1.0%, Po0.001). AQP2 abundance in the renal medulla is altered in response to TDF treatment

Abundance of a-ENaC in the renal cortex is unchanged in TDF-treated rats

Figure 3 shows representative immunoblots of renal cortex homogenates probed with the antibody against the a-subunit Kidney International (2008) 74, 910–918

As can be seen in Figure 4b, treatment with TDF reduced AQP2 abundance significantly (67.5±2.5 vs 99±1, Po0.01). This can partially explain the reduced urinary concentrating ability. 913

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%

a

160 140 120 100 80 60 40 20 0

AB Libo´rio et al.: Rosiglitazone reverses tenofovir effects

Control

protein abundance was comparable with that observed in control rats (93.3±4.4 vs 100±1%, P ¼ NS). The RSG group rats presented with no significant difference in serum pH or NHE3 expression in comparison with the control group rats (RSG: 108±7.3%).

•

•

Lo-TDF Lo-TDF+RSG

RSG

• P < 0.001 vs control and RSG NKCC2

Actin Control

%

b

Lo-TDF

180 160 140 120 100 80 60 40 20 0

Lo-TDF+RSG

RSG

∞ •

a-ENaC protein expression is significantly increased in rats treated with TDF and RSG

Figure 3 shows that a-ENaC expression was greater in the Lo-TDF þ RSG-treated rats than in the Lo-TDF-treated rats (145±2.9 vs 108.3±1.7%, Po0.001). In addition, a-ENaC expression was greater in the Lo-TDF þ RSG-treated rats than in control rats (145±2.9 vs 100%, Po0.001). In the RSG group, a-ENaC expression was increased in comparison with the control group (RSG: 155±2.9%, Po0.001). In addition, there was a significant difference between the RSG group and the Lo-TDF þ RSG group in terms of a-ENaC expression, which was greater in the RSG group (Po0.05).

•×

NKCC2 protein expression is increased in rats treated with TDF and RSG Control

Lo-TDF Lo-TDF+RSG

RSG

• P < 0.001 vs Lo-TDF+RSG and RSG × P < 0.01 vs control ∞ P < 0.05 vs control AQP2

Figure 4a shows that Lo-TDF þ RSG rats presented with levels of NKCC2 expression comparable with those seen in Lo-TDF group rats (145±2.9 vs 136±2.2%, P ¼ NS). Expression of NKCC2 was significantly higher in the Lo-TDF þ RSG and Lo-TDF group rats than in the control rats and in the RSG rats (Po0.001). In the RSG group, NKCC2 expression did not differ significantly from that observed for the controls (RSG: 111±5.2%).

Actin Control

Lo-TDF

Lo-TDF+RSG

RSG

Figure 4 | Semiquantitative immunoblotting of membrane fractions prepared from kidney medulla. Densitometric analysis of all samples from control, low-dose TDF (Lo-TDF), Lo-TDF þ rosiglitazone (Lo-TDF þ RSG), and rosiglitazone (RSG) rats. (a) The Lo-TDF and Lo-TDF þ RSG rats presented increased expression of the bumetanide-sensitive Na–K–2Cl cotransporter (NKCC2) in relation to controls. Immunoblots reacted with antiNKCC2 revealing a band of 146–176 kDa (centered at 161 kDa). (b) Levels of AQP2 expression were completely restored in response to RSG, also increasing in relation to controls. Immunoblots reacted with anti-AQP2 revealing 29-, 35-, and 50-kDa AQP2 bands.

AQP2 protein expression is significantly increased in rats treated with TDF and RSG

Figure 4b shows that Lo-TDF þ RSG rats presented greater AQP2 expression than that seen in the Lo-TDF group rats (143.3±7.2 vs 67.5±2.5%, Po0.001). In addition, AQP2 abundance was greater in Lo-TDF þ RSG rats than in control rats (143.3±7.2 vs 99±1%, Po0.001). In the RSG group, AQP2 expression increased in comparison with that observed in the control group (RSG group: 165±5.4%, Po0.001). In addition, there was a significant difference between the RSG group and the Lo-TDF þ RSG group in terms of AQP2 expression, which was higher in the RSG group (Po0.05).

RSG reverses the TDF-induced downregulation of NaPi-IIa

DISCUSSION

In rats receiving low-dose TDF, RSG restored NaPi-IIa expression (Figure 2a), explaining the normalization of phosphaturia observed in the Lo-TDF þ RSG group. There was no significant difference between the RSG group rats and the controls in terms of NaPi-IIa expression (RSG: 89.3±2.3%).

Treatment with TDF induced a broad spectrum of nephrotoxicity, including renal failure, proximal tubular injury (hyperphosphaturia and renal tubular acidosis), and reduced urinary concentrating ability. The dose-dependent deterioration in renal function was associated with renal vasoconstriction and reduced abundance of eNOS. We also demonstrated downregulation of membrane transporters (NaPi-IIa, NHE3, and AQP2). Most importantly, RSG reversed all TDFinduced renal alterations. It has been demonstrated that changes in the expression of membrane protein transporters can occur in many forms of acute kidney injury, including those caused by ischemia or nephrotoxicity.21,22 However, it

RSG ameliorates TDF-induced renal tubular acidosis by restoring NHE3 abundance

In the Lo-TDF þ RSG-treated rats, NHE3 protein expression was higher than that seen in the Lo-TDF group rats (93.3±4.4 vs 59±2.1, Po0.01; Figure 2b), and NHE3 914

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remains unknown whether these alterations are specific to injury caused by TDF administration or are simply characteristic of how renal tubular cells react under stress (for example, ischemia) Nevertheless, in this study, alterations of this type occurred in rats treated with tenofovir. Renal failure induced by TDF is dose dependent and is associated with reduced production of NO

In experimental studies involving isolated human proximal tubular cells, TDF presented with lower cytotoxicity than other nucleoside reverse transcriptase inhibitors.23 However, there are studies showing that patients using TDF-containing HAART regimens present with renal dysfunction, whereas control HIV patients do not.24 In other reports, the incidence of renal failure associated with TDF-containing HAART regimens is no different from that associated with other HAART regimens.25,26 For treatment-naı¨ve patients, as well as for treatmentexperienced patients, TDF has rapidly become a favored nucleoside component of antiretroviral regimens. In this study, rats receiving high-dose TDF developed severe renal failure accompanied by intense renal vasoconstriction, neither of which was observed in those receiving the low dose. It has been suggested that TDF causes direct proximal tubular damage, leading to renal failure.27 We have demonstrated herein that another mechanism contributes to TDF-induced renal failure, namely diminished production of NO. In previous studies conducted by our group in patients and in animal models, we found that indinavir also decreases NO production.28,29 RSG reverses TDF-induced downregulation of eNOS and ameliorates renal function

Various studies have shown that PPAR-g agonists are efficacious in slowing the progression of glomerulosclerosis (diabetic and nondiabetic), ischemia–reperfusion injury, and cisplatin nephrotoxicity.19,30–32 Polikandriotis et al.33 demonstrated that RSG increased NO production via distinct PPARdependent signaling pathways. Song et al.20 studied normal rats receiving RSG and showed that eNOS expression in the renal tissue was increased, blood pressure was lowered, and creatinine clearance was reduced. In this study, we demonstrated that 15 days of treatment with RSG did not alter creatinine clearance. In addition, eNOS upregulation was found to be associated with improved renal function in rats receiving TDF. We speculate that, under conditions of renal vasoconstriction, such as that resulting from the administration of TDF, RSG-induced vasodilatation has beneficial effects. Tubular proximal damage induced by TDF can be selective to sodium transporters

In the Lo-TDF experiments, we found tubular alterations and minor (not statistically significant) changes in creatinine clearance. It has been demonstrated that renal tubular dysfunction, with or without a decrease in glomerular Kidney International (2008) 74, 910–918

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filtration rate, occurs in patients.34 It has been demonstrated that TDF use provokes proximal tubular dysfunction, including glycosuria, tubular proteinuria, hyperphosphaturia, and renal tubular acidosis. Kinai and Hanbusa34 studied patients initiating a TDF-containing HAART regimen and reported that 12 out of 17 presented with high levels of urinary b-2 microglobulin accompanied by significant decreases in tubular reabsorption of phosphate. In our experimental model of TDF nephrotoxicity, the effects on the proximal tubules appeared to be specific to sodium transporters (NHE3 and NaPi-IIa), as no glycosuria or increased proteinuria was observed. It is possible that, in rats, prolonged exposure to TDF would cause generalized proximal tubular dysfunction such as that described in humans. One of the most consistent findings in patients suffering from TDF nephrotoxicity is hypophosphatemia.12 The rats in the Lo-TDF group presented with hyperphosphaturia, which is the most likely cause of the hypophosphatemia seen in those same rats. It is of note that, despite presenting with metabolic acidosis, these animals developed hypophosphatemia. In the Lo-TDF group rats, expression of the NaPi-IIa cotransporter was downregulated. This transporter is located in the proximal tubule and is responsible for nearly 80% of phosphate reabsorption along the nephron. The mechanism by which TDF downregulates NaPi-IIa expression remains unexplained. However, the high TDF levels in the proximal tubules might account for this tubular cellular dysfunction. The nonanion-gap metabolic acidosis observed in the rats studied is consistent with the proximal type of renal tubular acidosis, as the pH of urine remained low. Located in the proximal tubule, the NHE3 countertransporter is the principal agent of final bicarbonate generation and reabsorption. We found that NHE3 was downregulated in renal cortex tissue, implicating NHE3 reduction in TDF-induced metabolic acidosis. The possibility that other transporters (basolateral carbonic anhydrase II and IV or sodiumbicarbonate cotransporter (NBC1)) are involved in TDFrelated metabolic acidosis cannot be ruled out. Although TDF-treated rats presented with a marked reduction in proximal sodium transporter abundance, there was no difference between TDF-treated rats and the corresponding control rats in terms of urinary sodium excretion. As a consequence of the decreased protein expression of sodium transporters (especially NHE3), fluid delivery to the distal nephron might be increased. It is possible that the marked increase in NKCC2 band density presented by the TDF-treated rats represents a compensatory response to the greater delivery of sodium chloride and water. It has been shown that sodium transporter abundance is upregulated in response to chronic diuretic treatment in rats.35 This compensatory mechanism could explain the absence of increased kaliuresis in this model of nephrotoxicity, despite the fact that hypokalemia has been reported in patients receiving TDF.36 915

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Tubular toxicity induced by TDF is not restricted to proximal cells

Several cases of NDI have been described in patients receiving TDF.7,13,14 In all cases, the patients also received ritonavir or lopinavir, which have been implicated in altered water–electrolyte balance. Multidrug resistance protein 1 is expressed in the basolateral membranes of cells in the proximal tubules, in Henle’s loop and in the cortical collecting duct. Inhibition of multidrug resistance protein 1 leads to reduced renal tubular responsiveness to vasopressin and can, at least in part, be the cause of NDI. In addition, it is known that ritonavir inhibits multidrug resistance protein 1. Therefore, it has been hypothesized that the TDF–ritonavir combination induces NDI in some patients.27 However, we have demonstrated herein that TDF has the direct effect of reducing the abundance of AQP2, thereby impairing urinary concentrating ability and leading to a state of partial NDI. In a previous study, Andrade et al.21 found that another antiviral agent, acyclovir, induced hyperphosphaturia and hypophosphatemia, which were accompanied by polyuria as well as by increased urinary excretion of sodium, potassium, and magnesium. Similar to tenofovir, acyclovir induced downregulation of Na-Pi-IIa and AQP2 expression. However, unlike tenofovir, acyclovir downregulated NKCC2 expression. As acyclovir is the drug of choice for the treatment of herpes virus simplex encephalitis and varicella-zoster infection in immunocompromised hosts, it is not unusual for the combination of acyclovir and tenofovir to be used in HIV patients, thereby increasing the risk of nephrotoxicity. Treatment with RSG completely reverses TDF-induced tubular toxicity

It is known that PPAR-g is expressed in renal tubular cells, especially those in the collecting duct and also those in the proximal tubule.19 Song et al.20 studied the effects of RSG on the renal tubules of rats. The authors showed that treatment with RSG significantly increased renal abundance of NHE3, NaPi-IIa, NKCC2, and AQP2. These data provide the basis for the theory that RSG reverses TDF-induced tubular toxicity. In addition to its protective effect in TDF-induced renal failure, RSG completely reverses the downregulation of NaPiIIa, NHE3 countertransporter, and AQP2, thereby reverting hyperphosphaturia, metabolic acidosis, and NDI. It is possible that the higher glomerular filtration rate in the Hi-TDF þ RSG group rats resulted from the extracellular volume expansion caused by RSG. However, body weight, which is a measure of extracellular volume expansion, was not significantly higher in the Hi-TDF þ RSG or Lo-TDF þ RSG groups in comparison with the TDF-only and control groups. However, a significant weight gain was observed in the RSG-only group. Glomerular filtration and active tubular secretion partially eliminate TDF. In the latter case, organic anion transporters carry TDF through the basolateral membrane, after which it is secreted into the lumen. This second route of elimination leads to a high TDF concentration in proximal tubular cells, which can increase its toxicity.37 916

AB Libo´rio et al.: Rosiglitazone reverses tenofovir effects

As RSG is extensively metabolized, the unadulterated form of the drug is not excreted in urine. The major forms of metabolism are N-demethylation and hydroxylation, followed by conjugation with sulfate and glucuronic acid. All of the circulating metabolites of RSG are considerably less potent than the parent.38 It has been shown that, following oral or intravenous administration of [14C] RSG maleate, approximately 64 and 23% of the dose is eliminated in the urine and in the feces, respectively.38 There are convincing data in the literature demonstrating RSG-induced increases in the protein expression of sodium transporters and of AQP220,39,40 However, there are few data demonstrating how such increases occur. It is not completely understood how RSG affects the expression of all renal transporters, and there are few data available on this topic. Guan et al.36 demonstrated that thiazolidinediones increase the mRNA expression of Scnn1g, which encodes the gamma subunit of ENaC, indicating that PPAR-g has a direct effect on that gene. An increased risk of myocardial infarction and death from cardiovascular causes has been attributed to RSG use.41 In addition, 10–15% of patients treated with thiazolidinediones develop edema, which often requires the discontinuation of therapy.42 In our study, body weight and urinary osmolality were both higher in the RSG group than in the corresponding control group. The RSG group also presented a significant increase in the urinary excretion of sodium, potassium, and phosphorus in comparison with the corresponding control group. We hypothesize that the greater urinary excretion of these ions is associated with extracellular volume expansion, caused by an increase in a-ENaC and AQP2 protein expression. Considering these recent findings, the clinical use of RSG should be evaluated with caution; patients and providers should bear in mind that treatment with RSG can have serious adverse effects. In conclusion, TDF caused renal failure in a dosedependent matter, leading to a great reduction in eNOS expression and intense renal vasoconstriction. In addition, we found that TDF-induced tubular toxicity involved downregulation of NHE3, NaPi-Iia, and AQP2. We found no proteinuria or glycosuria, although such disorders might appear after prolonged use of TDF. Furthermore, the upregulation of NKCC2 in rats treated with TDF appears to be a compensatory mechanism triggered by greater sodium delivery to the distal nephron segments. Moreover, in this animal model, RSG reversed all mechanisms of TDF-induced nephrotoxicity, re-establishing the expression of eNOS, NHE3, NaPi-Iia, and AQP2, as well as normalizing biochemical parameters. Further studies are needed to determine whether these findings are specific and representative of the effects of TDF use in humans. MATERIALS AND METHODS Animals For the purposes of this study, male Wistar rats, weighing 200–230 g, were obtained from the University of Sa˜o Paulo School of Medicine Kidney International (2008) 74, 910–918

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AB Libo´rio et al.: Rosiglitazone reverses tenofovir effects

animal facilities. The Hi-TDF experiments were designed on the basis of a previous study in rats.43 However, the Hi-TDF dosage caused severe impairment of renal function. We used this dosage to assess creatinine clearance, RBF, renal vascular resistance, and whole-kidney eNOS expression. The Lo-TDF experiments allowed us to assess tubular function without provoking significant impairment of creatinine clearance. The Ethics Committee of the University of Sa˜o Paulo School of Medicine approved the experimental protocol. Metabolic cage studies and renal function studies At 30 days, rats were moved to metabolic cages (one rat per cage), maintained on a 12-h light/dark cycle and given free access to drinking water. The rats were acclimated to the housing conditions for 1 day before the experimental procedures, which began with the collection of 24-h urine samples. Rats in the Hi-TDF groups were anesthetized with intraperitoneal injections of sodium pentobarbital (50 mg/kg body weight) and placed on a temperature-regulated surgical table. A tracheotomy was performed, and a PE50 catheter was inserted into the left femoral artery to record mean arterial pressure. An ultrasonic flow probe (T-106; Transonic Systems Inc., Ithaca, NY, USA) was placed at the left renal artery to monitor RBF. Final blood samples were obtained, and the animals were then killed with a lethal dose of sodium pentobarbital. Analysis of blood and urine samples The volume of each 24-h urine sample was measured gravimetrically. Urine samples were centrifuged in aliquots to remove suspended material, and the supernatants were analyzed. Urine osmolality was measured with a vapor pressure osmometer (5520; Wescor, Logan, UT, USA). Urinary levels of sodium and potassium were measured by flame photometry, and the molybdate method was used to measure urinary phosphate. A colorimetric assay (Labtest, Lagoa Santa, Brazil) was used to quantify urinary levels of glucose, protein, magnesium, and creatinine. Creatinine clearance (in ml/min/100 g body weight) was determined using the following formula: urine creatinineðurine volume ½ml=time ½1440 minÞ =serum creatinine Urinary pH was also measured (MP225 pH meter; Mettler Toledo, Greifensee, Switzerland). Membrane protein isolation Kidneys were dissected to separate cortices and medullae. Cortex and medulla samples were homogenized in ice-cold isolation solution (200 mmol/l mannitol, 80 mmol/l HEPES, 41 mmol/l KOH, pH 7.5) containing a protease inhibitor cocktail (Sigma, St Louis, MO, USA) using a Teflon-pestle glass homogenizer (Schmidt and Co., Frankfurt am Main, Germany). The homogenates were centrifuged at low speed (3000 g) for 15 min at 4 1C to remove nuclei and cell debris. Subsequently, the supernatants were spun at 100,000  g for 1 h at 4 1C (70 Ti rotor, Beckman Coulter, Fullerton, CA, USA) to produce a pellet containing membrane fractions enriched with plasma membranes and intracellular vesicles. The pellets were suspended in isolation solution with protease inhibitors. Protein concentrations were determined using the Bradford assay method (Bio-Rad Protein Assay kit; Bio-Rad Laboratories, Hercules, CA, USA). Kidney International (2008) 74, 910–918

Preparation of renal samples for eNOS protein expression assays The Teflon-pestle glass homogenizer was used to homogenize kidney samples in ice-cold isolation solution (200 mM mannitol, 80 mM HEPES, 41 mM KOH, pH 7.5) containing the protease inhibitor cocktail. The homogenates were centrifuged at low speed (3000 g) for 15 min at 4 1C to remove nuclei and cell debris. The pellets were suspended in isolation solution with protease inhibitors. Protein concentrations were determined using the Bradford assay method (Bio-Rad Protein Assay kit; Bio-Rad Laboratories). Electrophoresis and immunoblotting Immunoblots of membrane fraction samples were run on polyacrylamide minigels: 12.5% gels for AQP2; 10% gels for NaPi-IIa, NHE3, and the a-ENaC; and 8% gels for NKCC2. After electroelution to nitrocellulose membranes (PolyScreen, PVDF Transfer; Life Science Products, Boston, MA, USA), the blots were blocked for 1 h with 5% milk and 0.1% Tween-20 in phosphate-buffered saline (NaCl 8.7 g/l, dibasic phosphate 7.2 mmol/l, and monobasic phosphate 2.8 mmol/l). The blots were then incubated with one of the following: anti-AQP2 antibody (1:2000), NHE3 antibody (1:500), a-ENaC antibody (1:1000); NKCC2 antibody (0.12 mg/ ml); or NaPi-IIa antibody (0.54 mg/ml). The labeling was visualized with horseradish peroxidase-conjugated secondary IgG antibody (anti-rabbit, 1:2000, Sigma, or anti-goat, 1:5000, Sigma) by using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK). The specific polyclonal antibodies to NKCC2, NHE3 and NaPi-IIa were kindly supplied by Dr Mark Knepper (NHLBI/NIH, Bethesda, MD, USA). The specific polyclonal antibodies to AQP2, a-EnaC, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For evaluation of the eNOS isoform, 100 mg of total protein from each sample were separated on an 8% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed with eNOS antibody diluted 1:2000 in Tris-buffered saline and Tween 0.1%. Immunodetection was accomplished using the appropriate anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000 in Tris-buffered saline and 0.1% Tween) and the enhanced chemiluminescence system. To control for loading, the blots were incubated with actin antibody (1:3000, Santa Cruz Biotechnology). The labeling was visualized with horseradish peroxidase-conjugated secondary IgG antibody (anti-goat, diluted 1:5000; Sigma). Quantification of kidney levels of proteins The enhanced chemiluminescence films presenting bands within the linear range were scanned using a video imager (ImageMaster VDS; Pharmacia Biotech, Uppsala, Sweden). All densitometry values were normalized to those obtained for actin protein abundance. Statistical analysis All quantitative data are expressed as mean±s.e.m. Differences among the means of multiple parameters were analyzed by one-way analysis of variance followed by the Student–Newman–Keuls test or Tukey’s test. Values of Po0.05 were considered as statistically significant. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

Sources of support: the Fundac¸a˜o de Pesquisa do Estado de Sa˜o Paulo (FAPESP, Foundation for the Support of Research in the state of 917

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AB Libo´rio et al.: Rosiglitazone reverses tenofovir effects

20.

Sa˜o Paulo); the Laborato´rio de Investigac¸a˜o Me´dica (LIM, Laboratory for Medical Research); and the Fundac¸a˜o Faculdade de Medicina (FFM, School of Medicine Foundation). A.B.L., T.R.C.S. and A.C.S. are recipients of grants from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, National Council for Scientific and Technological Development).

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