Vitamin D deficiency aggravates nephrotoxicity, hypertension and dyslipidemia caused by tenofovir: role of oxidative stress and renin-angiotensin system

Abstract

Vitamin D deficiency (VDD) is prevalent among HIV-infected individuals. Vitamin D has been associated with renal and cardiovascular diseases because of its effects on oxidative stress, lipid metabolism and renin-angiotensin-aldosterone system (RAAS). Tenofovir disoproxil fumarate (TDF), a widely used component of antiretroviral regimens for HIV treatment, can induce renal injury. The aim of this study was to investigate the effects of VDD on TDF-induced nephrotoxicity. Wistar rats were divided into four groups: control, receiving a standard diet for 60 days; VDD, receiving a vitamin D-free diet for 60 days; TDF, receiving a standard diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days; and VDD+TDF receiving a vitamin D-free diet for 60 days with the addition of TDF for the last 30 days. TDF led to impaired renal function, hyperphosphaturia, hypophosphatemia, hypertension and increased renal vascular resistance due to downregulation of the sodium-phosphorus cotransporter and upregulation of angiotensin II and AT1 receptor. TDF also increased oxidative stress, as evidenced by higher TBARS and lower GSH levels, and induced dyslipidemia. Association of TDF and VDD aggravated renovascular effects and TDF-induced nephrotoxicity due to changes in the redox state and involvement of RAAS.

Figure 1. Representative photomicrographs of the tubular injury.

(a) control, (b) VDD, (c) TDF and (d) VDD+TDF. Magnification, 400x. (e) Score of tubular injury of C, VDD, TDF and VDD+TDF. Values are means ± SEM. ^ap<0.001 vs. C; ^bp<0.001 vs. TDF.

Figure 2. Semiquantitative immunoblotting of kidney fractions.

(a) A densitometric analysis of samples from control (n = 4), VDD (n = 6), TDF (n = 6) and VDD+TDF (n = 6) rats is shown. (b) Immunoblots reacted with anti-angiotensinogen revealing a 60-kDa band. Values are means ± SEM. ^ap<0.05 vs. C.

Figure 3. Expression of Angiotensin II in renal tissue.

Immunostaining for angiotensin II (brown) in kidney cortex samples from C (a), VDD (b), TDF (c) and VDD+TDF (d). Magnification, ×400. Note that the staining is more extensive and intense in the sample from the VDD, TDF and VDD+TDF.

Figure 4. Semiquantitative immunoblotting of kidney fractions.

(a) A densitometric analysis of samples from control (n = 4), VDD (n = 6), TDF (n = 6) and VDD+TDF (n = 6) rats is shown. (b) Immunoblots reacted with anti-AT1 revealing a 43-kDa band. Values are means ± SEM. ^ap<0.05 vs. C; ^bp<0.01 vs. C and ^cp<0.05 vs. TDF.

Figure 5. Bar graph representation of the renal mRNA expression of components of renin-angiotensin system in C (n = 5), VDD (n = 6), TDF (n = 5) and VDD+TDF (n = 6).

Figure 6. Semiquantitative immunoblotting of kidney fractions.

(a) A densitometric analysis of samples from control (n = 4), VDD (n = 6), TDF (n = 6) and VDD+TDF (n = 6) rats is shown. (b) Immunoblots reacted with anti-eNOS revealing a 140-kDa band. Values are means ± SEM. ^ap<0.001 vs. C; ^bp<0.001 vs. TDF.

Figure 7. Semiquantitative immunoblotting of kidney fractions.

(a) A densitometric analysis of samples from control (n = 4), VDD (n = 6), TDF (n = 6) and VDD+TDF (n = 6) rats is shown. (b) Immunoblots reacted with anti-NaPi-IIa revealing a 85-kDa band. Values are means ± SEM. ^ap<0.001 vs. C; ^bp<0.001 vs. TDF.

Figure 8. Serum and urinary TBARS.

(a) Serum TBARS concentration of C, VDD, TDF and VDD+TDF groups and (b) urinary TBARS excretion of C, VDD, TDF and VDD+TDF groups. Values are means ± SEM. ^ap<0.05, ^bp<0.001 vs. C; ^cp<0.01 vs. TDF.

Figure 9. Whole blood glutathione concentration of C, VDD, TDF and VDD+TDF groups.

Values are means ± SEM. ^ap<0.05, ^bp<0.01, ^cp<0.001 vs. C; ^dp<0.01 vs. TDF.