Regression of albuminuria and hypertension and arrest of severe renal injury by a losartan-hydrochlorothiazide association in a model of very advanced nephropathy

Abstract

Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization.

Figure 1. Percent Survival in Protocol 1 (a) and Protocol 2 (b).

S, Sham-operated; Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan and hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. *, p<0.05 vs. Sham; ^#, p<0.05 vs. Nx+V; ^& p<0.05 vs. Nx+L and ^¢, p<0.05 vs. Nx+LH.

Figure 2. Tail-cuff pressures (TCP, mmHg) in Protocol 1 (a) and Protocol 2 (b).

S, Sham-operated (clear circles); Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V (filled circles), untreated Nx; Nx+L (triangles), losartan-treated Nx; Nx+LH (diamonds), Nx treated with losartan+hydrochlorothiazide; Nx+AHHz (squares), Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 3. Urinary albumin excretion rates (U_albV, mg/24 h) in Protocol 1 (a) and Protocol 2 (b).

S, Sham-operated (clear circles); Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V (filled circles), untreated Nx; Nx+L (triangles), losartan-treated Nx; Nx+LH (diamonds), Nx treated with losartan+hydrochlorothiazide; Nx+AHHz (squares), Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 4. Plasma aldosterone (ALDO, pg/mL) in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 5. Representative microphotographs of renal tissue from Nx_pre (60 days after renal ablation) and from all other groups (150 days after renal ablation) in Protocol 1.

PAS, Periodic Acid-Schiff; %INT, percent cortical area occupied by interstitium in sections stained with Masson trichrome; α-SMA, alpha-smooth muscle actin.

Figure 6. Representative microphotographs of renal tissue from Nx_pre (120 days after renal ablation) and from all other groups (210 days after renal ablation) in Protocol 1.

PAS, Periodic Acid-Schiff; %INT, percent cortical area occupied by interstitium in sections stained with Masson trichrome; α-SMA, alpha-smooth muscle actin.

Figure 7. Percent glomerulosclerosis (%GS) in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 days or 120 after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 8. Quantitative analysis of percent renal area occupied by collagen I in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 9. Percent cortical alpha-smooth muscle actin (α-SMA) in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.

Figure 10. Representative microphotographs of renal tissue obtained for Nx_pre (60 days after renal ablation) and for all other groups (150 days after renal ablation) in Protocol 1.

AII, tubulointerstitial cells staining positively for AII; PCNA, proliferating-cell nuclear antigen; NCC, sodium-chloride cotransporter, specific for distal convoluted tubule (DCT). Arrowheads in Figs. 10c, f, i, l, o and r (double staining for PCNA and NCC) indicate examples of PCNA-positive cells in DCT.

Figure 11. Representative microphotographs of renal tissue obtained for Nx_pre (120 days after renal ablation) and for all other groups (210 days after renal ablation) in Protocol 2.

AII, tubulointerstitial cells staining positively for AII; PCNA, proliferating-cell nuclear antigen; NCC, sodium-chloride cotransporter, specific for distal convoluted tubule (DCT). Arrowheads in Figs. 11c, f, i, l, o and r (double staining for PCNA and NCC) indicate examples of PCNA-positive cells in DCT.

Figure 12. PCNA (Proliferating-cell nuclear antigen)-positive cells in glomerular (dotted areas), tubular (dark grey areas) and interstitial (light grey areas) compartments in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH. Letters denoting significance are placed immediately above the corresponding bar area.

Figure 13. Frequency of proliferating cells in distal convoluted tubule, evaluated by double staining for PCNA (Proliferating-cell nuclear antigen) and NCC (distal convoluted tubule-specific sodium-chloride cotransporter) in Protocol 1 and Protocol 2.

S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.